Construction and characterization of an n-butanol...

Construction and characterization of an n-butanol synthetic pathway in Escherichia coli

by

Brooks Bennett Bond-Watts

A dissertation submitted in partial satisfaction of the

requirements for the degree of

Doctor of Philosophy in

Chemistry

in the

Graduate Division

of the

University of California, Berkeley

Committee in charge:

Professor Michelle C. Y. Chang, Chair

Professor Matthew B. Francis

Professor Susan Marqusee

Fall 2013


© 2013

by Brooks Bennett Bond-Watts

Abstract


by

Brooks Bennett Bond-Watts

Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Michelle C. Y. Chang, Chair

A diverse array of molecular functions has evolved in biological systems. The ability to

reorganize and utilize these remarkable capabilities in order to design pathways for in vivo chemical synthesis requires expanding on foundational biochemical principles in order to better understand the function of entire pathways, thereby allowing for de novo design. Furthermore, in order to integrate these synthetic pathways effectively into organisms such that they are capable of working at the desired high flux, it is necessary to understand the principles affecting pathway flux and pathway interactions with the native metabolism.

To this end, we have engineered Escherichia coli to synthesize n-butanol from acetyl-CoA. In design of a high flux de novo pathway, we have demonstrated that using the enzymatic reaction mechanism of the reduction of the enoyl-CoA intermediate as a kinetic control element can drive flux to the final product. This is in constrast to high yield pathways that use a thermodynamic control element to drive the pathway to completion, generally decarboxylation or sequestration of an insoluble product. These methods have the disadvantages of using ATP and limiting the range of target molecules, respectively. Using the enoyl-CoA reduction to drive pathway flux, we have constructed a chimeric pathway capable of producing titers of n-butanol competitive with native fermentative pathways in E. coli (4650 ± 720 mg/L).

The enoyl-CoA reductase, tdTer, plays a key role in the production of high titers of n-butanol. We characterized this enzyme structurally and biochemically to examine its potential for use with other substrates. The crystal structure of tdTer was determined to 2.00 Å resolution and shows the enzyme is highly similar to members of the FabV family of enoyl-CoA reductases. Biochemical studies show that similar to other enoyl-CoA (ACP) reductases, the enzyme utilizes an ordered bi-bi reaction mechanism initiated by binding of the NADH redox cofactor. Analysis of the activity and inhibition of the C4, C6, and C12 substrates and products with a variety of binding loop mutants suggests the region is important in discrimination of chain length and points to the major portal as a target for modifying chain length specificity.

The introduction of tdTer as the enoyl-CoA reductase was shown to be a key facor in the high yield of n-butanol from our synthetic pathway. In order to study the flux through this step, we implemented a protein scaffold system to colocalize enzymes in the pathway via fusing the enzymes of interest to ligand binding domains. We colocalized tdTer with the enzyme catalyzing the previous step in the biosynthetic pathway and separately with enzyme catalyzing the next

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n-butanol production, and that the fusion of an affinity tag with the AdhE2 enzyme is capable of increasing specificity of the enzyme. Unfortunately, the deleterious effects of introducing the scaffolding system and the lack of reproducibility in the results prevents the use of the system to effectively probe flux through the n-butanol synthetic pathway.

In order to expand the ability of the pathway to probe the metabolism of E. coli, the pathway was modified to draw on the malonyl-CoA pool in addition to acetyl-CoA. To do so, the first enzyme was replaced with NphT7, which condenses one malonyl-CoA and one acetyl-CoA to one acetoacetyl CoA. The introduction of NphT7 creates a significant bottleneck primarily related to malonyl-CoA availability. Increasing the intracellular malonyl-CoA concentration through genetic engineering and heterologous expression was capable of increasing n-butanol production, but not to the levels seen in the acetyl-CoA dependent pathway. We would like to use the modified pathway to identify changes to NphT7 or the host metabolism capable of increasing flux through malonyl-CoA. In order to identify desired mutations to nphT7 and/or the E. coli genome, a strain was developed in which growth rate was correlated with n-butanol production. Use of this strain allowed for the selection of mutated strains with increased n-butanol production; however, low n-butanol yield under selecting conditions and contamination of high yield strains have thus far prevented the idenification of high yield strains. Additional method development allowing for new types of library design is likely necessary in order to identify mutants resulting in high yield malonyl-CoA based pathways.

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Table of Contents Table of Contents i

List of Figures, Schemes, and Tables iii

List of Abbreviations viii

Acknowledgments x

Chapter 1: Introduction 1.1 Introduction 2

1.2 Production of short-chain alcohols from branched amino acids 3

1.3 Production of short-chain alcohols from acetyl-CoA 5

1.4 Production of diesel targets from acetyl-CoA 8

1.5 Conclusions 10

1.6 References 11

Chapter 2: Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways 2.1 Introduction 16

2.2 Materials and methods 17

2.3 Results 26

2.4 Discussion 38

2.5 References 39

Chapter 3: Biochemical and Structural Characterization of the trans-Enoyl-CoA Reductase from Treponema denticola 3.1 Introduction 45


3.3 Results and discussion 51

3.4 Conclusions 66

3.5 References 66

i

Chapter 4: Analysis of pathway flux utilizing co-localization via a protein scaffold 4.1 Introduction 72



4.4 Conclusions 83

4.5 References 84

Chapter 5: Design and optimization of a malonyl-CoA dependent pathway for production of n-butanol 5.1 Introduction 88



5.4 Conclusions 109

5.5 References 109

Appendices Appendix 1: Oligonucleotides maps and primer sequences for gene synthesis 113

of genes used in discussed experiments

Appendix 2: The complete list of plasmids and strains used in this study 177

Appendix 3: The complete list of plasmids and strains generated 181

Appendix 4: Oligonucleotides used for cloning and genetic engineering 196

Appendix 5: Oligonucleotides used for sequencing 205

ii

List of Figures, Schemes, and Tables

Chapter 1

Figure 1.1 The production of advanced biofuels 2

Figure 1.2 The production of alcohols from amino acid metabolism 4

Figure 1.3 The production of n-butanol from acetyl-CoA 6

Figure 1.4 The production of biodiesel from acetyl-CoA 9

Chapter 2

Table 2.1 E. coli strains containing n-butanol pathway variants 19

Scheme 2.1 Comparison of a high-yielding polyhyrdoxyalkanoate pathway with 26

the design of a chimeric n-butanol pathway

Figure 2.1 In vivo production of E. coli strains containing various synthetic 27

n-butanol biosynthetic pathways

Figure 2.2 Detection of n-butanol by GC-MS 29

Figure 2.3 Standard curves for quantitation of n-butanol by GC-MS and 29

GC-FID

Figure 2.4 Monitoring the dependence of n-butanol titers on concentration of 30

soluble intracellular Ccr

Figure 2.5 The consumption of butyryl-CoA by E. coli cell lysate 30

Figure 2.6 In vitro characterization of His6-Ter from T. denticola 31

Figure 2.7 The consumption of n-butanol pathway intermediates in cell lysate 32

Table 2.2 PhaA, PhaB/Hbd, and Crt specific activities in cell lysate 32

Figure 2.8 In vitro characterization of Ccr-His6 from S. collinus 34

Figure 2.9 In vitro characterization of Strep-Crt from C. acetobutylicum 34

Scheme 2.2 Optimization of the synthetic Ter-dependent pathway for n-butanol 35

production

Table 2.3 Quantitation of NAD(P)H and NAD(P)+ n-butanol producing strains 35

Table 2.4 His6-Ter kinetics with respect to crotonyl-CoA and isocrotonyl-CoA 36

Table 2.5 Specific activity of the PDHc in cell lysate 37

Figure 2.10 Time course of production with Ter based n-butanol pathway 37

iii

with and without heterologous PDHc expression

Figure 2.11 Production with Ccr based n-butanol pathway with and without 37

heterologous PDHc expression

Chapter 3

Figure 3.1 Compressed phylogenetic tree of Ter, FabV, and FabI homologs 52

Figure 3.2 Uncompressed phylogenetic tree of Ter, FabV, and FabI homologs 52

Figure 3.3 SDS-PAGE analysis of purifications of tdTer variants 53

Figure 3.4 Size-exclusion chromatograms of purified tdTer variants 53

Table 3.1 Data collection and refinement statistics for GTGA-Ter structures 54

Figure 3.5 Crystal structure of tdTer 55

Figure 3.6 View of the Rossman fold of tdTer 56

Figure 3.7 Orientation of Lys247 and Lys249 in the tdTer active site 57

Figure 3.8 Comparison of the ACP binding site of FabV from Y. pestis 58

to tdTer

Figure 3.9 Docking model of tdTer with NADH 59

Figure 3.10 Docking model of tdTer with NADH and crotonyl-CoA 59

Figure 3.11 Docking model of tdTer with the most favored ternary complexes 61

Figure 3.12 Steady state characterization of tdTer with enoyl-CoA substrates 61

Table 3.2 Kinetic constants measured for enoyl-CoA reduction by wild-type 62

and mutant TdTers

Figure 3.13 Amino acids mutated in tdTer variants 63

Figure 3.14 Substrate inhibition of tdTer 64

Table 3.3 Kinetic constants for the product inhibition of tdTer 64

Figure 3.15 Product inhibition of tdTer 65

Chapter 4

Figure 4.1 The design of the protein scaffold system 72

Table 4.1 Oligonucleotides used for cloning in this study 74

Table 4.2 The complete list of plasmids used in this study 75

Figure 4.2 Variation in n-butanol production due to culture vessel 79

Figure 4.3 Variation in n-butanol production due to culture seal 79

iv

Figure 4.4 The relative amount of n-butanol production in cells expressing 80

enzymes fused to binding ligands

Figure 4.5 n-Butanol production levels using the Crt_PDZ and/or SH3_Ter 80

fusion enzymes and titrated expression of the protein scaffold

Figure 4.6 The ethanol and n-butanol production levels for the biosynthetic 82

system using the Ter_PDZ and/or SH3_AdhE2 fusion enzymes

Table 4.3 Michaelis-Menten kinetic parameters for AdhE2 and SH3_AdhE2 82

Figure 4.7 SDS-PAGE analysis of AdhE2 and SH3_AdhE2 purifications 83

Chapter 5

Figure 5.1 The fate of carbon from glucose through central metabolism 88

Figure 5.2 Design of a malonyl-CoA dependent pathway for production of 89

n-butanol

Table 5.1 Plasmids and strains used in this study 91

Table 5.2 Oligonucleotides used for cloning and gene disruption 92

Table 5.3 Oligonucleotides used for sequencing 95

Table 5.4 Analysis of mutation frequency for nphT7 libraries generated 95

by ePCR

Figure 5.3 Oligonucleotides used to perform saturation mutagenesis in the 96

NphT7 enzyme using the ligation based method

Table 5.5 Oligonucleotides used to perform saturation mutagenesis in the 97

NphT7 enzyme using the homologous recombination method

Figure 5.4 Design of an E. coli knockout strain for studying n-butanol 101

production with NphT7

Figure 5.5 Promoter and RBS optimization of n-butanol production 102

using an nphT7 based pathway

Table 5.6 Specific activity of other pathway enzymes 102

Figure 5.6 Examining the effect of ACS and ACCase overexpression on 103

n-butanol production using an nphT7 based pathway

Figure 5.7 Examining the effect of malonic acid feeding with MatB 105

expression using an nphT7 based pathway

Figure 5.8 Production of n-butanol using the nphT7 based pathway in 105

v

cultures mutagenized the EMS

Figure 5.9 Optimization of production of n-butanol under selecting 106

growth conditions

Figure 5.10 Growth of cultures expressing ePCR generated nphT7 mutants 107

in the n-butanol producing pathway

Figure 5.11 Selecting residues in NphT7 for saturation mutagenesis 108

Table 5.7 Summary of selection efforts for optimizing an nphT7 108

dependent pathway

Appendix 1

Table A1.1 Oligonucleotide list for accA3 gene synthesis 114

Table A1.2 Oligonucleotide list for accBC gene synthesis 116

Table A1.3 Oligonucleotide list for accD4 gene synthesis 118



Table A1.6 Oligonucleotide list for accE gene synthesis 124

Table A1.7 Oligonucleotide list for tbACS4 gene synthesis 125

Table A1.8 Oligonucleotide list for aldh593 gene synthesis 127

Table A1.9 Oligonucleotide list for alkK gene synthesis 129

Table A1.10 Oligonucleotide list for atfA gene synthesis 131

Table A1.11 Oligonucleotide list for bdhA gene synthesis 133

Table A1.12 Oligonucleotide list for bdhB gene synthesis 135

Table A1.13 Oligonucleotide list for ccr from Streptomyces 137

cinnamonensis gene synthesis

Table A1.14 Oligonucleotide list for dtsR1 gene synthesis 139

Table A1.15 Oligonucleotide list for jFar gene synthesis 141

Table A1.16 Oligonucleotide list for hbd gene synthesis 143

Table A1.17 Oligonucleotide list for nphT7 gene synthesis 144

Table A1.18 Oligonucleotide list for phaJ gene synthesis 145

Table A1.19 Oligonucleotide list for ppPhaJ4 gene synthesis 146

Table A1.20 Oligonucleotide list for phaZ1 gene synthesis 147

Table A1.21 Oligonucleotide list for egTer gene synthesis 149

vi

Table A1.22 Oligonucleotide list for tdTer gene synthesis 151

Figure A1.1 Oligonucleotide maps for the assembly of synthetic genes 153

Appendix 2

Table A2.1 Plasmids and strains that have been previously published 178

Table A2.2 Plasmids and strains used in unpublished experiments 179

Appendix 3

Table A3.1 The complete list of strains engineered 182

Table A3.2 The complete list of plasmids cloned 183

Appendix 4

Table A4.1 Oligonucleotides used for molecular cloning and strain 197

engineering

Appendix 5

Table A5.1 Oligonucleotides used for sequencing 206

vii

List of Abbreviations

3MB 3-methyl-1-butanol

ABE acetone, n-butanol, and ethanol

ACCase acetyl-CoA carboxylase

ACP acyl carrier protein

ADH alcohol dehydrogenase

aTet anhydrotetracycline

ATP adenosine-5´-triphosphate

CoA coenzyme A

Cb carbenicillin

Cm chloramphenicol

CRP catabolite repressor protein

DCW dry cell weight

DIEA N,N-diisopropylethylamine

dNTP deoxynucleotide triphosphate

DMAPP dimethylallyl diphosphate

DMF dimethylformamide

DMSO dimethyl sulfoxide

DNase deoxyribonuclease

DTT diothiothreitol

dTTP deoxythymidine triphosphate

DXP deoxyxylulose-5-phosphate

EDTA ethylenediaminetetraacetic acid

ePCR error-prone polymerase chain reaction

ESI-MS electron spray ionization mass spectrometry

FAEE fatty acid ethyl esters

FPLC fast protein liquid chromatraphy

FPP farnesyl diphosphate

GC-FID gas chromatography-flame ionization detection

GC-MS gas chromatography-mass spectrometry

HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

IPP isopentenyl dephosphate

IPTG isopropyl β-D-1-thiogalactopyranoside

viii

KCM KCl, CaCl2, MgCl2

KDC 2-keto acid decarboxylase

Km kanamycin

LB Luria broth with Miller’s modification

LC-MS liquid chromatography- mass spectrometry

MAD multiwavelength anomalous diffraction

MOPS 3-(N-morpholino)propanesulfonic acid

NMR nuclear magnetic resonance

OD600 optical density at 600 nm

PCR polymerase chain reaction

PDHc pyruvate dehydrogenase complex

PHA polyhydroxyalkanoate

PMSF phenylmethanesulfonyl fluoride

RMSD root-mean-square deviation

RP-HPLC reversed phase-high performance liquid chromatography

rpm revolutions per minute

SDS-PAGE sodium dodecyl sulfate - polyacrylamide gel electrophoresis

SEC size exclusion chromatography

Sp spectinomycin

TCEP tris(2-carboxyethyl)phosphine

TEA triethylamine

TEMED N,N,N′,N′-tetramethylethane-1,2-diamine

Tet tetracycline

TEV tobacco etch virus

TB terrific broth

TCEP tris(2-carboxyethyl)phosphine

Tris trisaminomethane

ix

Acknowledgments

There are many people to thank for helping me complete this work. Foremost, I would like to

thank Dr. Michelle Chang, my advisor, for her guidance throughout my time in graduate school. Without her help, I would have learned and accomplished a small fraction of what I have during my time at UC Berkeley. I would also like to thank my committee members, Dr. Susan Marqusee and Dr. Matt Francis for their support.

I also owe a great debt for the help provided by all of the members of the Chang lab for their help and comradery over the years. I especially would like to thank the butanol subgroup, Matt Davis, Dr. Jeff Hanson, and Dr. Miao Wen. In addition, my classmates Dr. Amy Weeks, Dr. Mark Walker, and Dr. Maggie Brown have time and again proved to be important supports, helping me through graduate school. I would also like to thank Bob Bellerose and Tim Roth, two undergraduates who worked with me. In addition, I have worked with a number of gifted rotation students who helped me explore a variety of areas of the project: Matt Davis, Ben Thuronyi, Kat Hirano, Meera Atreya, Mark Grabiner, Mike Blaisse, Vimalier Reyes, Minxi Rao, Peter Robinson, and Luke Latimer.

Finally I would like to thank my friends and loved ones who supported, encouraged, discouraged, and dealt with me, as well as the weird habits and hangups that my journey through Berkeley entailed.

x

Chapter 1: Introduction

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1.1. Introduction

Living systems offer a unique solution to targeted chemical synthesis, providing a diverse array of transformations to build inexpensive and sustainable routes to the production of fine and commodity chemicals from simple renewable carbon sources like glucose [1-3]. Indeed, many classes of compounds, such as amino acids, vitamins, and antibiotics, are currently produced commercially by fermentation of their native host organism [4-6]. One major area in which microbial synthesis has been tapped is the production of liquid transportation fuels from renewable plant biomass carbon sources. In this case, the domestication of yeasts throughout human history has allowed the establishment of strains that produce ethanol from glucose at near quantitative yield. Since net economic, energy, and carbon balance for fuels are tied directly to yield from glucose, ethanol has been the dominant biofuel in use to date based on its robust and efficient production as well as our expertise in its manufacture [7,8]. However, the molecular properties of ethanol as a fuel product create major limitations in developing a sustainable biofuel pipeline [9,10]. Its high miscibility with water and low energy density compared to gasoline significantly increases the energy usage for its separation from aqueous fermentations and transportation by truck while also preventing high percentage blends with gasoline for use in our current transportation fleet. Thus, the development of microbial strains with the ability to produce drop-in gasoline or diesel additives and replacements – such as longer chain alcohols, esters, and alkanes – would allow us to begin stepping beyond these issues (Figure 1.1A).

However, a major roadblock in the development of new fermentation processes is that naturally-occurring microbial hosts that combine production of desirable gasoline and diesel targets with the speed, efficiency, and low cost of yeast fermentation have yet to be identified. In this regard, advances in metabolic engineering and synthetic biology offer us the opportunity to engineer pathways for the production of advanced biofuel targets in a well-studied and

Figure 1.1. The production of advanced biofuels. (A) Structures of advanced fuel targets compared to ethanol. (B) Pathways to advanced biofuels from building blocks derived from central metabolism.

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industrially-relevant host [11-14]. One challenge is that the metabolic logic underlying the design of high-flux pathways for these advanced targets is quite different from the production of ethanol, which has several advantageous features for high yield. First, ethanol is made directly from pyruvate as a fermentation product that balances the redox requirements of glycolysis (Figure 1.1B). As a result, ethanol production is required for survival of the host under anaerobic conditions by allowing glycolysis to continue as the sole source of ATP, which generates a selective pressure for quantitative yield from glucose and limits the spread of carbon through the metabolic network. Furthermore, the first step in its biogenesis is the decarboxylation of pyruvate, creating an irreversible sink to drive the pathway equilibrium to completion and immediately commit carbon to its production. In contrast, next-generation fuels are assembled from building blocks derived from pathways that extend beyond glycolysis, which makes their biosynthesis more complex as this aspect greatly expands both the number of metabolic transformations and competing pathways that are involved (Figure 1.1B). In addition, the biosynthetic pathways for the production of these advanced targets require many more steps than for ethanol and therefore more components to manage in order to engineer high flux to the end product. In this context, we will discuss the construction of several different engineered pathways for the production of advanced biofuel targets in Escherichia coli.

1.2. Production of short-chain alcohols from branched amino acids

The addition of two carbons to ethanol to make a C4 alcohol greatly reduces its solubility in water (~7 g/L) while also increasing energy density [11]. Thus, C4 and other short-chain alcohols are currently regarded as the next-generation biofuel to replace ethanol. Besides the energetic benefits from separations and transport related to their low water solubility, they can also be blended at high percentage with gasoline to meet future government mandates or perhaps even be used as a drop-in fuel for traditional combustion engines [15,16]. Some of these short-chain alcohols, such as isobutanol, have been observed as trace products of microbial amino acid metabolism through the Erlich degradation pathway for 2-keto acids, which are converted to alcohols in two steps (Figure 1.2A) [17,18]. Like for ethanol production, this particular pathway has the advantage that it can also be driven forward by an irreversible decarboxylation. Work from both academic [19] and industrial groups [20] has shown that this pathway can be exploited to produce a broad range of products by engineering endogenous amino acid metabolism as well as the degradation of 2-keto acids.

The initial insertion of the Erlich degradation pathway consisting of a broad specificity 2-keto acid decarboxylase (KDC, kivd from Lactococcus lactis) and alcohol dehydrogenase (ADH, adh2 from Saccharomyces cerevsiae) resulted in the production of 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol in E. coli [19]. The increase in yields and product selectivity upon feeding of 2-keto acid precursors suggested that flux was limited by the availability of intracellular 2-keto acids rather than by the downstream KDC-ADH pathway. Thus, isobutanol titers were improved by overexpressing genes converting pyruvate to 2-ketoisovalerate (alsS and ilvCD), the precursor to isobutanol (Figure 1.2A). Upon deletion of pyruvate-formate lyase (pflB) to decrease competition for pyruvate, the engineered E. coli host was able to produce 22 g/L of isobutanol at a yield of 86% of the theoretical maximum [19]. In addition to isobutanol, n-butanol and n-propanol can be produced from 2-ketovalerate by engineering its production from threonine using thrABC (Figure 1.2A) [21].

3

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4

The high efficiency of this pathway opens the door for expanded engineering applications, such as new host organisms [22,23] or alcohol products [24]. Since the chain length of the alcohols derived from this pathway is limited by the carbon number in the amino acid pathways, a “+1” pathway derived from the extension of 2-ketoisovalerate to 2-ketoisocapronate used in leucine biosynthesis (leuABC) has been used to increase the size of the 2-keto acid precursors for alcohol production (Figure 1.2B) [25]. Another interesting advantage of using amino acid precursors for fuel production is that these pathways provide an alternative use for the protein hydrolysates resulting from microbial fermentations. In order to access these substrates, the carbon skeleton must be released from the amino acid rather than being trapped within nitrogen metabolism pathways. In this regard, the keto acid pathway was used to demonstrate the feasibility of fuel synthesis from amino acids by engineering nitrogen flux [26]. To start, amino acids were denitrified by the introduction of three different transamination and deamination cycles, thereby releasing high levels of ammonia (Figure 1.2C). To divert carbon flux into alcohol synthesis, a metabolic driving force is created by blocking ammonia from re-entry into cell metabolism. By creating this sink, a mixture of alcohols (isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol (3MB)) can be produced at high titers (4.0 g/L) and yields (56% of theoretical yield) from protein sources.

The Ehrlich pathway has also been introduced into other organisms to produce isobutanol in alternative host organisms. The Ehrlich is naturally seen in S. cerevisiae, but produces low levels of fuel molecules, typically on the order of 10 mg/L [27,28]. The pathway flux is complicated by the upstream part of the pathway (the generation of valine or α-ketoisovalerate confined to the mitochondria while the KDC and ADH are present in the cytoplasm. Overexpression of the upstream pathway enzymes in the cytoplasm has been shown to cause an increase in biofuel production up to 630 mg/L [29]. The introduction of the KDC and ADH enzymes to the mitochondria has been shown to be much more successful in generating high biofuel titers, with titers seen as high as 850 mg/L [28,30]. The pathway has also been introduced into R. eutrophus in order to couple fuel production with CO2 and electricity as the sole carbon and energy inputs. Through the reduction of CO2 to formate which fed the production of isobutanol and 3MB production of up to 140 mg/L of biofuels could be achieved [23].

1.3. Production of short-chain alcohols from acetyl-CoA

In addition to the production of isobutanol from amino acids, n-butanol can be synthesized directly from acetyl-CoA in an analogous fashion to fatty acid biosynthesis (Figure 1.3). n-Butanol is naturally produced by Clostridium acetobutylicum as part of its native fermentation pathways as a mixture of acetone, n-butanol, and ethanol (ABE) [31]. The ABE fermentation was first designed in the 1920s and has been optimized for either acetone or n-butanol over time. In an effort to improve industrial production, this pathway was transplanted directly into yeast and E. coli but is limited to n-butanol production yields of <1% to 10% [13,32-34], which is significantly lower than the yield in the native butanologenic host (45%). These results suggested that the enzymatic pathway from C. acetobutylicum is limited in its ability to produce n-butanol at high flux, with different studies implicating the enoyl-CoA reduction step as the bottleneck in the pathway [33,35].

The dependence of n-butanol production on the enoyl-CoA reduction step led our group to further evaluate its role in controlling pathway flux [35]. Interestingly, E. coli native metabolism does not appear to react with either crotonyl-CoA or butyryl-CoA, which are respectively the

5

Figure 1.3. The production of n-butanol from acetyl-CoA. A synthetic pathway for the production of n-butanol based on the clostridial pathway.

6

substrate and product of the enoyl-CoA reductase. These results indicate that enoyl-CoA reduction may be important for trapping carbon in the synthetic pathway and pulling it away from intermediates that can dissipate to other parts of the host metabolic network. This function is especially important given the rapid and high reversibility of several pathway components, such as the thiolase (PhaA) (Keq ~ 10-5) and the crotonase (Crt) (Keq ~ 10-1) (Figure 1.3). Indeed, feeding of crotonyl-CoA in the presence of the heterologously-expressed pathway led to reversion to its precursor, 3-hydroxybutyryl-CoA instead. We therefore reasoned that the enoyl-CoA reduction was particularly important for drawing carbon away from the reversible and non-committed steps early in the pathway and that an effectively irreversible step was required at this node to drive the pathway equilibrium to completion. Since the thermodynamics of the enoyl-CoA reduction step could not be changed, we decided to utilize a kinetic trap instead by replacing the native enoyl-CoA reductase from C. acetobutylicum with a mechanistically distinct enzyme with a high kinetic barrier to the back reaction related to its use of a single NADH co-factor [35]. We selected and tested the trans-enoyl-CoA reductase (Ter) from Treponema denticola as a potential kinetic trap and found that titers could be amplified 1,000-fold compared to our original pathway design to >4 g/L (41% of theoretical yield). To evaluate the contribution of Ter as a kinetic trap, we took advantage of its ability to biochemically distinguish trans- and cis-butenoyl-CoA as substrate. Although the catalytic efficiency of Ter with respect to cis-butenoyl-CoA is 105-fold lower compared to trans-butenoyl-CoA, the n-butanol titer only drops by 10-fold with the incorrect stereochemistry, which demonstrates that the kinetic trapping behavior appears to be the key feature of Ter for increasing flux through the synthetic pathway. This effective irreversibility can be further used to amplify n-butanol titers through anaerobic production in engineered E. coli strains with fermentation pathways knocked out, leading to product titers of 30 g/L under fed-batch conditions [36].

Pathways based on the C. acetobutylicum ABE pathway have also been introduced into other hosts for n-butanol production. Introduction of the pathway into S. cerevisiae by the Keasling group has resulted in low levels of n-butanol produced. Through the expression of genes from C. acetobutylicum and comparisons with expression of homologous genes from other organisms, they were able to reach titers of up to 2 mg/L n-butanol [37]. The Prather group has engineered Pseudomonas putida and Bacillus subtilis to produce n-butanol at titers of 122 and 24 mg/L, respectively, again using a pathway dependent on the butyryl-CoA dehydrogenase from C. acetobutylicum. They were able to demonstrate that production of n-butanol in P. putidia depended on expression of the bcd-etfAB genes [38]. An ABE like pathway using the Ter enzyme has also been introduced into the cyanobacteria Synechococcus elongatus, an organism natively capable of growing photosynthetically with CO2 as the sole carbon source. The production of n-butanol from reduced carbon compounds generated photosynthetically was achieved and optimization of transcription levels and enzyme activity resulted in yields up to 13 mg/L [39].

n-Butanol can also be produced by engineering the native metabolism of E. coli and deregulating pathways already present in the host. In this regard, the native -oxidation machinery has been engineered to proceed in reverse to synthesize rather than degrade fuel-like molecules, including primary alcohols and fatty acids [40]. As such, this pathway is quite similar in its reaction chemistry to the pathways inspired by C. acetobutylicum described above, which are also ATP-independent based on the use of a thiolase to avoid the utilization of malonyl-CoA. In order to activate the enzyme activities required for n-butanol biosynthesis, the transcriptional

7

regulators of -oxidation pathway, FadR and AtoC, were mutated. In addition, the native catabolite repressor protein (CRP) was replaced by a cAMP-insensitive mutant to alleviate catabolite repression in the presence of glucose. Despite these regulatory changes, this strain was not found to produce detectable levels of n-butanol. Based on enzyme activities, it appeared as if this bottleneck was related to extremely low butyryl-CoA dehydrogenase activity, which could be overcome by overexpression of endogenous aldehyde dehydrogenases (YqhD or FucO). Titers were further improved by overexpression of various enzymes in the pathway (YqeF and FucO), increasing n-butanol titer to 2 g/L. It is interesting to note that the catalytic activity at each node along the n-butanol pathway is approximately the same, which implies that the build up of pathway intermediates is prevented. In combination with the thermodynamic driving force from diffusion of butanol into bulk media, the carbon is pulled into the engineered n-butanol pathway despite the reversibility of first thiolase-dependent reaction.

1.4. Production of diesel targets from acetyl-CoA

Beyond gasoline targets, longer-chain products in the diesel range (C12-C20) provide the largest benefit for increasing energy density as well as reducing the energetic cost for product separation from aqueous fermentation. Two major metabolic routes to hydrocarbons of this chain length have been engineered from fatty acid biosynthesis and the isoprenoid pathway, both of which utilize the acetyl-CoA building block to build the backbone (Figure 1.1B). However, each pathway offers different advantages with respect to fuel production. For example, fatty acids are made at higher theoretical yields than isoprenoids are from the mevalonate pathway, based on lower carbon loss and ATP consumption, but also require tailoring steps for conversion to the fuel product (Figure 1.4). In comparison, the isoprenoid pathway offers greater diversity of structure and also produces a fully deoxygenated hydrocarbon as its initial product. In this section, we will review work utilizing these two pathways for biodiesel production in E. coli.

Fatty acid esters derived from plant oils are already in use today as biodiesels, but are costly to produce from an agricultural perspective [41]. Thus, microbial fermentation provides an option with greater potential for long-term sustainability. Overall, the engineering of fatty acid-derived biodiesels breaks down into two different phases, fatty acid biosynthesis and the tailoring of the fatty acid to produce the desired product. The chemistry of fatty acid biosynthesis is highly conserved, utilizing the acetyl-CoA building block for iterative cycles of chain elongation, reduction, and dehydration to extend the alkyl chain two carbons at a time on an acyl carrier protein (ACP) (Figure 1.2A). However, the behavior and regulation of the fatty acid biosynthetic machinery differs from host to host and is tightly controlled for physiological purposes. In E. coli, nine different proteins are involved in the production of fatty acids – FabABDFGHIZ and ACP – and generate fatty acyl-ACPs in the C14 to C18 size range as the end product [42] (Figure 1.4A). Since the acyl-ACP is primarily converted to membrane phospholipids in E. coli, fatty acid biosynthesis is highly regulated at several different points. One of the most important control points is the feedback inhibition by fatty-acyl ACPs of acetyl-CoA carboxylase (ACCase), which catalyzes the ATP-dependent carboxylation of acetyl-CoA to form the activated malonyl-CoA building block as the first committed step in fatty acid biosynthesis [42]. Two approaches have been taken to increase the malonyl-CoA pool by overexpressing the ACCase [43] as well as an acyl-ACP thioesterase to release the free fatty acid product [43,44]. Since free fatty acids are also rapidly degraded back to acetyl-CoA, it is also important to block the -oxidation pathway by deletion of either fadD or fadE. The productivity of these engineered

8

strains has been found to increase the free fatty acid content of E. coli by 20- to 40-fold to produce 1,200-1,500 mg/L of product at yields that are 6-14% of the theoretical limit [43,44]. Although these genetic modifications substantially increase the free fatty acid content, additional cell-free [45] and in vitro [46] studies of fatty acid biosynthesis have the potential to provide new insight into additional strategies to amplify fatty acid titers and yields.

A variety of strategies have been explored for converting fatty acids into different biofuels, such as alcohols, esters, ketones and alkanes (Figure 1.1A). As fatty acid esters are already currently in use, initial efforts have focused on engineering their production in vivo rather relying on post-fermentation chemical processing. In this regard, a pathway for the production of fatty acid ethyl esters (FAEE) has been incorporated into fatty acid overproduction strains by insertion of an acyl-CoA ligase (faa2 from Saccharomyces cerevisiae) and a broad-specificity acyltransferase (atfA from Acinetobacter baylyi) [47,48] along with a two-step pathway consisting of pyruvate decarboxylase and alcohol dehydrogenase (Figure 1.1B) to provide ethanol for transesterification (Figure 1.4A) [44]. Since extension of the pathway to the FAEE drops productivity two-fold to 700 mg/L, it is likely that metabolic imbalance related to rapid accumulation of ethanol and secretion of free fatty acids is limiting in this system. Therefore, a dynamic sensor-regulator system was designed in order to tune the expression of the ethanol and

Figure 1.4. The production of biodiesel from acetyl-CoA. (A) Biosynthesis and tailoring of fatty acids. (TE, thioesterase; ACL, acyl-CoA ligase) (B) Engineering the isoprenoid pathway for the production of biodiesels.

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FAEE production modules in response to acyl-CoA availability. Upon introduction of a FadR-based biosensor, the extracellular accumulation of free fatty acid decreased and led to an overall increase of FAEE production from 9.4% to 28% of the maximum theoretical yield (1,500 mg/L) [49]. To expand the promise of utilizing engineered E coli for biodiesel production on industrial relevant scale, a consolidated bioprocess has been developed to allow the FAEE-producing E. coli to use cellulose and hemicellulose from ionic liquid-pretreated switchgrass as carbon source at 80% of estimated theoretical yield [50]. Beyond fatty acid esters, the fermentation product that would most closely reproduce diesel is a mixture of alkanes. Alkanes have been reported in a diversity of microorganisms, including cyanobacteria, and are found to contain an odd number of carbons. Since heptadecane is the most abundant alkane reported, it appears that alkanes are derived from the “n-1” decarbonylation of aldehydes. The two genes involved in this process were found in Synechococcus elongatus by comparative genome analysis and used to engineer the alkane production in E. coli [51]. In conjunction with the identification of a gene involved in “n+1” pathways [52], these discoveries provide promise for our ability to access deoxygenated hydrocarbons through this pathway.

Isoprenoids are another class of hydrocarbons made in biological systems that could serve as biodiesels. In contrast to fatty acids, these compounds are made through the assembly of the C5 building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which can be made through two different pathways. Bacteria, such as E. coli, typically utilize the deoxyxylulose-5-phosphate (DXP) pathway rather than the mevalonate pathway, which is found in eukaryotes. In order to bypass the regulation of the native pathway, a heterologous mevalonate pathway initiated from the condensation of three acetyl-CoA molecules was constructed to drive the production of isoprenoids in E. coli (Figure 1.4B) [53]. Using an optimized version of this pathway, the production of sesquiterpene (C15) products can be produced from farnesyl diphosphate (FPP) precursor at 27 g/L under fed-batch fermentation conditions [54]. Because of the modularity of isoprenoid biosynthesis, this strain can also be utilized for the production of any C15 product by simply changing the terpene synthase. In this regard, bisabolane, the hydrogenated product of bisabolene, has been demonstrated to be biosynthetic alternative to D2 diesel fuel (Figure 1.4B) [55]. Since terpene synthases catalyze the cyclization of FPP with loss of pyrophosphate, they have been found to act as an irreversible sink that controls product titers. Thus, five bisabolene synthases from plants were screened for their productivity for bisabolene. Upon codon-optimization of the best candidate, bisabolene could be produced at 900 mg/L and isolated for reduction to the biodiesel product. Because of the strong influence of the terpene synthase on productivity, further discovery or engineering efforts to optimize their activity will allow the construction of strains with higher fuel yields.

1.5. Conclusions

Advanced biofuels offer significant advantages in terms of fuel properties and process design, however these pathways remain challenging to engineer with the efficiency and robust performance of yeast ethanol production. Towards this goal, high-flux pathways for production of both gasoline and diesel range fuels can be constructed with the introduction of a sink or driving force to push the pathway equilibrium to completion. In addition, advances in downstream chemical processing of shorter chain products can allow upgrading of these fuels for biodiesel production [56,57].

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This was written in collaboration with the following person: Dr. Miao Wen.

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Chapter 2: Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways

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2.1. Introduction

The ability to manipulate and control molecular structure through chemical synthesis has transformed society in many important ways, from production of modern materials to development of new human therapeutics. Like traditional synthetic chemistry, living systems also provide a general approach to designing new chemistry, offering a vast selection of reactions, in the form of enzymes and metabolites that can be mixed in series and in parallel to generate a target structure [1-3]. In fact, biological systems can often provide a more cost-effective and direct approach to the industrial production of both fine [4] and commodity chemicals [5,6] or remediation of toxins and pollutants [7]. In addition to a basic reaction set shared by most organisms, nature has also uncovered many unusual solutions to exploit and adapt to the niches found in diverse biospheres. The result of this selective pressure to find new uses for surrounding resources is the ability to carry out unique chemical transformations that could be useful if redesigned for human purposes, such as production of a biodegradable plastic or a defectless mineral. However, our capability to disconnect these biochemical components from their native context and reconnect them in new ways for in vivo chemical synthesis is limited by our understanding of the general design principles for de novo pathway construction when the individual catalysts have evolved in different pathways and organisms.

We have focused specifically on the design of pathways for the biosynthesis of second-generation biofuels because their production presents a particularly unique set of problems that illustrate the roadblocks involved in development of living cells as a “green” reactor for scalable chemical synthesis [8]. In contrast to fine chemicals and other commodity chemicals, the titers and yields of fuels from microbial fermentation are intimately tied not only to cost but also to achieving the positive energy balance and net reduction of carbon emissions essential to maintaining a sustainable alternative energy source. Consequently, the production levels that are needed to meet our goals in this arena require that the carbon assimilated by the host be channeled at unusually high efficiency into targeted small molecule synthesis and outcompete any off-pathway reactions, including those driven by the cell’s evolutionary impetus to grow and divide. Although ethanol has poor molecular characteristics as a gasoline additive or replacement, pre-existing expertise in its industrial fermentation at high yield from sugars (>90%) has allowed it to lead as the main biofuel in use today. However, the high water solubility and low energy density of ethanol creates a substantial energy and carbon penalty in its production and transport and longer-chain advanced fuels with enhanced fuel properties are needed to improve the outlook for biologically-derived gasoline and diesel replacements [8,9].

One of the major challenges for these goals is that naturally-occurring systems combining desirable target structures with the rapid, high-yielding, and robust production afforded by ethanologenic yeasts have yet to be identified. Thus, the development of sustainable solutions to the industrial production of liquid transportation fuels requires the construction of new pathways [10-12] and hosts [13,14] to merge these two necessary traits synthetically in a single organism. For example, n-butanol has been tapped as a possible gasoline replacement, as the addition of two carbons to ethanol significantly decreases its water solubility and volatility while increasing energy density [15,16]. However, n-butanol is produced in a slow-growing native host, Clostridium acetobutylicum (and related clostridia), as a mixture with acetone and ethanol in a biphasic fermentation [15,16]. Advances in genetic manipulation of clostridia have led to large increases in titer and productivity [16-19]; however, the regulation of solvent production is highly complex and cannot yet be achieved in a single-stage fermentation at yields that compete

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with yeast ethanol production. The seven-gene clostridial pathway has also been used as a basis to engineer more industrially-tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for n-butanol production with yields ranging from 2.5 mg/L to 580 mg/L in lab-scale shake-flask growths [20-22]and 60 mg/L/OD in higher cell-density fermentations [23].

Our group is interested in using n-butanol production as a system to explore the basic biochemical principles underlying the design of high-flux synthetic pathways inspired by ethanol fermentation. Like other longer-chain fuel targets, n-butanol is also built from the condensation of acetyl coenzyme A (CoA) monomers in a reaction sequence that is fully reversible. In contrast to these long-chain fuels, the production of soluble fuel targets in the gasoline size range, like n-butanol, cannot rely on sequestration of the products in insoluble lipid bodies to drive the pathway equilibrium to completion. In this work, we describe the design and analysis of a robust synthetic pathway for the production of n-butanol that utilizes enzyme chemical mechanism rather than a change in physical state to amplify n-butanol yields to titers of 4,650 mg/L (740 mg/L/OD) in lab-scale shake-flask experiments.

2.2. Materials and methods

Commercial materials. Terrific Broth (TB), LB Broth Miller (LB), LB Agar Miller, sulfuric acid, glacial acetic acid, potassium phosphate monobasic, and glycerol were purchased from EMD Biosciences (Darmstadt, Germany). Isopropyl-β-D-1-thiogalactopyranoside (IPTG), D-glucose, dithiothreitol (DTT), Tris-HCl, phenylmethanesulfonyl fluoride (PMSF), carbenicillin (Cb), streptomycin sulfate, magnesium chloride hexahydrate, sodium phosphate monobasic monohydrate, sodium chloride, reagent grade triethylamine, quinoline, and HPLC-grade acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). Potassium phosphate dibasic, d-desthiobiotin, 50% poly(ethyleneimine) solution, sodium pyruvate, thiamine pyrophosphate, L-arabinose, chloramphenicol (Cm), kanamycin sulfate (Km), coenzyme A (CoA), acetyl-CoA, acetoacetyl-CoA, crotonyl-CoA, butyryl-CoA, butyraldehyde, 2-butynoic acid, Lindlar catalyst, N,N-diisopropylethylamine (DIEA), N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (HATU), N,N,N′,N′-tetramethylethylenediamine (TEMED), NADH, NADPH, NAD+, and NADP+, were purchased from Sigma-Aldrich (St. Louis, MO). Imidazole was purchased from Acros (Geel, Belgium). Acrylamide/bis-acrylamide solution (30%, 37.5:1), Protein Assay reagent, electrophoresis grade sodium dodecyl sulfate (SDS), and ammonium persulfate were purchased from Bio-Rad Laborabories (Hercules, CA). PageRuler Plus Prestained Protein Ladder, PageRuler Prestained Protein Ladder, and DNase were purchased from Fermentas (Glen Burnie, MD). EnzyChrom NAD+/NADH Assay Kit and EnzyChrom NADP+/NADPH Assay Kit were purchased from BioAssay Systems (Hayward, CA). Deoxynucleotides (dNTPs) and Platinum Taq High-Fidelity DNA polymerase (Pt Taq HF) were purchased from Invitrogen (Carlsbad, CA). All restriction enzymes, antarctic phosphatase, polynucleotide kinase, T4 polymerase, T4 DNA ligase, and Phusion polymerase were purchased from New England Biolabs (Ipswich, MA). DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (QIAGEN; Valencia, CA) as appropriate. pET29a and pET16b were purchased from Novagen (San Diego, CA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and resuspended at a stock concentration of 100 μM in 10 mM Tris-HCl, pH 8.5. Codon-optimization and back-translation for synthetic gene construction were carried out using Gene Designer 2.0 (DNA 2.0; Menlo Park, CA). All synthetic genes and

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inserts were sequenced using the sequencing primers for the appropriate gene(s) following plasmid construction by the UC Berkeley Sequencing Facility, Sequetech (Mountain View, CA), or Quintara Biosciences (Berkeley, CA). All absorbance readings were taken on a DU-800 spectrometer (Beckman-Coulter; Fullerton, CA) or a SpectraMax M2 plate reader (Molecular Devices; Toronto, Canada). LC-MS data was collected on an Agilent 1200 series HPLC coupled to a diode-array detector and 6130 Single-Quadrupole ESI-MS. 1H-NMR spectra were collected in D2O (Cambridge Isotope Laboratories; Cambridge, MA) at 25ºC on a Bruker AV-600 spectrometer at the College of Chemistry NMR Facility at the University of California, Berkeley. All chemical shifts are reported in the standard δ notation of parts per million and annotated based on literature assignments and numbering for CoA [24]. High-resolution mass spectral analyses were carried out at the QB3/Chemistry Mass Spectrometry Facility.

Gene synthesis. Synthetic genes encoding PhaA, PhaB, Crt, Ccr, and AdhE2 were optimized for E. coli class II codon usage and obtained from Epoch Biosciences (Sugar Land, TX). The synthetic genes encoding Hbd, Ter, and PhaJ were also optimized for E. coli class II codon usage and synthesized using PCR assembly (Appendix 1). Gene2Oligo (http://berry.engin.umich.edu/gene2oligo) was used to convert the gene sequence into primer sets using default optimization settings (Appendix 1). To assemble the synthetic gene, each primer was added at a final concentration of 1 μM to the first PCR reaction (50 μL) containing 1 × Pt Taq HF buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4), MgSO4 (1.5 mM), dNTPs (250 μM each), and Pt Taq HF (5 U). The following thermocycler program was used for the first assembly reaction: 95ºC for 5 min; 95ºC for 30 s; 55ºC for 2 min; 72ºC for 10 s; 40 cycles of 95ºC for 15 s, 55ºC for 30 s, 72ºC for 20 s plus 3 s/cycle; these cycles were followed by a final incuabation at 72ºC for 5 min. The second assembly reaction (50 μL) contained 16 μL of the unpurified first PCR reaction with standard reagents for Pt Taq HF. The thermocycler program for the second PCR was: 95ºC for 30 s; 55ºC for 2 min; 72ºC for 10 s; 40 cycles of 95ºC for 15 s, 55ºC for 30 s, 72ºC for 80 s; these cycles were followed by a final incubation at 72ºC for 5 min. The second PCR reaction (16 μL) was transferred again into fresh reagents and run using the same program. Following gene construction, the DNA smear at the appropriate size was gel purified and used as a template for the rescue PCR (50 μL) with Pt Taq HF and rescue primers (Hbd F1 and R1, TdTer F1 and R1, PhaJ F1 and R1) under standard conditions. The resulting rescue product was either inserted directly into the appropriate vector or first cloned into pCR2.1-TOPO using a TOPO TA Cloning Kit from Invitrogen.

Bacterial strains. E. coli BL21(de3), W3110(de3), MG1655(de3), DH10B(de3), DH5α(de3), DH1(de3), and DH1 were used for protein and n-butanol production studies. DH10B-T1R was used for construction of plasmids and manipulation of DNA. W3110 (ATCC No. 39936), MG1655 (ATTC No. 700926), DH10B-T1R (Invitrogen), DH5α, and DH1 (ATCC No. 33839) were lysogenized using a λDE3 Lysogenization Kit (Novagen) and screened by ability to express protein from a T7 promoter.

Construction of plasmids. Standard molecular biology techniques were used to carry out plasmid construction using E. coli DH10B-T1R as the cloning host. All PCR amplifications were carried out with Phusion polymerase or Pt Taq HF using the oligonucleotides listed in Appendix 6. Annealed inserts were generated by phosphorylating each primer (1.5 pmol) individually with polynucleotide kinase in T4 DNA ligase buffer at 37ºC for 30 min and heat inactivation at 65ºC for 20 min. The phosphorylated primers were then mixed in annealing buffer (100 mM NaCl, 50 mM HEPES, pH 7.4) and annealed using the following program and

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used immediately after the reaction reached 25ºC: 90ºC for 4 min, 70ºC for 10 min, ramped to 37ºC at 0.5ºC/s, 37ºC for 15 min, ramped to 25ºC at 0.5ºC/s.

pCR2.1-phaA2.phaB. Since the initial synthetic phaA gene showed issues with recombination near the 5´-end, the codon usage in this stretch was altered by amplification of phaAB using the phaA F2 and phaB R1 primers. The PCR product was inserted into pCR2.1-TOPO using TA cloning to produce pCR2.1-phaA2.phaB. All plasmids in this study utilize the phaA2 sequence but are designated phaA for simplicity.

pBAD33-Bu1. The phaA-phaB-crt operon was amplified from pBSK-Bu1 using the phaA F5 and crt R2 primers and inserted directly into the XbaI-HindIII restriction sites of pBAD33.

pBT33-Bu1. The phaAB operon was amplified from pCR2.1-phaA2.phaB using the phaA2 F2 and phaB R2 primers and inserted into the SacI-XbaI restriction sites of pBAD33 to generate pBAD33-phaAB. The pTrc99a-crt cloning intermediate was made by inserting the synthetic crt gene into the NcoI-XmaI restriction sites of pTrc99a using the crt F2 and crt R2 primers to amplify the insert. The resulting PTrc.crt.rrnB cassette was amplified from pTrc99a-crt using the pTrc99a F4 and pTrc99a R4 primers and inserted non-directionally into the BglI site of pBAD33-phaAB to produce pBT33-phaAB-crt, which was designated pBT33-Bu1. Sequencing showed the coding strand of the phaAB operon was on the same strand as the crt gene.

strain E. coli host plasmid 1 plasmid 2 plasmid 3

1 BL21(de3) pBad33-Bu1 pET-ccr.adhE2 —

2 W3110(de3) pBad33-Bu1 pET-ccr.adhE2 —

3 MG1655(de3) pBad33-Bu1 pET-ccr.adhE2 —

4 DH10B(de3) pBad33-Bu1 pET-ccr.adhE2 —

5 DH5α(de3) pBad33-Bu1 pET-ccr.adhE2 —

6 DH1(de3) pBad33-Bu1 pET-ccr.adhE2 —

7 DH1(de3) pBad33-Bu1 pBad-ccr.adhE2 —

8 DH1(de3) pBad33-Bu1 pTrc-ccr.adhE2 —

9 DH1(de3) pBad33-Bu1 pCWori-ccr.adhE2 —

10 DH1 pBT33-Bu1 pCWori-ccr.adhE2 —

11 DH1 pTT33-Bu1 pCWori-ccr.adhE2 —

12 DH1 pT5T33-Bu1 pCWori-ccr.adhE2 —

13 DH1 pBT33-Bu2 pCWori-ccr.adhE2 —

14 DH1 pBT33-Bu1 pCWori-ter.adhE2 —

15 DH1 pBT33-Bu1 pCWori-ter.adhE2 pBBR1




19 DH1 pBT33-Bu2 pCWori-ter.adhE2 pBBR1-aceEF.lpd

20 DH1 pBT33-Bu3 pCWori-ter.adhE2 pBBR1-aceEF.lpd

Table 2.1. E. coli strains containing variants of the synthetic n-butanol pathway used in this study.

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pTT33-Bu1. The lacIq gene and PTrc promoter were amplified from pTrc99a using the pTrc99a F7 and pTrc99a R6 primers and inserted into the NsiI-SacI restriction sites of pBT33-Bu1.

pT5T33-Bu1. The lacIq gene was amplified from pTrc99a using the lacIq F1 and lacIq R1 primers and inserted non-directionally into the AatII site of pQE-30Xa (Qiagen) to generate the cloning intermediate pQE-30bXa-lacIq. Sequencing showed the coding strand of lacIq was on the same strand as the ampicillin resistance marker. The lacIq gene and PT5 promoter were amplified from pQE-30bXa-lacIq using the lacIq R2 and T5 R1 primers and inserted into the NsiI-SacI restriction sites of pBT33-Bu1.

pBT33-Bu2. The pCR2.1-phaA.hbd cloning intermediate was constructed by amplification of the synthetic hbd gene from pCR2.1-hbd with the hbd F1 and hbd R1 primers and insertion into the EcoRI-HindIII restriction sites of pCR2.1-phaA2.phaB. The phaAB operon of pBT33-phaAB-crt was replaced with a new multiple cloning site by digestion with NdeI and XhoI and insertion of a linker using sequence and ligation independent cloning (SLIC). The insert was made by amplifying the rrnB terminator from pBAD33 using the rrnB SLIC F1 and rrnB SLIC R1 primers. The amplified fragment and digested vector were independently treated with 0.5 U T4 polymerase for 30 min and the reaction was quenched with the addition of dATP. The insert and vector were incubated in 1× ligation buffer for 30 min at 37ºC and transformed immediately. The phaA-hbd operon was amplified using the phaA2 F2 and hbd R100 primers and inserted in the NdeI-XhoI restriction sites of the newly generated cloning intermediate. The plasmid pBT33-phaA.hbd-crt was designated pBT33-Bu2.

pBAD33-Bu2. The phaA-hbd-crt operon was constructed using splicing by overlap extension (SOE) PCR. The phaA-hbd operon was amplified from pBT33-Bu2 using the phaA F5 and HBD/crt SOE R1 primers and the crt gene was amplified using the HBD/crt SOE F1 and crt R2 primers. The two PCR products were gel purified and both PCR products were amplified together using phaA F5 and crt R2. The resulting phaA-hbd-crt operon was inserted directly into the XbaI-HindIII restriction sites of pBAD33.

pBT33-Bu3. The Ptrc-phaJ fragment was constructed by SOE PCR by first individually amplifying the Trc promoter from pBT33-Bu2 and the phaJ gene using the pTrc99a F11/phaJ SOE R1 and PhaJ SOE F1/phaJ R100 primer sets, respectively. The two products were gel purified and both PCR products were amplified together using pTrc99a F11 and phaJ R100. The resulting Ptrc-phaJ operon was inserted into the EagI-XmaI restriction sites of pBT33-Bu1.

pBT33-Bu4. The Ptrc-phaJ operon was assembled as described for pBT33-Bu3 and inserted directly into the EagI-XmaI restriction sites of pBT33-Bu2.

pET29a-ccr.adhE2. The ccr gene was amplified using the ccr F1 and ccr R2 primers and inserted into the NdeI-EcoRI restriction sites of pET29a to generate the pET29a-ccr intermediate. pET29-ccr.adhE2 was constructed by insertion of the adhE2 gene into the EcoRI-SacI restriction sites of pET29a-ccr after amplification using the adhE2 F1 and adhE2 R2 primers.

pBAD33-ccr.adhE2. The ccr-adhE2 operon was amplified from pET29a-ccr.adhE2 using the ccr F1 and adhE2 R17 primers, digested with NdeI and XhoI, and inserted into the NdeI-SalI restriction sites of pBAD33-phaAB.

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pTrc99a-ccr.adhE2. The ccr-adhE2 operon was amplified from pET29a-ccr.adhE2 using the ccr F15 and adhE2 R2 primers and inserted into the NcoI-SacI restriction sites of pTrc99a.

pCWOri-ccr.adhE2. The ccr-adhE2 operon was amplified from pET29a-ccr.adhE2 using the ccr F1 and adhE2 R1 primers and inserted into the NdeI-HindIII restriction sites of pCWOri.

pCWOri-ccr.Stag. The ccr gene was amplified using the ccr F1 and ccr R13 primers and inserted into the NdeI-XbaI restriction sites of pCWOri.

pET29a-ccr.Stag-adhE2. The ccr.Stag gene was amplified using the ccr F1 and pCWori-Stag R4 primers and inserted into the NdeI-EcoRI restriction sites of pET29a-ccr.adhE2.

pCWori-ccr.Stag-adhE2. The ccr.Stag gene was amplified using the ccr F1 and pCWori-Stag R4 primers and inserted into the NdeI-EcoRI restriction sites of pCWOri-ccr.adhE2.

pTrc99a-ccr.Stag-adhE2. The ccr.Stag gene was amplified using ccr F1 and pCWori-Stag R4 and inserted into the NdeI-EcoRI restriction sites of pTrc99a-ccr.adhE2.

pCWori-ccr-His6. The ccr gene was amplified using the ccr F1 and ccr R11 primers and inserted into the NdeI-XbaI restriction sites of pCWOri.

pET16b-His6-ter. The ter gene was amplified from the gene synthesis using the TdTer F1 and TdTer R101 primers and inserted directly into the NdeI-XhoI restriction sites of pET16b.

pCWOri-ter.adhE2. The ter gene was amplified using the TdTer F1 and TdTer R102 primers and inserted directly into the NdeI-EcoRI restriction sites of pCWOri-ccr.adhE2.

pET16b-His6-phaB. The phaB gene was amplified using the phaB F1 and phaB R1 primers and inserted into the NdeI-BamHI restriction sites of pET16b.

pET16b-His6-hbd. The hbd gene was amplified using the hbd F100 and hbd R102 primers and inserted into the NdeI-BamHI restriction sites of pET16b.

pET16b-Strep-crt. The crt gene was amplified using the crt F4 and crt R2 primers and inserted directly into the NdeI-HindIII restriction sites of pCWOri.

pBBR1-aceEF.lpd. The aceEF-lpd operon was amplified from E. coli DH10B genomic DNA using the aceE F2 and lpd R2 primers and inserted into the XhoI-SacI restriction sites of pBBR1MCS2.

Cell culture. E. coli strains were transformed by electroporation using the appropriate plasmids. A single colony from a fresh transformation was then used to seed an overnight culture grown in Terrific Broth (TB) supplemented with 1.5% glucose and appropriate antibiotics at 37C in a rotary shaker (200 rpm). Antibiotics were used at a concentration of 50 μg/mL for strains with a single resistance marker. For strains with multiple resistance markers, kanamycin (Km) and chloramphenicol (Cm) were used at 25 μg/mL and carbenicillin (Cb) was used at 50 μg/mL.

In vivo production of n-butanol. Overnight cultures of freshly-transformed E. coli strains were grown for 12-16 h in TB at 37C and used to inoculate TB (50 mL) with 1.5% glucose replacing the standard glycerol supplement and appropriate antibiotics to OD600 = 0.05 in a 250 mL-baffled flask. The cultures were grown at 37C in a rotary shaker (200 rpm) and induced with IPTG (1.0 mM) and L-arabinose (0.2%) at OD600 = 0.35-0.45. At this time, the growth temperature was reduced to 30C and the culture flasks were sealed with Parafilm M (Pechiney

21

Plastic Packaging, Chicago, IL) to prevent n-butanol evaporation. Additional glucose (1%) was added concurrent with culture sampling after 1 d. Flasks were unsealed for 10 to 30 min every 24 h then resealed with Parafilm M after sampling. Samples from strains utilizing ccr were quantified after 6 d of cell culture while those using ter were analyzed after 3 d.

Extraction and quantification of n-butanol. Samples (2 mL) were removed from cell culture and cleared of biomass by centrifugation at 20817 g for 2 min using an Eppendorf 5417R centrifuge (Hamburg, Germany). The supernatant or cleared media sample was then mixed in a 9:1 ratio with an aqueous solution containing the isobutanol internal standard (10,000 mg/L). These samples were then analyzed on a Trace GC Ultra (Thermo Scientific; Waltham, MA) using an HP-5MS column (0.25 mm 30 m, 0.25 μM film thickness, J & W Scientific). The oven program was as follows: 75C for 3 min, ramp to 300C at 45C min-1, 300C for 1 min. n-Butanol was quantified by flame ionization detection (FID) (flow: 350 ml min-1 air, 35 ml min-1 H2, and 30 ml min-1 He). Samples containing n-butanol levels below 500 mg/L were re-quantified after extraction of the cleared media sample or standard (500 µL) with toluene (500 µL) containing the isobutanol internal standard (100 mg/L) using a Digital Vortex Mixer (Fisher) for 5 min set at 2000. The organic layer was then quantified using the same GC parameters with a DSQII single-quadrupole mass spectrometer (Thermo Scientific; Waltham, MA) using single ion monitoring (m/z 41 and 56) concurrent with full scan mode (m/z 35-80). Samples were quantified relative to a standard curve of 2, 4, 8, 16, 31, 63, 125, 250, 500 mg/L n-butanol for MS detection or 125, 250, 500, 1000, 2000, 4000, 8000 mg/L n-butanol for FID detection. Standard curves were prepared freshly during each run and normalized for injection volume using the internal isobutanol standard (100 or 1000 mg/L for MS and FID, respectively).

Enzyme assays. Cell cultures were prepared as described for n-butanol production studies and were harvested 20 h after induction by centrifugation at 15,316 g for 20 min at 4C (Sorvall Legend RT+; Thermo Scientific; Waltham, MA) and stored at -80C after removal of the supernatant. Frozen cell pellets were thawed and resuspended at 5 mL/g cell paste in 100 mM Tris-HCl, 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, pH 7.5. The cell suspension was homogenized and lysed by passage through a French Pressure Cell (Thermo Scientific) at 14,000 psi. The crude lysate was centrifuged at 15,316 g for 30 min at 4C to remove cell debris and insoluble protein. The supernatant was used for enzyme assays as described below. Protein concentration of samples was measured using Protein Assay Reagent (Bio-Rad Laboratories; Hercules, CA) relative to a standard curve of bovine serum albumin (New England Biolabs; Ipswich, MA; 50-900 μg/mL; ε280 = 0.66 ml/mg cm-1). Spectrophotometric assays for PhaA, PhaB, Ccr, Ter, and AdhE2 activity were carried out on a SpectraMax M2 plate reader (Molecular Devices; Sunnyvale, CA). The pathlength was calculated using the water constant protocol in order to convert readings to a 1 cm-pathlength. Absorbance measurements for Crt assays were collected with DU-800 spectrophotometer (Beckman-Coulter; Brea, CA). All assays were carried out at 30C except for Ter assays, which were performed at 25C.

Acetoacetyl-CoA synthase/thiolase. Thiolase activity was assayed in the forward direction by monitoring NADPH oxidation at 340 nm at 30ºC in a coupled assay with His6-PhaB [25,26]. The assay mixture contained acetyl-CoA (1 mM), NADPH (100 μM), and His6-PhaB (7.5 U/mL) in 100 mM Tris-HCl, 4.5 mM MgCl2, 1.5 mM DTT, pH 7.5. The reaction was initiated by addition of acetyl-CoA.

22

Acetoacetyl-CoA reductase. The reduction of acetoacetyl-CoA was assayed by monitoring the oxidation of NAD(P)H at 340 nm at 30ºC [27]. The assay mixture contained acetoacetyl-CoA (100 μM) and NAD(P)H (100 μM ) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of acetoacetyl-CoA.

3-Hydroxybutyryl-CoA dehydratase (crotonase). Dehydratase activity was measured in the reverse direction by monitoring hydration of the double bond at 263 nm at 30ºC [28]. The assay mixture contained crotonyl-CoA (30 μM) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of crotonyl-CoA.

Crotonyl-CoA reductase/trans-enoyl reductase. Crotonyl-CoA reduction activity was assayed in the forward direction by monitoring the oxidation of NAD(P)H at 340 nm at 30ºC [29]. The assay mixture contained crotonyl-CoA (50 μM) and NAD(P)H (100 μM) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of crotonyl-CoA. When assayed in the reverse direction, the assay mixture contained butyryl-CoA (50 μM -5 mM) and NAD(P)+ (100 μM-12.5 mM) in 100 mM Tris-HCl, pH 7.5.

Aldehyde dehydrogenase. The butyraldehyde dehydrogenase activity was assayed in the reverse direction by monitoring the reduction of NAD+ at 340 nm at 30ºC [30]. The assay mixture contained butyraldehyde (10 mM), CoA (400 μM), and NAD+ (400 μM) in 100 mM Tris-HCl, 0.5 mM DTT, pH 7.5. The reaction was initiated by addition of butyraldehyde.

Alcohol dehydrogenase. The butanol dehydrogenase activity was assayed in the forward direction by monitoring the oxidation of NADH at 340 nm at 30ºC [30]. The assay mixture contained butyraldehyde (10 mM), and NADH (100 μM) in 100 mM Tris-HCl, 0.5 mM DTT, pH 7.5. Background activity with NADH in the cleared cell lysate was measured by excluding butyraldehyde. Both reactions were initiated with addition of the cleared cell lysate.

Pyruvate dehydrogenase. The pyruvate dehydrogenase activity was assayed in the forward direction by monitoring the reduction of NAD+ at 340 nm at 30ºC [31]. The assay mixture contained sodium pyruvate (5 mM), CoA (0.13 mM), NAD+ (2 mM), thiamine pyrophosphate (0.5 mM), and MgCl2 (5 mM) in 50 mM potassium phosphate, 0.12 mM DTT, pH 7.9. The reaction was initiated by the addition of sodium pyruvate.

Characterization of crotonyl-CoA and butyryl-CoA utilization in cell lysates. Biomass was prepared as described in the main text for n-butanol production studies, except that cells were harvested 24 h after IPTG and L-arabinose induction by centrifugation at 15,316 × g for 8 min at 4ºC. Frozen cell pellets were thawed and resuspended at 5 mL/g cell paste using Buffer C (100 mM Tris-HCl, 5 mM DTT, 0.5 mM PMSF, pH 7.5 at 4ºC) and homogenized before lysis by passage through a French Pressure cell at 14,000 psi. Total lysate was centrifuged at 15,316 × g for 30 min at 4ºC to separate soluble and insoluble fractions. Either crotonyl-CoA (1 mM) or butyryl-CoA (1 mM) and NADH (5 mM), if used, was added to cleared cell lysate (1 µg/mL final total protein concentration by Bradford assay) and incubated in 100 mM Tris-HCl, pH 7.5 at 30ºC. Samples were analyzed by LC-MS after 10 min and 60 min using an Eclipse XDB C-8 column (3.5 µm, 3.0 x 150 mm, Agilent) using a 0-100% acetonitrile gradient with 20 mM triethylamine, 10 mM acetic acid, pH 4.0 as the mobile phase. MS data was collected in full spectral mode and the ion corresponding to the most abundant monoisotopic peak for the individual acyl-CoAs was extracted for plotting.

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Purification of affinity-tagged proteins. TB (1 L) containing carbenicillin (50 μg/mL) in a 2.8 L- Fernbach baffled shake flask was inoculated to OD600 = 0.05 with an overnight TB culture of freshly transformed E. coli BL21(de3) containing the appropriate overexpression plasmid (Ccr, pCWori-ccr-His6; Ter, pET16b-His6-ter; PhaB, pET16b-His6-phaB; HBD, pET16b-His6-hbd; Crt, pET16b-Strep-crt). The cultures were grown at 37°C at 200 rpm to OD600 = 0.6 to 0.8 before inducing with IPTG (1 mM) and dropping the temperature to 30°C. Cell pellets were harvested 4 h after IPTG induction by centrifugation at 15,316 × g for 20 min at 4ºC and stored at -80ºC.

For His6-tagged proteins, frozen cell pellets were thawed and resuspended at 5 mL/g cell paste using Buffer A (50 mM sodium phosphate, 300 mm sodium chloride, pH 8.0 at 4ºC) containing imidazole (10 mM), PMSF (0.5 mM) and DNAse (0.7 U/g cell paste) and homogenized before lysis by passage through a French Pressure cell (Thermo Scientific) at 14,000 psi. Total lysate was centrifuged at 15,316 × g for 30 min at 4ºC to separate soluble and insoluble fractions. The DNA was precipitated from the cleared lysate by addition of streptomycin sulfate (20%) over 10 min at 4ºC to a final concentration of 1%. The precipitated DNA was removed by centrifugation at 15,316 × g for 20 min at 4ºC. The supernatant was then passed through a Ni-NTA Agarose (Qiagen) column (7 mL) at a flow of <1 mL/min. The column was washed with 20 column volumes of Buffer A before elution with Buffer A containing 250 mM imidazole in 8 mL fractions.

For Strep II-tagged proteins, frozen cell pellets were thawed and resuspended at 5 mL/g cell paste using Buffer B (100 mM Tris-HCl, 150mM sodium chloride, pH 8.0 at 4ºC) containing DTT (5 mM), PMSF (0.5 mM), and DNAse (0.7 U/g cell paste) and homogenized before lysis by passage through a French Pressure cell (Thermo Scientific) at 14,000 psi. Total lysate was centrifuged at 15,316 × g for 30 min at 4ºC to separate soluble and insoluble fractions. The DNA was precipitated from the cleared lysate by addition of polyethyleneimine (15%) over 10 min at 4ºC to a final concentration of 0.5%. The precipitated DNA was removed by centrifugation at 15,316 × g for 20 min at 4ºC. The supernatant was passed through a StrepTactin Superflow Agarose (Novagen) column (4 mL) at a flow of <1 mL/min. The column was washed with 20 column volumes of Buffer B before elution with Buffer B containing 2.5 mM desthiobiotin in 4 mL fractions.

For all proteins, fractions were pooled based on protein content using Protein Assay reagent and concentrated with an Amicon filtration device using a YM10 membrane (Millipore Corporation; Billerica, MA). For His6-tagged proteins, the imidazole was then removed by passage through a Sephadex G-25 column (Sigma-Aldrich, bead size 50-150 μm, 200 mL) in 20 mM Tris, 5 mM EDTA, pH 8.0. The protein-containing fractions were concentrated to 2 mg/mL using calculated extinction coefficients (Ccr-His6: ε280 = 65,320 M-1 cm-1; His6-Ter: ε280 = 42,560 M-1 cm-1; His6-PhaB: ε280 =32,650 M-1 cm-1; His6-Hbd: ε280 = 12,570 M-1 cm-1; Strep-Crt: ε280 =7,450 M-1 cm-1) and stored at -20ºC (His6-PhaB) or -80ºC (Ccr-His6, His6-Ter, His6-Hbd, Strep-Crt).

LC-MS assay to monitor Ccr and Ter production distributions. Ccr-His6 (5 μg/ml) or His6-Ter (10 μg/mL) was incubated with crotonyl-CoA (1 mM), NADPH or NADH (2.5 mM) and 3 mM NaHCO3, if used, in 100 mM Tris-HCl, pH 7.5 buffer (500 µL) for 30 min at 30ºC. Samples were lyophilized and analyzed by LC-MS on an Eclipse XDB C-8 column (3.5 µm, 3.0 × 150 mm, Agilent) using a 0-100% acetonitrile gradient over 25 min with 20 mM triethylamine, 10 mM acetic acid, pH 4.0 as the mobile phase.

24

Synthesis of (S)- and (R)-3-hydroxybutyryl-CoA. His6-Hbd (35 μg/ml) or His6-PhaB (17.5 μg/ml) was incubated with acetoacetyl-CoA (12.5 mM) and NADH or NADPH (125 mM) in 100 mM Tris-HCl, pH 7.5 (300 μL) for 60 min at 30ºC to produce (S)- and (R)-3-hydroxybutyryl-CoA, respectively. Both products were isolated by RP-HPLC using an Eclipse XDB C18 column (5 µm, 9.4 × 250 mm, Agilent) using a 0-100% acetonitrile gradient over 25 min with 10 mM acetic acid, pH 4.0 as the mobile phase. The (S)-3-hydroxybutyryl-CoA was further purified by RP-HPLC using an Eclipse XDB C-8 column (3.5 µm, 3.0 × 150 mm, Agilent) using a 0-100% acetonitrile gradient over 25 min with 20 mM triethylamine, 10 mM acetic acid, pH 4.0 as the mobile phase. Purified (S)- and (R)-3-hydroxybutyryl-CoA were lyophilized following each purification step and analyzed by LC-MS using an Eclipse XDB C18 column (5 µm, 4.6 × 150 mm, Agilent) using a 0-100% acetonitrile gradient over 25 min with 10 mM acetic acid, pH 4.0 as the mobile phase. ESIMS (M-H) calcd for C25H41O18N7P3S m/z, 852.1, found 852.1 ((S)-3-hydroxybutyryl-CoA) and 852.1 ((R)-3-hydroxybutyryl-CoA).

Crt kinetic characterization. Activity of Strep-Crt in the forward direction was measured in a coupled assay with His6-Ter by monitoring the oxidation of NADH at 340 nm at 30ºC. The assay mixture contained Strep-Crt (0.2 μg/mL), His6-Ter (500 μg/mL), and NADH (200 μM) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by the addition of either (S)-3-hydroxybutyryl-CoA at concentrations of 25, 50, 100, 150, 200, and 300 μM or (R)-3-hydroxybutyryl-CoA at concentrations of 50, 250, 500, 750, 1000, and 2000 μM. Kinetic parameters (kcat and KM) were determined by fitting the data using Origin 6.1 (OriginLab Corporation; Northampton MA) to the equation: Vo = Vmax [S]/(KM + [S]), where Vo is the initial rate and [S] is the substrate concentration.

Synthesis of 2-butynoyl-CoA. 2-Butynoyl-CoA was made according to a standard literature procedure [32,33]. 2-Butynoic acid (6 mg, 0.071 mmol) and HATU (28.7 mg, 0.075 mmol) were added to a mixture of 50% aqueous DMF (650 µL) containing DIEA (0.29 M) and stirred for 5 min before adding CoA (50 mg, 0.063 mmol). The reaction was stirred under N2 for 2 h at room temperature and purified by RP-HPLC on an Eclipse XDB C-18 column (5 µm, 9.4 × 250 mm, Agilent) using a 0-100% acetonitrile gradient over 30 min with water as the mobile phase. The reaction was run six times and the isolated 2-butynoyl-CoA was pooled and lyophilized (48.7 mg 15% yield). 1H-NMR (600 MHz, D2O, 25 °C): δ = 8.50 (s, 1H, H8), 8.21 (s, 1H, H2), 6.10 (d, J = 6.6 Hz, 1H, H1′), 4.79 (m, 2H, H2′ and H3′) , 4.51 (s, 1H, H4′), 4.15 (s, 2H, H5′), 3.95 (s, 1H, H3″), 3.76 (m, 1H, H1a″), 3.48 (m, 1H, H1b″), 3.37 (t, J = 6.48 Hz, 2H, H5″), 3.28 (t, J = 6.24 Hz, 2H, H8″), 2.99 (t, J = 6.36 Hz, 2H, H9″), 2.35 (t, J = 6.48 Hz, 2H, H6″), 1.97 (s, 3H, -CH3), 0.83 (s, 3H, H10″), 0.69 (s, 3H, H11″). HR-ESIMS (M-H) calcd for C25H37O17N7P3S m/z, 832.119, found 832.116.

Synthesis of isocrotonyl-CoA. (Z)-2-Butenoyl-CoA (isocrotonyl-CoA) was synthesized by hydrogenation of 2-butynoyl-CoA to the cis-alkene using the Lindlar catalyst. 2-Butynoyl-CoA (48.7 mg, 0.058 mmol), Lindlar catalyst (10 mg), and quinoline (10 µL, 0.085 mmol) were added to methanol (1 mL) and stirred for 24 h at room temperature under H2. The product was purified by RP-HPLC on an Eclipse XDB C-18 column (5 µm, 9.4 × 250 mm, Agilent) using a 0-100% acetonitrile gradient over 30 min with water as the mobile phase. Isocrotonyl-CoA of >95% purity by ESI-MS was pooled (0.002 mmol, 4% yield) and contained <1% (E)-2-butenoyl-CoA (crotonyl-CoA) based on 1H-NMR. Isocrotonyl-CoA and crotonyl-CoA can be distinguished by the chemical shift of the acyl group H3 (=CHCH3: crotonyl-CoA, δ = 6.80; isocrotonyl-CoA, δ = 6.14). 1H-NMR (600 MHz, D2O, 25 °C): δ = 8.43 (s, 1H, H8), 8.16 (s, 1H, H2), 6.14 (m, 1H,

25

=CHCH3), 6.06 (d, J = 6.0 Hz, 1H, H1′), 6.01 (m, 2H, =CHCO), 4.78 (m, 2H, H2′ and H3′) , 4.48 (s, 1H, H4′), 4.15 (t, J = 11.7 Hz, 2H, H5′), 3.92 (s, 1H, H3″), 3.74 (m, 1H, H1a″), 3.48 (m, 1H, H1b″), 3.35 (t, J = 6.72 Hz, 2H, H5″), 3.25 (t, J = 6.18 Hz, 2H, H8″), 2.91 (t, J = 6.24 Hz, 2H, H9″), 2.33 (t, J = 6.42 Hz, 2H, H6″), 1.92 (dd, J1 = 6.00 J2 = 1.32, 3H, -CH3), 0.80 (s, 3H, H10″), 0.67 (s, 3H, H11″). HR-ESIMS (M-H) calcd for C25H39N7O17P3S, m/z, 834.134, found 834.133.

Ter kinetic characterization. Activity of His6-Ter was measured by monitoring the oxidation of NADH at 340 nm at 25ºC. The assay mixture (125 μL) contained 400 μM or 200 μM NADH for crotonyl-CoA and isocrotonyl-CoA assays, respectively, in 100 mM potassium phosphate, pH 6.2. The reaction was initiated by the addition of either crotonyl-CoA at concentrations of 0.500, 0.375, 0.250, 0.200, 0.175, 0.150, 0.125, 0.100, 0. 88, 0.075, 0.050, 0.025 mM or isocrotonyl-CoA at concentrations of 1.000, 0.500, 0.350, 0.250, 0.170, 0.125, 0.100, 0.550, 0.050 mM. The generation of butyryl-CoA from isocrotonyl-CoA was further confirmed by LC-MS. Kinetic parameters were determined as described above for Crt.

Measurement of intracellular NAD(P)H and NAD(P)+ levels. Biomass was prepared as described in the main text for n-butanol production studies but harvested at 24 h after induction by centrifugation at 8,600 × g for 8 min at 4ºC. The measurement of intracellular NAD(P)H and NAD(P)+ levels was performed using the EnzyChrom NAD+/NADH Assay Kit and EnzyChrom NADP+/NADPH Assay Kit as described in the manufacturer-supplied protocol with an additional 10 s sonication on ice following resuspension in lysis buffer. Measurements were made immediately after harvesting.

2.3. Results

Design of a synthetic pathway for n-butanol production. n-Butanol can be assembled from acetyl-CoA units in an analogous fashion to fatty acids by the condensation of two monomers to produce the C4 backbone, followed by subsequent rounds of reduction and dehydration to yield butyryl-CoA (Scheme 2.1). As one of the major design challenges is the reversibility of this

Scheme 2.1. Comparison of a high-yielding polyhydroxybutyrate pathway with the design of a chimeric n-butanol pathway. A high-yielding pathway for production of polyhydroxyalkanoates (PHAs) in R. eutrophus and the design of a chimeric pathway for fuel synthesis derived from genes from three different organisms. (blue, Ralstonia eutrophus; red, Clostridium acetobutylicum; black, Streptomyces collinus; phaA, acetoacetyl-CoA thiolase/synthase; phaB, 3-hydroxybutyryl-CoA dehydrogenase; phaC, PHA synthase; crt, crotonase; ccr, crotonyl reductase; adhE2, bifunctional butyraldehyde/butanol dehydrogenase).

26

Figure 2.1. In vivo production of E. coli strains containing various synthetic n-butanol biosynthetic pathways Strains utilizing Ccr for the conversion of crotonyl-CoA to butyryl-CoA were cultured for 6 d while those incorporating Ter were cultured for 3 d. n-Butanol was quantified by GC-MS for titers less than 500 mg/L and by GC-FID for titers above 500 mg/L. Data are mean ± s.d. (n = 3). Production was optimized for E. coli host selection (1-6), ccr.adhE2 promoter (6-9), phaAB and crt promoters (9-12), enoyl-coA reductase and ketoreductase/enoyl-CoA hydratase selection (10, 13-18), and overexpression of PDHc (19, 20).

27

pathway, we focused on selecting enzyme components that normally work in the biosynthetic rather than the degradative direction. The initial synthetic pathway construction was inspired by the production of polyhydroxyalkanoates (PHAs) in engineered E. coli by transplantation of a three-gene pathway from Ralstonia eutrophus for monomer biosynthesis (phaAB) and polymerization (phaC) to yield a biodegradable plastic that can be produced at 50% dry cell weight at near theoretical yields (Scheme 2.1) [34]. In this system, the PhaA-dependent C-C bond forming reaction, whose equilibrium strongly favors the reactants in vitro [35,36], is driven to completion in vivo by the depletion of the (R)-3-hydroxybutyryl-CoA monomer by PhaC, which allows its physical sequestration as a polymer. Thus, we reasoned that PhaA and PhaB would be capable of supporting high flux in the forward direction if a similar effectively irreversible physical step took place later in the reaction sequence, such as the release of free CoA or secretion of the n-butanol product from the cell. We chose the crotonase (Crt) from C. acetobutylicum to catalyze the dehydration of 3-hydroxybutyryl-CoA to the corresponding enoyl-CoA because it is highly active and should also function physiologically in the biosynthetic direction [37,38]. For the next step, we sought to replace the multi-protein oxygen-sensitive system for reduction of crotonyl-CoA found in C. acetobutylicum consisting of butyryl-CoA dehydrogenase (Bcd) and two redox partners (EtfAB) [38] with crotonyl-CoA reductase (Ccr), a single-enzyme biosynthetic alternative from Streptomyces collinus [29] that had been tested previously for n-butanol production [20,21]. The bifunctional butyraldehyde/butanol dehydrogenase (AdhE2) from C. acetobutylicum was selected for the two-step conversion of butyryl-CoA to n-butanol because of its specificity toward a C4 substrate [30].

We assembled the synthetic genes encoding PhaA, PhaB, and Crt as a single operon driven by the relatively weak arabinose promoter (pBAD33-Bu1, Table 2.1) because we decided that low expression of these three enzymes would suffice based on their high in vitro activity [37,39] and limit the protein overexpression burden. A second plasmid containing an operon comprised of ccr and adhE2 was then constructed using a strong T7lac promoter (pET-ccr.adhE2) with the goal of achieving high expression of enzymes that could push the pathway equilibrium towards formation of the cell-permeable product. This two-plasmid system was tested for its ability to produce n-butanol in an E. coli BL21(de3) host. Gas chromatography-mass spectrometry analysis showed that the synthetic pathway was competent for fuel synthesis but functioned at low flux (quantified relative to a linear standard curve 3.0 mg/L) (Figure 2.1, 1) (Figure 2.2)(Figure 2.3). With a working pathway in hand, we were able to titrate through different promoter strengths for both operons as well as optimize host strain selection to yield an overall 30-fold increase in n-butanol titers to 95 mg/L (Figure 2.1, 2-12). The marked improvement in product yield resulted from decrease of the promoter strength for the ccr-adhE2 operon and indicated that a major bottleneck in the pathway is most likely the solubility and productivity of Ccr in vivo. Indeed, we visualized soluble Ccr levels using a C-terminal S-tag, which allows protein quantification in complex mixtures, and showed that a direct relationship exists between soluble intracellular Ccr levels and n-butanol titers (Figure 2.4).

Control of pathway flux by chemical reaction mechanism. The dependence of n-butanol titers on Ccr activity in combination with a previous report of this step as a bottleneck [20] led us to evaluate enoyl-CoA reduction as a possible point in the pathway where overall flux could be controlled. In order to test this possibility, we incubated butyryl-CoA in crude E. coli cell lysate to monitor its possible dissipation through native cellular pathways. There was no substantial change in butyryl-CoA levels compared to inactivated cell lysate (Figure 2.5), leading us to conclude that any butyryl-CoA formed in an engineered host could eventually be channeled to n-

28

Figure 2.3. Standard curves for quantitation of n-butanol by GC-MS and GC-FID. Representative standard curves for GC quantification of n-butanol using EI-MS with concentrations of 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 mg/L n-butanol with 100 mg/L isobutanol added as an internal standard (A) and FID with concentrations of 125, 250, 500, 1000, 2000, 4000, and 8000 mg/L n-butanol with 1000 mg/L isobutanol added as an internal standard (B) detection. Samples were normalized for injection volume based on an isobutanol internal standard.

Figure 2.2. Detection of n-butanol by GC-MS. n-Butanol production in E. coli BL21(de3), which was genetically modified to express the n-butanol biosynthetic pathway (phaA, phaB, crt, ccr, and adhE2). Comparison of the product of the engineered n-butanol-producing strain (sample) with an authentic n-butanol standard (standard) with respect to retention time (left) and EI mass spectrum (right) confirm the identity of the fermentation product ad n-butanol. Samples include isobutanol as an internal standard (IS) for normalization.

29

Figure 2.4. Monitoring the dependence of n-butanol titers on concentration of soluble intracellular Ccr. (A) n-Butanol production levels (quantified by GC-MS) compared to levels of soluble Ccr-Stag protein measured using the S-Tag Rapid Assay Kit, (Novagen) from E. coli DH1(de3) pBT33-Bu1 harboring ccr-Stag.adhE2 plasmids with varying promoters. (Trc, pTrc99a-ccr-Stag.adhE2; Tac, pCWori-ccr-Stag.adhE2; T7, pET29a-ccr-Stag.adhE2). (B) SDS-PAGE of soluble and insoluble protein extracts from E. coli DH1(de3) pBT33-Bu1 pCWori-ccr-Stag.adhE2: MW ladder (lane 1), pre-induction (lane 2), post-induction (lane 3), soluble protein fraction (lane 4), insoluble protein fraction (lane 5).

Figure 2.5. The consumption of butyryl-CoA by E. coli cell lysate. Monitoring the consumption of butyryl-CoA in inactive (1) and active E. coli DH1 cell lysates. (2-5). (A) The reactions were analyzed by RP-HPLC at 10 min and 60 min and are plotted as the sum of m/z = 809 (acetyl-CoA), 835 (crotonyl-CoA), 837 (butyryl-CoA), 851 (acetoacetyl-CoA), and 853 (3-hydroxybutyryl-CoA) collected in negative-ion mode. (B) The peak corresponding to butyryl-CoA was integrated and is reported relative to the inactivated cell lysate control.

30

butanol even if the AdhE2-catalyzed steps were significantly slower. Thus, selection of the appropriate enzyme to catalyze crotonyl-CoA reduction could potentially serve as a second filter to trap carbon in the synthetic pathway given that physical events, such as product secretion or cleavage of the CoA-thioester, were insufficient to amplify flux to the target goals.

The enoyl-CoA reductase activity is fairly ubiquitous and several divergent enzyme families need to be considered. The most well-studied family of enzymes that catalyze this transformation are typically associated with fatty acid biosynthesis and could be difficult to disengage from their native context because of the higher-order organization of fatty acid synthases. The acyl-CoA dehydrogenases involved in β-oxidation can be found with various acyl-CoA chain-length specificities, but utilize FAD/FADH2 for redox chemistry without recycling the NAD(P)H equivalents produced from glucose catobolism. The native clostridial Bcd/EtfAB system can achieve higher n-butanol titers compared to our Ccr-based pathway [20,22], but is complicated by oxygen sensitivity and dependence on interactions with incompletely defined downstream redox partners [40]. Most importantly, even if NAD(P)H were to act as the terminal reductant, all of these three systems depend on flavin cofactors to catalyze the double-bond reduction, which allows reversible reactivity as illustrated by its biological use in the opposing acyl-CoA β- oxidation pathway. Therefore, we focused on a smaller but mechanistically-distinct class of enoyl-CoA reductases that were found to catalyze the reduction of enoyl-CoAs using NAD(P)H in the absence of a flavin mediator [41,42]. This direct hydride transfer from NAD(P)H to the enoyl-CoA substrate increases the barrier to the reverse oxidation reaction based on standard reduction potentials and could potentially kinetically trap crotonyl-CoA in the synthetic n-butanol pathway. A crotonyl-CoA specific trans-enoyl-CoA reductase (Ter) from this family was identified in mitochondrial extracts of the photosynthetic flagellate Euglena gracilis, which interestingly do not show the ability to catalyze the reverse oxidation of butyryl-CoA to crotonyl-CoA in the presence of NAD+ or NADP+ [41]. This result indicates that direct NAD(P)H reduction of the double bond may indeed enforce an effectively irreversible step based on chemical rather than physical reaction mechanism.

Figure 2.6. In vitro characterization of His6-Ter from T. denticola. (A) SDS-PAGE of BL21(de3) pET16b-His6-Ter purification: MW ladder (lane 1), pre-induction (lane 2), post-induction (lane 3), purified His6-Ter (lane 4). (B) In vitro product distribution with and without addition of 3 mM sodium bicarbonate using crotonyl-CoA as a substrate. The reaction was monitored at 260 nm and is plotted as the sum of m/z = 837 (butyryl-CoA) and 881 (ethylmalonyl-CoA) collected in negative-ion mode.

31

Figure 2.7. The consumption of n-butanol pathway intermediates in cell lysate. Monitoring consumption of pathway intermediates in E. coli DH1 cell lysates expressing the synthetic butanol pathway (pBT33-Bu1 and pCWori-ter.adhE2, 3-6) compared to inactive (boiled, 1) and empty vector (2) controls. The reactions were analyzed by RP-HPLC at 10 min and 60 min and are plotted as the sum of m/z = 809 (acetyl-CoA), 835 (crotonyl-CoA), 837 (butyryl-CoA), 851 (acetoacetyl-CoA), and 853 (3-hydroxybutyryl-CoA) collected in negative-ion mode. (A) Addition of crotonyl-CoA to the empty vector control sample (2) led to very little dissipation of the reactant. In lysates containing active pathway (3, 4), substantial reverse reaction to form 3-hydroxybutyryl-CoA was observed with low levels of residual crotonyl-CoA. With the addition of 5 mM NADH (4, 5), the Ter-catalyzed reduction of crotonyl-CoA appears to trap increasing levels of butyryl-CoA over time. (C-CoA, crotonyl-CoA; 3-HB-CoA, 3-hydroxybutyryl-CoA; B-CoA, butyryl-CoA). (B) Addition of butyryl-CoA to the empty vector control (2) and active pathway (3, 4) samples led to very little dissipation of the reactant. With the addition of 5 mM NADH (4, 5), a low level of butyryl-CoA loss is observed.

PhaA (U) PhaB/Hbd (U) Crt (U) n-butanol (mg/L)

pBad33-Bu1 pCWori-ccr.adhE2

0.336 (0.016)

0.389 (0.089)

7.4 (1.9)

52 (13)

pBT33-Bu1 pCWori-ccr.adhE2

0.452 (0.140)

0.254 (0.208)

111 (25.1)

95 (12)

pTT33-Bu1 pCWori-ccr.adhE2

1.73 (0.073)

0.819 (0.105)

93.7 (24)

81 (3)

pT5T33-Bu1 pCWori-ccr.adhE2

4.20 (0.753)

1.84 (0.237)

114 (11)

51 (4)

Table 2.2. PhaA, PhaB, and Crt specific activities in cell lysate. The enzymatic specific activities in crude cell lysates of E. coli DH1 harboring variants of the Ccr-dependent pathway and n-butanol titers after 3 days of production. Data are mean (s.d.) (n = 3).

32

Monomeric orthologs of Ter have also been found in prokaryotes and we consequently decided to test the NADH-dependent crotonyl-CoA Ter from Treponema denticola [42] as a replacement for Ccr and Bcd/EtfAB. A codon-optimized ter gene was synthesized by primer assembly and inserted into an expression vector with an N-terminal His6-tag. Purification and biochemical characterization of His6-Ter indicated that it was highly soluble and active when heterologously xexpressed in E. coli (Figure 2.6). Furthermore, no reverse oxidation reaction was observed in vitro with butyryl-CoA concentrations up to 100-fold higher than used in the standard assay for crotonyl-CoA reduction and up to 125-fold higher NAD+ concentrations compared to NADH, which is approximately five-fold higher than the expected total intracellular concentration of NAD(H) in E. coli [43]. Monitoring crotonyl-CoA and butyryl-CoA consumption in cell lysate expressing the active synthetic pathway showed that Ter is also potentially irreversible in vivo as well. The feeding experiments with crotonyl-CoA resulted in considerable reversion to 3-hydroxybutyryl-CoA by Crt (Keq ~ 10-1) [37] in cell lysate; however, the remaining crotonyl-CoA can be pulled forward to form butyryl-CoA with addition of NADH (Figure 2.7). Again, no substantial change in the spiked butyryl-CoA levels is observed, even in the presence of active Ter, which also most likely means that AdhE2 activity is low in the engineered strains.

We next replaced ccr with ter in the two-plasmid system in order to assess alterations in n-butanol productivity. We were surprised to find that this replacement led to only a 3.5-fold increase (340 mg/L) in n-butanol titers (Figure 2.1, 10, and 14). While the overall yield is significant, it was smaller than expected especially given the more recent reports that the Ccr-catalyzed reduction of crotonyl-CoA to butyryl-CoA is a side product of the native reductive carboxylation reaction to form ethylmalonyl-CoA [44]. Without the addition of bicarbonate, we found the in vitro product distribution for the Ccr from S. collinus to favor ethylmalonyl-CoA (65%) over butyryl-CoA (35%) formation, which confirms that Ccr can provide a route for carbon to exit the synthetic n-butanol pathway (Figure 2.8). From these results, we concluded that Ter was possibly competent to provide a kinetic control element to drive flux in the forward direction, but that its behavior could be masked by other bottlenecks.

Unmasking kinetic control in a high-flux pathway. We then looked to the development of new upstream bottlenecks in the synthetic pathway that could have been hidden by the limitations in Ccr productivity. One issue is the stereochemical difference between the PhaB product ((R)-3-hydroxybutyryl-CoA) and Crt substrate ((S)-3-hydroxybutyryl-CoA). However, in vitro characterization of Crt had showed that the kcat/Km for the incorrect stereoisomer was well within the physiologically-relevant regime (2.0 × 107 M-1 s-1) and only 30-fold lower than the pseudo-second-order rate constant for the correct stereoisomer (6.5 × 108 M-1 s-1) (Figure 2.9). Indeed, in the pathway containing Ccr, there is no substantial difference in n-butanol production when PhaB is substituted with the NADH-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) from C. acetobutylicum (Figure 2.1, 2.13) or Crt activity is increased over 10-fold (Figure 2.9). Given the higher production potential of the Ter-containing pathway, we carried out the same gene replacement and discovered that n-butanol titers were increased by almost an order of magnitude to 2,950 mg/L (Figure 2.1, 2.16), which is 25-fold higher than the Ccr pathway analog containing HBD (Figure 2.1, 2.13). These results indicate that n-butanol yields are amplified by Ter beyond the difference in specific activity between Ccr and Ter measured in cell lysates prepared from the individual production strains.

We then sought to explore the origin of the latent bottleneck unmasked by the switch from PhaB to HBD in the presence of Ter, which could result from either change in cofactor

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Figure 2.9. In vitro characterization of Strep-Crt from C. acetobutylicum. (A) SDS-PAGE of purified proteins: MW ladder (lane 1), His6-PhaB (lane 2), His6-Hbd (lane 3), Strep-Crt (lane 4), His6-TdTer (lane 5). (B) RP-HPLC trace of (R)- and (S)-3-hydroxybutyryl-CoA monitoring the absorbance at 260 nm. (C) Crt kinetic constants. Data are mean ± s.e. (n = 3) as determined from non-linear curve-fitting. Error in the kcat/KM parameter was obtained from propagation of error from the individual kinetic terms.

Figure 2.8. In vitro characterization of Ccr-His6 from S. collinus. (A) SDS-PAGE of BL21(de3) pCWori-ccr-His6 purification: MW ladder (lane 1), pre-induction (lane 2), post-induction (lane 3), soluble protein fraction (lane 4), insoluble protein fraction (lane 5), purified Ccr-His6 (lane 6). (B) In vitro product distribution with and without addition of 3 mM sodium bicarbonate. The reaction was monitored at 260 nm and is plotted as the sum of m/z = 837 (butyryl-CoA) and 881 (ethylmalonyl-CoA) collected in negative-ion mode.

34

Scheme 2.2. Optimization of the synthetic Ter-dependent pathway for n-butanol production. (a) Stereochemical outcomes for different front-end pathways (Bu1-4). Proposed bottlenecks are indicated with a crossed arrow. (blue, R. eutrophus; red, C. acetobutylicum; black, T. denticola; light blue, Aeromonas caviae; hbd, 3-hydroxybutyryl-CoA dehydrogenase; phaJ, crotonyl-CoA hydratase) (b) Comparison of ethanol and n-butanol biosynthesis. Redox balance from glucose to product in terms NAD+ usage and recycling allows high yields of ethanol during fermentative growth. The production of advanced biofuels from acetyl-CoA rather than pyruvate requires an additional oxidation reaction to generate reducing equivalents for the assembly of saturated targets. Although several routes to acetyl-CoA are possible, the pyruvate dehydrogenase complex (PDHc) achieves redox balance with the synthetic n-butanol pathway. (pdc, pyruvate decarboxylase; adh, alcohol dehydrogenase; aceEF.lpd, PDHc).

NAD+

(nmol/mg DCW) NADH

(nmol/mg DCW) NADP+

(nmol/mg DCW) NADPH

(nmol/mg DCW)

pBAD33-0 pCWori-0 pBBR1-0

37.48 (5.36) 16.57 (2.29) 1.45 (0.35) 1.63 (0.23)

pBT33-Bu1 pCWori-ter.adhE2 pBBR1-0

34.52 (6.12) 15.61 (2.53) 1.70 (0.76) 1.06 (0.19)


41.34 (5.31) 18.70 (1.91) 1.46 (0.27) 0.99 (0.11)


36.37 (3.76) 18.51 (1.46) 2.03 (0.98) 1.67 (0.96)


46.08 (7.69) 21.88 (6.67) 1.59 (0.30) 1.09 (0.18)

pBAD33-0 pCWori-0 pBBR1-aceE.aceF.lpd

38.51 (4.67) 25.95 (2.62) 1.90 (0.01) 2.06 (0.12)

pBT33-Bu1 pCWori-ter.adhE2 pBBR1-aceE.aceF.lpd

40.22 (2.73) 26.85 (1.82) 2.16 (1.25) 1.20 (0.06)


35.45 (7.03) 29.61 (9.21) 1.93 (0.34) 1.40 (0.09)


30.73 (2.80) 28.10 (4.89) 1.65 (0.13) 1.11 (0.17)


49.83 (3.63) 29.08 (4.71) 2.66 (0.30) 1.21 (0.17)

Table 2.3. Quantitation of NAD(P)H and NAD(P)+ n-butanol producing strains. Measurements of NAD(P)H and NAD(P)+ levels in E. coli DH1 harboring variants of the Ter-dependent pathway compared to empty vector controls. Data are mean (s.d.) (n = 3). DCW, dry cell weight.

35

specificity to NADH or by stereochemical issues related to the geometry of the 3-hydroxybutyryl-CoA or enoyl-CoA products [45] produced with these different upstream pathways (Scheme 2.2A). Although previous metabolic engineering studies have demonstrated that redox factor usage can significantly affect the yield of high-flux pathways because of differential regulation of NADH and NADPH pools [46,47], measurement of NAD(H) and NADP(H) levels did not show appreciable differences between low- and high-producing strains (Table 2.3). In contrast, replacement of Crt with PhaJ from Aeromonas caviae [48], an R-specific enoyl-CoA hydratase that produces the trans- rather than cis-enoyl-CoA product (isocrotonyl-CoA), led to a similar n-butanol amplification as observed for replacement of PhaB with Hbd (Scheme 2.2A; Figure 2.1, 2.16, and 2.17). PhaJ also appears to be more stringent than Crt with respect to its substrate specificity, which is consistent with its reported in vitro behavior [48], as the corresponding mismatched specificities led to a nearly inactive pathway (Scheme 2.2A; Figure 2.1, 14 and 18). Further in vitro characterization of Ter with respect to isocrotonyl-CoA indicated that the kcat for reduction of the cis-isomer is reduced ~104-fold compared to the trans-isomer (crotonyl-CoA), although the KM remains similar (Table 2.4). From these data, we concluded that n-butanol yields in our synthetic pathway are more sensitive to the rate of Ter turnover compared to redox cofactor usage.

Linking synthetic fuel production with host metabolism. With a robust pathway from acetyl-CoA to n-butanol constructed, we can begin to look to ethanol fermentation for inspiration. Although we typically consider acetyl-CoA to be the cellular building block for advanced fuel synthesis, pyruvate, as the end product of glycolysis, is the more biologically-relevant starting material when pathway context is taken under consideration (Scheme 2.2B). Ethanol fermentation in yeast and other ethanologenic hosts occurs at high yields because it provides precise redox balance with glycolysis in terms of NAD+/NADH recycling based on the presence of a non-oxidative pyruvate decarboxylase. In contrast, E. coli fermentation results in the production of mixed organic acids that favor the direct reduction of pyruvate to lactate and the secretion of acetate as a product of glycolytic “overflow”. Although there are several routes to acetyl-CoA under anaerobic and aerobic conditions, the pyruvate dehydrogenase complex (PDHc: aceEF.lpd) provides the additional reducing equivalents to balance the production of the saturated n-butanol product from glucose (Scheme 2.2B). In order to increase the availability of acetyl-CoA and NADH made through PDHc, we overexpressed the aceEF-lpd operon, which increased PDHc activity levels by 3-fold compared to the empty control plasmid (Tables 2.3 and 2.5). Analysis of n-butanol production showed that the enhancement of PDHc activity resulted in

kcat (s-1) KM (µM) kcat/KM (M-1 s-1)

crotonyl-CoA (3.59 ± 0.12) × 105 79 ± 8 (4.54 ± 0.48) × 109

isocrotonyl-CoA (1.11 ± 0.12) × 101 235 ± 61 (4.73 ± 1.33) × 104

Table 2.4. His6-Ter kinetic with respect to crotonyl-CoA and isocrotonyl-CoA. Data are mean ± s.e. (n = 3) as determined from non-linear curve-fitting. Error in the kcat/KM parameter was obtained from propagation of error from the individual kinetic terms.

36

PDHc (U)

pBBR1 (empty) 0.440 (0.044)

pBBR1-aceE.aceF.lpd 1.31 (0.287)

Table 2.5. Specific activity of the PDHc in cell lysate. PDHc specific activities in crude cell lysates of E. coli DH1 pBT33-Bu2 pCWori-ter.adhE2 expressing aceEF.lpd compared to an empty plasmid control. Data are mean (s.d.) (n = 3).

Figure 2.10. Time course of production with Ter based n-butanol pathway with and without heterologous PDHc expression. Daily product titers of n-butanol produced by E. coli DH1 harboring the pBT33-Bu2, pCWori-ter.adhE2, either pBBR1 (empty) or pBBR1-aceEF.lpd plasmids. Data are mean ± s.d. (n = 3).

Figure 2.11. Protudction with a Ccr based n-butanol pathway with and without heterologous PDHc expression. n-Butanol production of E. coli DH1 pBT33-Bu1 pCWOri-ccr.adhE2 with either pBBR1 (empty) or pBBR1-aceEF.lpd after 3 days. Data are mean ± s.d. (n = 3).

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a 1.6-fold improvement in yield to 4,650 mg/L (Figure 2.1, 19, Figure 2.10), representing an increase from the initial n-butanol pathway design of over 1,500-fold (Scheme 2.1). Interestingly, similar overexpression of PDHc in strains utilizing Ccr showed no effect on n-butanol titers (Figure 2.11), which indicates that Ter can support large amplifications in product titers despite the low AdhE2 activity observed in the butyryl-CoA feeding studies. Moreover, these experiments also point to upstream cellular pathways and decisions in carbon fate as the current limitation to n-butanol production rather than the synthetic pathway itself.

2.4. Discussion

In addition to the practical applications of synthetic biology to scalable chemical synthesis, the de novo construction of pathways in model host organisms such as E. coli offers a platform to reconstitute and study the behavior of complex enzyme systems in vivo. Using this approach, we have shown that significant carbon channeling can be achieved through a synthetic pathway constructed from a highly reversible reaction sequence by taking advantage of the chemical mechanism of an enzyme component to drive the equilibrium to completion without relying on physical mechanisms, such as a phase change of the product [39], tethering of the substrate [49-51], formation of a multienzyme complex [49,51-54], or enzyme-substrate compartmentalization [55].

Although general classes of irreversible reactions - such as those involving the loss of CO2 - are well known to promote high flux, the overall transformation of enoyl-CoA reduction catalyzed by an unusual flavin-independent Ter remains reversible. However, the product of Ter appears to be kinetically trapped compared to the more ubiquitous classes of enoyl-CoA reductases based on differences in enzymatic reaction mechanism involving direct hydride transfer from NADH to the substrate without use of a flavin mediator. As a result of this effectively irreversible step, the n-butanol titers and yields for our pathway constructed from Ter (2,950 mg/L) significantly exceed those engineered from the flavin-dependent Bcd/EtfAB system derived from the native butanologenic clostridial host, which produce titers from 150 to 200 mg/L in lab-scale growths in the absence of additional host engineering [20,22]. Interestingly, this observation holds true even if flux through Ter is reduced by several orders of magnitude when isocrotonyl-CoA is provided as a substrate by the pathway based on phaB and crt (Bu1). In this case, the Ter-dependent pathway still produced 340 mg/L of n-butanol even though its activity is limited by the severe ~105 defect in the kcat/KM parameter for the cis-isomer and/or the slow racemization of (R)-3-hydroxybutyryl-CoA catalyzed by Crt (10-3 s-1) to produce crotonyl-CoA [56]. These results further suggest that the role of Ter in limiting the back reaction for enoyl-CoA reduction is more important in vivo than its overall turnover rate.

Analysis of pathway variants showed that the kinetic control element provided by the Ter reaction mechanism is key to building a robust synthetic pathway for n-butanol production. By using a synthetic pathway design that incorporates an effectively irreversible step at the initial committed step in the pathway, which was identified as formation of crotonyl-CoA, we can draw the pathway equilibrium towards product formation despite the high reversibility of thiolase (PhaA) (Keq ~ 10-4 to 10-5) and enoyl-CoA hydratase (Crt and PhaJ) [35,36] enzymes by trapping at the keto reduction (PhaB or Hbd, Keq ~ 500 [36,57]) or enoyl-CoA reduction steps, respectively. When the equilibrium is drawn forward in this manner, intermediates can still be channeled to product in the presence of subsequent bottlenecks (AdhE2) if the host does not express cellular pathways for its rapid dissipation and loss from the pathway.

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We have also demonstrated in an engineered host that a chimeric biosynthetic pathway can be constructed from enzyme components from different organisms that exceeds the production by 8- [20,22] to 12-fold [23] of pathways based on the native clostridial pathway that have been optimized for function in E. coli. More importantly, the synthetic n-butanol pathway can achieve exceptionally high product titers (4,650 mg/L) and yields from added glucose (28%) at the lab-scale that are comparable to yields produced on the lab-scale by the native n-butanol fermentation host (18%) [58] and within 3- to 4-fold of industrially-optimized ethanol production from yeast. Like ethanol, n-butanol production from pyruvate can also serve to provide redox balance with glycolysis to regenerate NAD+ for ATP synthesis and thus offers the potential to realize similar yields from glucose as yeast ethanol if the host can be engineered to utilize n-butanol over native fermentation pathways under anaerobic conditions.

2.5. References

Portions of this work were performed in collaboration with the following persons: Studies on the quantification and measurement of n-butanol production and Ccr-Stag levels were assisted by Mr. Robert Bellerose.

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Chapter 3: Biochemical and Structural Characterization of the trans-Enoyl-CoA Reductase from Treponema denticola

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3.1. Introduction

The construction of hydrocarbon backbones through the fatty acid metabolic machinery plays a key role in cellular maintenance [1,2] as well as in the development of engineered pathways for the production of advanced biofuels [3-8]. Although the chemistry of fatty acid synthesis is highly conserved, there is a range of diversity in the enzyme systems which catalyze these reactions that is important for their physiological function [9-12]. Indeed, the choice between individual FAS components with different biochemical behavior is important at the cellular level for controlling product titers from engineered biofuel pathways. For example, the switch from the native flavin-dependent enoyl-CoA reductase used in the production of n-butanol, a key second-generation biofuel, to a flavin-independent trans-enoyl-CoA reductase from Treponema denticola (tdTer) [13] leads to an order of magnitude increase in product yield in engineered Escherichia coli [14,15]. Interestingly in this case, the Ter appears to allow the pathway flux to be driven forward without the use of malonyl-CoA and ATP by introducing a kinetic trap at the enoyl-CoA reduction step [14].

The Ter family of reductases was first identified from Euglena gracilis, a photosynthetic algae with five different FAS systems including a type II malonyl-CoA-independent pathway in the mitochondria that uses CoA rather than ACP as the acyl carrier [16-19]. The egTer enzyme was isolated and characterized from E. gracilis mitochondrial extracts and was proposed to serve as the enoyl-CoA reductase in this system [20,21]. In terms of phylogeny, Ters are distinct from members of the FabI family, which represents the major enoyl-ACP reductase used in type II FAS systems, as well as from the FabK and FabL isozymes [9,10]. However, they are quite similar to FabV enzymes, which is a relatively new class of enoyl-ACP reductases that was discovered in Vibrio cholera [22]. Based on the ability of Ters to amplify fuel titers in engineered CoA-linked pathways, we set out to further structurally and biochemically characterize tdTer. We now report the crystal structure of tdTer as well as mutagenesis studies to examine the function of the substrate binding loop in determining chain length specificity.

3.2. Materials and Methods

Commercial materials. Terrific Broth (TB), LB Broth Miller (LB), LB Agar Miller, reagent grade triethylamine (TEA), and glycerol were purchased from EMD Biosciences (Darmstadt, Germany). Isopropyl β-D-1-thiogalactopyranoside (IPTG), D-glucose, dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), sodium phosphate, sodium chloride, streptomycin sulfate, Tris-HCl, Tris base, hydrogen chloride, sodium hydroxide, HPLC grade methylene chloride, HPLC grade acetonitrile, and carbenicillin (Cb) were purchased from Fisher Scientific (Pittsburgh, PA). Crotonyl-CoA, butyryl-CoA, hexanoyl-CoA, lauroyl-CoA, trans-2-hexenoic acid, coenzyme A hydrate, N,N-dimethylformamide (DMF), ethyl chloroformate, N,N,N´,N´-tetramethylethylenediamine (TEMED), NADH, NADPH, NAD+, and NADP+ were purchased from Sigma-Aldrich (St. Louis, MO). Trans-2-dodecenoic acid was purchased from Oakwood Products, Inc. (West Columbia, SC). Imidazole and formic acid were purchased from Acros Organics (Geel, Belgium). Potassium bicarbonate was purchased from Mallinckrodt (St. Louis, MO). Polyacrylamide (30%, 37.5:1), electrophoresis grade sodium dodecyl sulfate (SDS), and ammonium persulfate were purchased from Bio-Rad Laborabories (Hercules, CA). PageRuler Plus Prestained Protein Ladder and DNase were purchased from Fermentas (Glen Burnie, MD). Deoxynucleotides (dNTPs) and Platinum Taq High-Fidelity polymerase (Pt Taq HF) were purchased from Invitrogen (Carlsbad, CA). Complete Protease Inhibitor tablets were purchased

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from Roche Applied Science (Indianapolis, IN). All restriction enzymes, antarctic phosphatase, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). Phusion polymerase was purchased from Finnzymes (Lafayette, CO). Ni-NTA Agarose resin was purchased from Qiagen (Valencia, CA). pET16b was purchased from Novagen (San Diego, CA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and resuspended at a stock concentration of 100 µM in 10 mM Tris-HCl, pH 8.5. DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (Qiagen) as appropriate. All absorbance readings were taken on a DU-800 spectrometer (Beckman-Coulter; Fullerton, CA) or a SpectraMax M2 plate reader (Molecular Devices; Toronto, Canada). RP-HPLC purifications were performed on an Agilent 1200 series HPLC coupled to a diode-array detector. LC-MS data were collected on an Agilent 1290 series. High-resolution mass spectral analyses were carried out at the College of Chemistry Mass Spectrometry Facility.

Bacterial strains. Escherichia coli DH10B-T1R (Invitrogen) and BL21(de3)-T1R (New England Biolabs) were used for construction of plasmids and protein production, respectively.

Phylogenetic analysis. Using sequences of characterized Ter (Treponema denticola [13] and Euglena gracilis [21]) and FabV enzymes (from Vibrio cholera [22], Burkholderia mallei [23], Pseudomonas aeruginosa [24], Xanthomonas orzyae [25], and Yersinia pestis [26]) as well as FabI from E. coli, the UniRef50 database [27,28] was searched to yield sequences that share less than 50% sequence identity. For each homology search the top 10 scoring entries were selected, redundant sequences were removed, and the sequences were aligned with MEGA 5 [29] using the MUSCLE algorithm [30]. The alignment output was analyzed over 394 positions using the maximum likelihood method in MEGA with a nearest-neighbor-interchange strategy, while allowing for deletion of gaps that exist in less than 50% of the sequences and 500 bootstrap replicates to evaluate the confidence.

Construction of plasmids. pET16b-His10-Ter was constructed by amplification of the synthetic ter gene with the TdTer F1 and TdTer R101 primers and insertion into the NdeI-XhoI restriction sites of pET16b. The Y240F mutant was constructed by splicing by overlap extension PCR using standard methods with TdTer F1/TdTer SOE R1 (5´-end), TdTer SOE F1/TdTer R101 (3´-end) and inserted into the NdeI-XhoI restriction sites of pET16b (Table 3.1). All other mutants were generated by site-directed mutagenesis using Quikchange (Table 3.1). pET23a-His10-Tev-Ter was constructed by amplification of the synthetic ter gene with the TdTer F102 and TdTer R101 primers and insertion into the SfoI-XhoI restriction sites of a pET23a derivative, which was modified to encode a His10-tag and TEV protease cleavage site (Macrolab, UC Berkeley). All plasmids were verified by sequencing following construction (Quintara Biosciences; Berkeley, CA).

Expression of Ter Variants. TB containing carbenicillin (50 µg/mL) was inoculated to OD600

nm = 0.05 with an overnight TB culture of E. coli BL21(de3)-T1R, freshly transformed with the expression plasmid. The cultures were grown at 37°C at 200 rpm to an OD600 nm = 0.6 to 0.8 before induction with IPTG (1mM) and dropping the temperature to 30°C. Cells were harvested 4 h after IPTG addition by centrifugation at 12,300 × g for 7 min at 4°C and stored at -80°C.

Expression of selenomethionine His10-Tev-Ter. 50 mL of LB containing carbenicillin (50 µg/mL) was inoculated to OD600 nm = 0.05 with an overnight LB culture of E. coli BL21(de3)-T1R, freshly transformed with pET23a-His10-Tev-Ter. The cultures were grown at 37°C at 200

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rpm to an OD600 nm = 0.6 to 0.8. The culture was spun down (12,300 × g, 7 min) and washed in 50 mL of M9-MOPS media (1 × M9 salts, 100 mM MOPS pH 7.4, 1% D-glucose, 2 mM MgSO4, 0.1 mM CaCl2, 0.1 µg/mL thiamine, 10 µM ferrous sulfate, 0.1 µM zinc sulfate, 0.8 µM manganese chloride, 0.15 µM cupric sulfate, 0.3 µM cobalt chloride, 4 µM boric acid, 30 nM ammonium molybdate) containing carbenicillin (50 µg/mL). The cells were centrifuged (12,300 × g, 7 min) and resuspended in M9-MOPS Cb media (50 mL). 1 L of pre-warmed (37ºC) M9-MOPS Cb media was then inoculated with the cell suspension (10 mL). The culture was grown at 37°C at 200 rpm to an OD600 nm = 0.4 to 0.8 and supplemented with a filter-sterilized amino acid mixture (leucine, isoleucine, valine, 50 mg/L final concentration; phenylalanine, lysine, threonine, 100 mg/L final concentration; selenomethionine, 75 mg/L final concentration). After an additional incubation at 15 min, the cells were cooled on ice with shaking for 30 min before induction with IPTG (1 mM). The culture was then grown overnight (12-16 h) at 16°C at 200 rpm. Cells were harvested by centrifugation at 12,300 × g for 7 min at 4°C and stored at -80°C.

Purification of Ter variants. The cell pellet was thawed and resuspended at 5 mL/g cell pellet in Buffer A (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0 at 4°C) containing PMSF (500 µM) and DNAse (0.7 U/g cell pellet). The cells were homogenized prior to lysis by passage through a French Pressure cell (Thermo Scientific) at 14,000 psi. The total lysate was centrifuged at 12,300 × g for 45 min at 4°C to separate the soluble and insoluble fractions. The DNA was precipitated from the cleared cell lysate with streptomycin sulfate (20% w/v) added dropwise over 10 min at 4 °C to a final concentration of 1%. The precipitated DNA was removed by centrifugation at 12,300 × g for 30 min at 4°C. The supernatant was loaded onto a Ni-NTA agarose column (3 to 5 mL) using an ÄKTA Purifier FPLC system (GE Healthcare) at a flow rate of 0.5 mL/min. The column was washed at a flow rate of 2 mL/min with 20 column volumes (cv) of Buffer A followed by 20 cv of 92% Buffer A and 8% Buffer B (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0 at 4 °C). The column was eluted with Buffer B. Protein-containing fractions were identified and pooled by their absorbance at 280 nm and used for downstream purification as described below. The concentration of purified protein was estimated using the extinction coefficient at 280 nm calculated by the ExPASy Peptide Properties Calculator for His10-Ter (42,560 M-1cm-1).

His10-Ter variants. The pooled fractions from the Ni-NTA agarose column were concentrated to <5 mL using an Amicon stirred ultrafiltration cell with an Ultracel 5,000 MWCO filter membrane (Millipore), loaded onto a G-25 column (100 mL), and eluted with Buffer C (20 mM Tris-HCl, 50 mM sodium chloride, pH 7.5 at 4°C). The protein-containing fractions were identified and pooled by their absorbance at 280 nm. After concentrating to ~5 mg/mL, glycerol (60% stock solution) was added to a final concentration of 5% (v/v). Proteins were then flash frozen in liquid nitrogen and stored at -80°C.

GTGA-Ter. The pooled fractions from the Ni-NTA agarose column were concentrated to <3 mL as previously described. TEV protease (1:50 mg TEV/mg Ter) and DTT (1 mM) were added and the mixture was dialyzed at 4ºC for 16 h against TEV cleavage buffer (3.5 L; 50 mM sodium phosphate, 300 mM sodium chloride, 1 mM DTT, pH 8.0) using 3,500-5,000 MWCO cellulose ester tubing. To remove the His10-tagged TEV protease and the His10-peptide, the dialysate was passed over the washed (20 cv Buffer A) Ni-NTA agarose column. The column flow-through and an additional wash (2 mL Buffer A) were collected and concentrated to 2 mL using a 3,000 MWCO Amicon Ultra-15. The concentrated protein was loaded onto a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) using an ÄKTA Purifier FPLC system (1 mL/min).

47

GTGA-Ter was eluted with Buffer C and concentrated to 30-60 mg/mL for immediate use in crystallization. For purification of selenomethionine GTGA-Ter, all solutions were supplemented with TCEP (0.5 mM).

Size-exclusion chromatography. Ter variants (~0.5 mg) were analyzed on a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) using an ÄKTA Purifier FPLC system (1 mL/min Buffer C, 500 µL sample loop). The elution volume:void volume ratio was compared to the standard proteins (Bio-Rad). Protein molecular weight was estimated by fitting the data using Excel (Microsoft, Redmond, WA) to the equation: log10MW = m × Ve/Vo + b where MW is the molecular weight, Ve is the elution volume, and Vo is the void volume.

Crystallization and structure determination of GTGA-Ter. Protein crystals were obtained using the hanging drop vapor diffusion method by combining equal volumes of protein solution (diluted to 20 mg/mL) and reservoir solution (0.1 M sodium citrate, pH 7.5, 25% polyethylene glycol 6000, 2.5% glycerol). Crystals grew within 2 d and were cryoprotected by briefly soaking in a solution containing 75% reservoir solution and 25% ethylene glycol followed by flash freezing in liquid nitrogen. Data were collected at Beamline 8.3.1 at the Advanced Light Source (Lawrence Berkeley National Laboratory). Datasets for native crystals were collected at a wavelength of 1.116 Å while selenomethionine-modified Ter crystals were analyzed with an inflection/peak wavelength of 0.979 Å and remote wavelength of 0.957 Å. Datasets were processed and merged with XDS and XSCALE. Experimental phases were determined from the location of selenium atoms using Phenix AutoSol [31] and used to build an initial model for selenomethionine-modified Ter with Phenix AutoBuild. This model was then used to solve the structure of the native protein by molecular replacement using Phenix AutoMR and AutoBuild to build a near-complete chain trace of each crystal. Iterative cycles of Phenix AutoRefine and manual refinement in Coot [32] were used to generate the final model. Co-crystallization with stoichiometric amounts of substrates and/or products (NADH, NAD+, NAD+/crotonyl-CoA, and NAD+/butyryl-CoA) was tested but insufficient electron density was observed to create a model for any bound ligand.

Modeling substrates. The docking model was generated using Glide SP [33-35] in the Maestro molecular visualization environment. Each substrate was first optimized and minimized without taking into account non-polar hydrogens using the Maestro Preparation Wizard. The NADH was docked first as a flexible ligand in the tdTer chain A active site in 20 poses by generating a scoring grid (size, 20 × 20 × 20 Å3; ligand search range, 14 × 14 × 14 Å3) encompassing the entire active site and correlated with the location of the bound NADH in the Y. pestis FabV crystal structure (PDB ID 3ZU3) [26]. The top 5 poses were analyzed and the docking model most closely resembling the Y. pestis FabV crystal structure was used to generate a receptor grid (size, 20 × 20 × 20 Å3; ligand search range, 14 × 14 × 14 Å3) centered around Y240 to dock the crotonyl-CoA substrate using the same approach.

Synthesis of acyl-CoAs. Acyl-CoAs were prepared using the mixed anhydride method [36]. Briefly, trans-2-hexenoic acid or trans-2-dodecenoic acid (52 µmol) was pre-incubated with triethylamine (1 eq, 52 µmol, 7.3 µL) for 30 min at room temperature in CH2Cl2 (1 mL). The reaction mixture was then cooled to 4°C before addition of ethyl chloroformate (1 eq, 52 µmol, 5.0 µL). After incubation for 2 h, CH2Cl2 was evaporated from the reaction with N2 at 4°C. The residue was resuspended in DMF (1.5 mL), transferred into a stirred solution of coenzyme A hydrate (0.5 eq, 26 µmol, 20 mg) in 0.4 M potassium bicarbonate, pH 8.4 (1.5 mL), and incubated at room temperature for 10 min. The reaction was then acidified to pH 3-4 with formic

48

acid, diluted to 50 mL with water, flash frozen with liquid N2, and lyophilized. After resuspension in water (1 mL), the acyl-CoA was purified RP-HPLC on an Eclipse XDB C-18 column (5 µm, 9.4 × 250 mm, Agilent) using a linear gradient from 0 to 50% acetonitrile over 18 min with water as the aqueous mobile phase (3 mL/min). The fractions containing product were pooled and lyophilized.

Trans-2-hexenoyl-CoA: The fractions of >85% purity were pooled and re-purified by RP-HPLC to obtain product of >95% purity. 1H-NMR (600 MHz, D2O, 25°C) δ (ppm) (alkyl chain numbered as CX* from carbonyl): 8.52 (s, 1H, H8), 8.22 (s, 1H, H2), 6.89 (m, 1H, C3*), 6.13 (m, 2H, H1' and C2*), 4.57 (m, 1H, H4'), 4.21 (m, 2H, H5'), 4.00, (s, 1H, H3''), 3.82 (m, 1H, H1a''), 3.53 (m, 1H, H1b''), 3.41 (t, J = 6.6 Hz, 2H, H5''), 3.31 (t, J = 6.3 Hz, 2H, H8''), 2.99 (t, J = 6.3, 2H, H9''), 2.39 (t, J = 6.6, 2H H6''), 2.13 (q, J = 7.2, 2H, C4*), 1.41 (sex, J = 7.3, 2H, C5*), 0.85 (m, 6H, H10'' and C6*), 0.73 (s, 3H, H11'').

13C-NMR (600 MHz, D2O, 25°C) δ (ppm) (alkyl chain numbered as CX* from carbonyl): 193.86 (C1*), 174.73 (C7''), 174.00 (C4''), 155.67 (C6), 152.60 (C2), 149.32 (C4), 148.59 (C8), 139.89 (C3*), 127.81 (C2*), 118.63 (C5), 86.34 (C1'), 83.52 (C4'), 74.23 (C2'), 74.20 (C3'), 74.09 (C3''), 73.80 (C1''), 71.86, 65.35 (C5''), 38.63 (C8''), 38.31 (C2''), 38.26, 35.38 (C5''), 35.31 (C6''), 33.67 (C4*), 27.74 (C9''), 20.83 (C10''), 20.50 (C5*), 18.11 (C11''), 12.86 (C6*). HR-ESIMS (M-H2) calcd for C27H42O17N7P3S m/z, 430.5791, found 430.5787.

Trans-2-dodecoyl-CoA: The fractions of >95% purity were pooled and used for enzyme assays. 1H NMR (600 MHz, D2O, 25°C) δ (ppm) (alkyl chain numbered as CX* from carbonyl): 8.54 (s, 1H, H8), 8.24 (s, 1H, H2), 6.92 (m, 1H, C3*), 6.14 (m, 2H, H1' C2*), 4.58 (m, 1H, H4'), 4.23 (m, 2H, H5'), 4.02, (s, 1H, H3''), 3.84 (m, 1H, H1a''), 3.76 (m, 1H), 3.63 (m, 1H, H1b''), 3.54 (m, 2H), 3.43 (t, J = 6.6 Hz, 2H, H5''), 3.35 (t, J = 6.6 Hz, 2H, H8''), 3.03 (t, J = 6.3, 2H, H9''), 2.41 (t, J = 6.3, 2H H6''), 2.15 (q, J = 7.2, 2H, C4*), 1.37 (sex, J = 7.3, 2H, C5*), 1.18 (m, 12H, C6*, C7*, C8*, C9*, C10*, C11*), 0.88 (s, 3H, H10''), 0.82 (t, J = 7.2, 3H, and C12*), 0.75 (s, 3H, H11'').

13C NMR (900 MHz, D2O, 25°C) δ (ppm) (alkyl chain numbered as CX* from carbonyl): 196.65 (C1*), 184.63, 177.46 (C7''), 176.74 (C4''), 158.33(C6), 155.56(C2), 152.10(C4), 151.82 (C8), 142.60 (C3*), 130.54 (C2*), 121.42 (C5), 89.10 (C1'), 86.31 (C4'), 77.00 (C2'), 76.88 (C3'), 76.71 (C3''), 74.83 (C1''), 74.63, 68.11, 65.26 (C5''), 41.42 (C8''), 41.11 (C2''), 38.23 (C5''), 38.18 (C6''), 34.37 (C9''), 33.89 (C10''), 31.32 (C4*), 31.14 (C10*), 31.11 (C6*), 30.89 (C7*), 30.58 (C5*), 29.73 (C8*), 26.47 (C9*), 24.80 (C11*), 23.66 (C10''), 20.96 (C11''), 16.19 (C12*). HR-ESIMS (M-2H)-2 calcd for C33H54O17N7P3S m/z, 472.6260, found 472.6251.

Steady-state kinetic measurements. Ter activity was measured by monitoring the decrease in absorbance of NAD(P)H at 340 nm using a modified literature protocol [14]. The specific conditions used for each tdTer variant are available in the Supporting Information. Data were analyzed by non-linear curve fitting to the Michaelis-Menten equation with data reported as mean ± s.e. (n ≥ 3). Error in kcat/KM was calculated by error propagation from the individual kinetic constants. The following conditions for each tdTer variant represent the amount of enzyme, assay volume, and substrate concentrations used for individual reactions.

His10-Ter: NADH (60 ng; 500 µL; 400 µM crotonyl-CoA; 1.5, 2.5, 5, 10, 25, 37.5, 50, 100, or 200 µM NADH), NADPH (600 ng; 500 µL; 400 µM crotonyl-CoA; 50, 100, 150, 200, 250, 275, 300, 400, 500, 750, 1000, 1250, 1500 µM NADPH), hexenoyl-CoA (20 ng; 250 µL; 100 µM NADH; 5, 10, 15, 25, 37.5, 50, 100 µM hexenoyl-CoA), dodecenoyl-CoA (8 ng; 250 µL; 100 µM NADH; 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 5.5, or 6 µM dodecenoyl-CoA).

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His10-Ter (Y240F): crotonyl-CoA (20 ng; 400 µL; 200 µM NADH; 25, 50, 75, 100, 125, 175, 250, or 500 µM crotonyl-CoA).

His10-Ter (I287A): crotonyl-CoA (200 ng; 250 µL; 100 µM NADH and 50, 100, 200, 300, 400, or 800 µM crotonyl-CoA). hexenoyl-CoA (200 ng; 250 µL; 100 µM NADH; 3, 5, 7.5, 10, 17.5, 25, 50, or 100 µM hexenoyl-CoA), dodecenoyl-CoA (200 ng, 250 µL; 100 µM NADH; 1, 1.5, 2, 2.5, 3, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 µM dodecenoyl-CoA).

His10-Ter (L291A): crotonyl-CoA (20 ng; 250 µL; 100 µM NADH; 50, 100, 150, 175, 200, 300, 400, or 800 µM crotonyl-CoA), hexenoyl-CoA (100 ng; 250 µL; 100 µM NADH; 5, 10, 17.5, 25, 30, 40, 50, 100 µM hexenoyl-CoA).

His10-Ter (F295A): crotonyl-CoA (20 ng; 250 µL; 100 µM NADH; 50, 100, 150, 200, 300, 400, or 800 µM crotonyl-CoA). hexenoyl-CoA (50 ng; 250 µL; 100 µM NADH; 5, 10, 17.5, 25, 30, 40, 50, 100, 150 µM hexenoyl-CoA).

His10-Ter (Y370A): crotonyl-CoA (20 ng; 250 µL; 100 µM NADH; 50, 100, 200, 250, 300, 400, or 800 µM crotonyl-CoA), hexenoyl-CoA (100 ng; 250 µL; 100 µM NADH; 10, 15, 20, 25, 37.5, 50, 100, or 150 µM hexenoyl-CoA).

His10-Ter (L276A V277A): crotonyl-CoA (200 ng; 250 µL; 100 µM NADH; 25, 50, 75, 100, 150, 200, or 400 µM crotonyl-CoA), hexenoyl-CoA (200 ng; 250 µL; 100 µM NADH; 3, 5, 7.5, 10, 12.5, 15, or 25 µM hexenoyl-CoA).

His10-Ter (L276A V277A F295A): crotonyl-CoA (200 ng; 250 µL; 100 µM NADH; 50, 100, 200, 300, 400, 600, 800, or 1000 µM crotonyl-CoA), hexenoyl-CoA (200 ng; 250 µL; 100 µM NADH; 5, 10, 15, 20, 25, 30, 40, 50, 100, or 150 µM hexenoyl-CoA), dodecenoyl-CoA (200 ng; 250 µL; 100 µM NADH; 1, 1.5, 2, 2.5, 3, 3.5, 4, or 5 µM dodecenoyl-CoA).

Ternary complex formation. Assays were performed as described for steady-state kinetics. Data (n ≥ 3) were plotted using a double reciprocal plot and analyzed using least-square regression linear fitting. 10 ng of His10-Ter were used per reaction (250 µL). Crotonyl-CoA was used at concentrations 5, 10, 25, 50 100 µM and NADH at concentrations of 3, 4, 5, 7.5, 10 µM. All crotonyl-CoA concentrations were run at all NADH concentrations.

Order of binding. Assays were performed as described for steady-state kinetics. The specific conditions for each reaction are available in the Supporting Information. Data (n ≥ 3) were plotted using a double reciprocal plot and analyzed using least-square regression linear fitting to analyze product inhibition patterns by NAD+ and butyryl-CoA. 20 ng of His10-Ter were used per reaction (250 µL). The following conditions for were used for order of binding experiments.

NAD+ (400 µM crotonyl-CoA): 0 or 500 µM NAD+ (3, 4, 5, 7.5, or 10 µM NADH) and 1000 µM NAD+ (4, 5, 7.5, 10, or 15 µM NADH).

NAD+ (100 µM NADH): 0 µM NAD+ (5, 10, 25, 50 100 µM crotonyl-CoA), 2.5 µM NAD+ (7.5, 10, 25, 50, or 100 µM with and 100 µM crotonyl-CoA), 1000 µM NAD+ (10, 25, 50, 100, or 200 µM crotonyl-CoA).

Butyryl-CoA (400 µM crotonyl-CoA): 0 µM butyryl-CoA (3, 4, 5, 7.5, or 10 µM NADH), 500 µM butyryl-CoA (4, 5, 7.5, 10, or 15 µM NADH), 1000 µM butyryl-CoA (10, 15, 25, 37.5, or 50 µM NADH).

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Butyryl-CoA (100 µM NADH): 0 or 500 µM butyryl-CoA (5, 10, 25, 50, or 100 µM crotonyl-CoA) and 1000 µM butyryl-CoA (10, 25, 50, 100, or 200 µM crotonyl-CoA).

Substrate inhibition. Assays were performed as described for steady-state kinetics. Data (n ≥ 3) were analyzed by non-linear curve fitting to the Michaelis-Menten equation to obtain Vmax, app and KM, app, which were used to calculate Ki and Ki'. The following substrate concentrations were used for His10-Ter (20 ng, 250 µL): hexenoyl-CoA (10, 100, or 200 µM) or dodecenoyl-CoA (5, 10, or 15 µM) with 1.5, 2.5, 5, 10, 25, 37.5, 50, 100, or 200 µM NADH.

Product inhibition. Assays were performed as described for steady-state kinetics. The specific conditions used for each tdTer variant are available in the Supporting Information. Data (n ≥ 3) were analyzed by non-linear curve fitting to the Michaelis-Menten equation to obtain Vmax, app and KM, app. The following conditions for were used for product inhibition experiments.

His10-Ter (20 ng; 250 µL; 100 µM NADH): 500 µM and 1000 µM butyryl-CoA (5, 10, 25, 50, 100, 200, 400, or 800 µM crotonyl-CoA), 50 µM hexanoyl-CoA (37.5, 50, 75, 100, 125, 200, 400, 800 µM crotonyl-CoA), 250 µM hexanoyl-CoA (50, 75, 100, 125, 150, 200, 400, 800 µM crotonyl-CoA), 1 µM lauroyl-CoA (25, 50, 100, 150, 200, 300, 400, or 800 µM crotonyl-CoA), 1.5 µM lauroyl-CoA (100, 150, 200, 300, 400, or 800 µM crotonyl-CoA).

His10-Ter (I287A) (200 ng, 250 µL; 100 µM NADH): 0.025 µM lauroyl-CoA (75, 100, 125, 150, 200, 300, or 400 µM crotonyl-CoA), 0.1 µM lauroyl-CoA (5, 100, 125, 150, 200, 300, or 400 µM crotonyl-CoA.

His10-Ter (L276A V277A F295A) (200 ng, 250 µL; 100 µM NADH): 5 µM lauroyl-CoA (100, 150, 200, 250, 300, 400, or 800 µM crotonyl-CoA), 10 µM lauroyl-CoA (100, 200, 250, 300, 350, 400, or 800 µM crotonyl-CoA).

3.3. Results and Discussion

Phylogeny of trans-enoyl-CoA reductases. In order to examine the relationship between the Ter and functionally-related FabV and FabI families, a phylogenetic tree was constructed using a maximum likelihood approach with 500 bootstrap replicates from a sequence alignment of the top ten homologs from the UniRef50 database of two characterized Ter enzymes (T. denticola [13] and Euglena gracilis [21]), five characterized FabVs (Vibrio cholera [22], Burkholderia mallei [23], Pseudomonas aeruginosa [24], Xanthomonas orzyae [25], and Yersinia pestis [26]), as well as the FabI from Escherichia coli [9,10] (Figure 3.1, Figure 3.2). Like the FabVs, Ters share little sequence identity with and are significantly larger in size than FabIs, which serve as the major enoyl-ACP reductase of canonical type II fatty acid synthase systems [9,10]. The Ter sequences were consequently found to cluster with FabVs on a separate branch compared to FabIs. In this analysis, the prokaryotic Ter enzyme from T. denticola (tdTer) lies on a different branch from the characterized FabVs (46-52% sequence identity); however, the eukaryotic Ter from E. gracilis (egTer) was found to cluster with the FabV branch (52-57% sequence identity with the functionally encoded protein). These observations suggest that Ter enzymes may potentially diverge from FabVs, but that the two families of reductases may overlap or be difficult to distinguish by sequence analysis or annotation alone.

Crystal structure of the trans-enoyl-CoA reductase from Treponema denticola. Based on the distinct phylogeny of tdTer and absence of any Ter crystal structures deposited in the Protein

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Figure 3.1. Compressed phylogenetic tree of Ter, FabV, and FabI homologs. Maximum likelihood analysis of characterized Ter, FabV, and FabI enzymes (red) and homologs with <50% sequence identity from the UniRef50 database. Bootstrap values are indicated at branchpoints.

Figure 3.2. Uncompressed phylogenetic tree for FabVs, Ters, and FabIs. An alignment of UniProt sequences from the UniRef50 database using the FabIs, FabVs, and Ters (red) was performed using the MUSCLE algorithm. A maximum likelihood-nearest neighbor interchange tree was then constructed in MEGA 5.0 and tested with 500 bootstrap replicates. Bootstrap values are listed near each branch. UniProtKB numbers are listed in parentheses.

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Figure 3.4. Size-exclusion chromatograms of tdTer variants. (A) Wild-type tdTer andY240F active site mutant. (B) Binding loop mutants.

Figure 3.3. SDS-PAGE analysis of purifications of tdTer variants. SDS-PAGE showing pre-induction (lane 1), post-induction (lane 2), soluble fraction (lane 3), Ni-NTA eluate (lane 4), G-25 eluate (lane 5) for His10-tdTer variants. (A) wild-type, (B) I287A mutant, (C) L291A mutant, (D) F295A mutant, (E) Y370A mutant, (F) L276A/V277A double mutant, and (G) L276A/V277A/F295A triple mutant.

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Selenomethionine-tdTer* tdTer

Data Collection

Space group P1 P1 P1

Unit cell parameters

a, b, c (Å) 62.7, 87.6, 92.4 62.4, 87.6, 91.6 62.05, 87.05, 91.46

�, β, γ (deg) 106.4, 109.6, 98.3 106.3, 109.9, 98.4 106.14, 109.72, 98.52

Wavelength(s) (Å) 0.979 0.957 (Se Remote) 1.116

Resolution (Å) 19.85-2.05 (2.12-2.05) 19.85-2.05 (2.12-22.05) 19.68-2.00 (2.071-2.00)

Rmerge (%) 15.1 (59.9) 16.9 (72.8) 13.5 (69.9)

<I/σ(I)> 20.67 (4.38) 13.29 (2.77) 15.61 (2.60)

Total reflections 438763 (70586) 472313 (75927) 222904 (14852)

Unique reflections 114220 (17931) 122645 (19279) 105852 (7127)

Completeness (%) 97.1 (96.1) 97.4 (96.0) 94.04 (88.17)

Multiplicity 3.8 (3.9) 3.9 (3.9) 2.1 (2.1)

Refinement

Rwork (%) 18.22 (23.92) 20.62 (30.66)

Rfree (%) 21.45 (27.29) 23.47 (35.02)

Number of atoms 12861 12957

Nonsolvent 12316 12316

Solvent 545 641

B-factors 39.90 37.70

Protein 39.90 37.70

Water 41.20 37.90

RMSD

Bond lengths (Å) 0.004 0.003

Bond angles (deg) 0.78 0.66

Ramachandran plot

Most favored (%) 98 97

Disallowed (%) 0 0.25

PDB ID 4GGP 4GGO

* Selenomethionine-tdTer datasets were merged in order to build the initial model.

Table 3.1. Data collection and refinement statistics for GTGA-Ter structures.

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Figure 3.5. Crystal structure of tdTer. (A) Cartoon representation of tdTer looking down at the active site (Y240 and K249) and Rossman fold (E80) as well as a view rotated 165° around y-axis. The major (helices 3, 4, 6, 8, 9, 10, 15 and strands 7 and 8) and minor (helices 1, 3, 12, and 15) portals and the substrate binding loop (residues T278-V286, including helix 10) are indicated. (B) View of the putative active site showing Y240 and K249 as well as the E80 and the NADH binding pocket formed by the Rossman fold. (C) Overlay of the tdTer crystal structure (grey) with the FabV from Y. pestis (blue). The differences in the main chain trace are highlighted in magenta (tdTer) or red (FabV).

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Databank, we set out to structurally characterize tdTer and to study its function in more detail. For structural studies, the synthetic gene encoding tdTer was expressed heterologously in E. coli with an N-terminal His10-tag and an intervening TEV protease site. tdTer was purified and cleaved with TEV protease to yield enzyme with a residual GTGA linker at the N-terminus (Figure 3.3). tdTer crystallized in an asymmetric unit containing four identical copies of the Ter monomer, which is in agreement with the apparent molecular weight measured in solution (Figure 3.4). Until the recent deposit of the X. orzyae [25] and Y. pestis [26] FabV structures, there were no structural homologs with sufficiently high similarity to tdTer to use as search models for molecular replacement. Therefore, the initial model for tdTer was built with phasing determined from a two-wavelength MAD data set of selenomethionine-substituted protein, with the most complete monomer serving as the molecular replacement model for the additional tdTer chains in the asymmetric unit (RMSD, 0.170 to 0.256 Å between monomers). Electron density was identified for all 401 native residues in each of the monomers but was not observed for the N-terminal GTGA linker, which was not included in the model. The crystal structure of native tdTer was then solved to 2.00 Å resolution by molecular replacement using the selenomethionine-modified structure and found to be in close agreement (RMSD, 0.246 Å for a single monomer) (Table 3.1).

The tdTer structure contains fifteen α-helices and ten β-strands comprising one six-stranded parallel β-sheet (strands 1-4 and 9-10) and two two-stranded anti-parallel β-sheets (strands 5-6, strands 7-8) (Figure 3.5A). Like other members of the SDR family, tdTer possesses a typical Rossman fold for nucleotide binding based on the presence of the G69-X-G71-XXX-G76 consensus motif. In the crystal structure, the Rossman fold is formed by helices 3 and 5 along with the six-stranded β-sheet and contains E80 in the cofactor binding pocket (Figure 3.5A, Figure 3.6), predicting specificity for NADH over NADPH [37-39], which is consistent with previous biochemical studies on tdTer [13]. The active site can be located by the putative catalytic tyrosine, Y240 (Figure 3.5B); however, several reductases along the tdTer branch, including tdTer, contain both a Y-X8-K and a Y-X6-K consensus sequence that are the respective signatures of FabVs [23] and the other more common families of enoyl-ACP reductases, such as FabI, FabK, and FabL [40]. From the relative orientations of K247 and K249 (Figure 3.7), tdTer also shares resistance to triclosan inhibition as observed for FabVs [22-24,26]. Surrounding the

Figure 3.6. View of the Rossman fold of tdTer. View from the major portal of tdTer showing the Rossman fold for binding of the NADH cofactor.

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active site, tdTer also has the major (helices 3, 4, 6, 8, 9, 10, 15 and strands 7 and 8) and minor (helices 1, 3, 12, and 15) portals into the active site and substrate binding loop (residues T278-V286, including helix 10) that are shared with structurally-characterized FabVs and FabIs.

Overall, the tdTer structure aligns well at the structural level with the recently reported FabV structures from X. orzyae (RMSD, 0.630 Å) and Y. pestis (RMSD, 0.691Å) (Figure 3.5C). One of the main differences in the main chain trace of tdTer is the absence of the final β-strand at the C-terminus found in the FabVs that forms part of the β-sheet in the Rossman fold (Figure 3.5C). These residues are situated directly next to the binding pocket for the adenine moiety of NADH, but do not appear to directly interact with the cofactor. The tdTer structure also contains an elongated loop (T40-K48), with an α-helix (helix 2) that is missing in the FabV structures. Although this loop is distal to the substrate binding pocket, it connects helix 1 to the six-stranded β-sheet and may play a role in the movement of one of the α-helices (helix 1) of the minor portal. In addition to these differences, there is a 3.3 Å shift in the substrate binding loop away from the minor portal that increases the size of the binding pocket in the tdTer structure compared to the FabV structures.

One possible functional difference between Ters and FabVs is that the Ter enzymes are proposed to be part of a FAS system that relies on CoA rather than ACPs [17,21], which serve as the acyl carrier in canonical type II FAS systems. The interaction of ACPs with the enzymes involved in fatty acid biosynthesis is mediated by the N-terminal α-helix of the ACP that contains negatively-charged residues surrounded by a hydrophobic region and associates with the corresponding positively-charged patch on its protein partners that is also surrounded by hydrophobic residues [41-43]. In the Y. pestis FabV, the ACP recognition domain is suggested to consist of the basic residues, K4, R6, and R8, and the hydrophobic residues, G9, F10, I11, V13, and A15, at the N-terminus [26]. In comparison, reductases in the tdTer branch lack the full complement of basic residues and several of the proposed hydrophobic areas are replaced with charged or polar residues instead (Figure 3.8A). For tdTer specifically, K4 and R8 overlay well with the corresponding residues in FabV but the exchange of R6 for M6 results in the formation of a hydrophobic center in this charged patch and a significant decrease in positive charge at the

Figure 3.7. Orientation of Lys247 and Lys249 in the tdTer active site. tdTer contains both a Y240-X6-K247 and Y240-X8-K249 consensus sequence around the catalytic tyrosine (Y240) similar to the FabIs and FabVs, respectively. Based on the orientation of these two lysines with respect to the active site tyrosine, K249 appears to be the catalytic lysine.

57

surface (Figure 3.8BC). In addition, G9 and F10 in the hydrophobic region are changed to polar amino acids, N9 and N10, thereby reducing the hydrophobicity of the N-terminal region of tdTer. Taken together, these changes could lead to a difference in acyl carrier specificity between the Ter and FabV families.

A docking model for NADH and enoyl-CoA binding. A docking model was generated in order to examine how the enoyl-CoA substrate and NADH cofactor might be oriented within the tdTer active site. The structures of the substrates were first minimized and then docked as flexible ligands using Glide [33-35,44]. The initial model was generated with NADH alone, as there are a significant number of crystal structures solved with a nicotinamide cofactor bound to a Rossman fold that could be used for model validation. The NADH was constrained to a 8,000 Å3 grid encompassing the entire active site and the ligand center was allowed to vary 14 Å from the center of the Rossman fold. The top five binding models were examined (Figure 3.7) with the top four models placing the nicotinamide in the predicted binding pocket with N6 of the adenine within hydrogen-bonding distance of D116 and the 3´-OH of the ribose sugar bound to E80. These results agree with the NADH binding mode found in other structures containing a Rossman fold, including the Y. pestis FabV. The second solution was selected for docking of the enoyl-CoA substrate because it was the most similar to the FabV structure with the nicotinamide ring adjacent to Y240 (Figure 3.10A, Figure 3.7).

The C4 substrate, crotonyl-CoA, which was previously reported to be the major substrate for tdTer [13], was then docked into the tdTer-NADH model using the same approach. In this case, the full acyl-CoA substrate and the substrate without the adenine or adenosine moiety were used for docking studies in order to control for the lack of adenosine-specific interactions typical to many CoA binding enzymes [45,46]. Regardless of the ligand, all models place the double bond of the butenoyl functional group between the docked NADH substrate and Y240 and the terminal

Figure 3.8. Comparison of the ACP binding site of FabV from Y. pestis to tdTer. (A) Sequence alignments of the N-terminal region of FabVs and Ters involved in ACP recognition (B) View and electrostatic map of the ACP binding surface of FabV. The electrostatic map was generated by ABPS with the scale indicating calculated potential in solution. (C) View and electrostatic map of the same region on tdTer.

58

Figure 3.10. Docking model of tdTer with NADH and crotonyl-CoA. (A) The initial model of tdTer with the docked NADH redox cofactor (magenta). Dotted lines represent distances less than 4 Å. (B) Docking model of the tdTer ternary complex with NADH (magenta) and crotonyl-CoA (blue). (C) View of the terminal carbon of the crotonyl-CoA acyl group (blue) with respect to the minor portal.

Figure 3.9. Docking models of tdTer with NADH. The top four models generated by Glide for flexible docking of NADH (blue) into the tdTer active site. Model 2 was selected to model the ternary complex based on the location of the nicotinamide ring and its similarity to the NADH-complex structure of the FabV from Y. pestis.

59

carbon of the acyl group at the entrance to the minor portal near a tightly-packed area formed by a strand containing G278 to V280 and helix 11 (Figure 3.10BC, Figure 3.9). Docking was also attempted with longer enoyl-CoA substrates but did not yield any models with substrate bound within the active site. We then used MOLE [47] to further examine the minor portal of tdTer and the FabVs from X. orzyae and Y. pestis to explore how these substrates might be accommodated. These studies show that while the minor portal is open in all three structures, it contains significant bottlenecks of 1.5 to 2 Å that would prevent binding of substrates larger than crotonyl-CoA to a static structure. A conformational change or dynamic movement in the minor portal would therefore be necessary to allow tdTer to interact with these substrates if they were to bind with the acyl moiety in the minor portal of the enzyme.

Biochemical characterization of Ter. Enzymes that catalyze similar reactions to tdTer, including the FabV from B. mallei [23], have generally been found to form a ternary complex using an ordered bi-bi reaction mechanism in which the redox cofactor is bound first and followed by the substrate [48-51].The docking model of tdTer with NADH and crotonyl-CoA appears to support this mechanism, as the binding site of NADH would be occluded if crotonyl-CoA were to bind first. Steady-state kinetic experiments with varying NADH and crotonyl-CoA confirm the formation of a ternary complex, with the double reciprocal plots showing a set of intersecting lines (Figure 3.12A). The selectivity of tdTer for NADH (KM, 5.2 ± 0.4 μM; kcat/KM, (1.6 ± 0.1) × 107 M-1s-1) over NADPH (KM, 190 ± 20 μM; kcat/KM, (3.9 ± 0.5) × 105 M-1s-1) was also confirmed at this time. The order of binding was then established by examining the effect of product inhibition on the steady state kinetics. These studies show that NAD+ acts as a competitive inhibitor for NADH binding and a mixed inhibitor with respect to crotonyl-CoA (Figure 3.12B), while butyryl-CoA serves as a mixed inhibitor of both substrates (Figure 3.12C). This pattern of product inhibition indicates that tdTer also uses an ordered bi-bi reaction mechanism with NADH binding first.

We next turned our attention to mutagenesis studies to elucidate the function of the putative catalytic tyrosine, Y240, as well as residues that might be involved in substrate selectivity. Introduction of the Y240F mutation leads to a 5,000-fold drop in catalytic efficiency with no significant change in KM (Table 3.2), which is consistent with a key role in catalysis as suggested for FabVs [23,26] and FabIs [10,49,52]. In terms of substrate specificity, the kcat/KM of tdTer for crotonyl-CoA is similar to the value measured for the FabV from V. cholera [22] (Table 3.2). These data are consistent with the proposal that Ters can service CoA-dependent FAS pathways; however, their activity with ACP-dependent substrates has not yet been fully explored in this family of enzymes.

Although tdTer had previously been reported to exclude the hexenoyl-CoA substrate [13], it has been used in the construction of synthetic fuel pathways for hexanol production, which shows that it is active in vivo on a C6 substrate [53]. Indeed, we find that tdTer is 7-fold and 25-fold more active in vitro with hexenoyl-CoA and dodecenoyl-CoA, respectively, as a result of both higher kcats and lower KMs. With this result in hand, we decided to explore the role of the substrate binding loop in influencing chain length specificity. The I287A, L291A, F295A, Y370A, L276A/V277A, and L276A/V277A/F295A mutations were selected because these residues appear to be involved in mediating the conformation of the binding loop (Figure 3.13). Three of the mutants, L291A, F295A, and Y370A, demonstrated relatively similar kinetic behavior to wild-type tdTer on crotonyl-CoA and hexenoyl-CoA (Table 3.2). Because of the significant drops in kcat with the remaining three mutants, I287A, L276A/V277A, and L276A/

60

Figure 3.11. Docking model of tdTer with the most favored ternary complexes. Four of the top five models generated by Glide for flexible docking of crotonyl-CoA (blue) into the active site of the tdTer-NADH complex.

Figure 3.12. Steady-state kinetic characterization of tdTer with enoyl-CoA substrates. (A) Double reciprocal plots varying NADH and crotonyl-CoA indicate ternary complex formation. (B) Double reciprocal plots monitoring NAD+ product inhibition with respect to NADH (top) and crotonyl-coA (bottom). Data are fit to a linear regression and represent mean ± s.d. (n ≥ 3) with propagated error. (C) Double reciprocol plots monitoring butyryl-CoA product inhibtion with respect to NADH (top) and crotonyl-CoA (bottom). Data are fit to a linear regression represent mean ± s.d. (n ≥ 3) with propagated error.

61

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62

V277A/F295, the data were normalized by calculating the ratios of kcat/KM for the C4 versus C6 substrate for each individual mutant. Using this analysis, we see that both the I287A single mutant and L276A/V277A/F295A triple mutant exhibit larger increases in catalytic efficiency on the longer hexenoyl-CoA substrate of 100- and 17-fold compared to wild-type (7-fold). These results suggest that these mutations may increase accessibility of the longer acyl chain to the active site pocket through perturbations of either the minor or major portal, which are both modulated by the substrate binding loop.

In addition to demonstrating higher catalytic efficiencies and lower KMs, the longer chain substrates also cause a significant level of inhibition of tdTer under saturating conditions. Neither hexenoyl-CoA and dodecenoyl-CoA change KM, NADH, which shows that the longer chain acyl-CoA substrates do not compete with NADH binding (Figure 3.14). In contrast, we observe strong product inhibition with the addition of hexanoyl-CoA and lauroyl-CoA (Table 3.3, Figure 3.15). The data were fit to a model for mixed inhibition based on the patterns observed in the double reciprocal plot where both kcat and KM, app are affected. Due to the order of binding as well as the absence substrate inhibition, which is consistent with previous studies [23], we interpret this data to mean that the acyl-CoA product can bind either tdTer-NADH or tdTer-NAD+. The two mutants with higher ratios of C6:C4 activity, I287A and L276A/V277A/F295A, were also characterized with respect to product inhibition by lauroyl-CoA (Table 3.3, Figure 3.15). Interestingly, Ki and Ki´ are much lower than wild-type for the I287A mutant but not the triple mutant. The correlation of C12 product inhibition with the increased C6:C4 activity ratio for the I287A mutation, which is located near the front of the binding loop, seems to suggest that the major portal is more important than the minor portal for recognition of longer chain substrates. In comparison, the weak preference of the L276A/V277A/F295A mutant for the C6 substrate along with the higher Ki and Ki´ versus wild-type tdTer seems to imply that relaxing of the minor portal can accommodate slightly larger substrates without increasing affinity for an extended acyl chain.

Figure 3.13. Amino acids mutated in tdTer variants. View of the positions mutated in the tdTer binding loop region.

63

Enzyme Inhibitor [Inhibitor] (µM) Ki (µM) Ki' (µM) wild-type butyryl-CoA 500 400 ± 100 750 ± 60 1000 400 ± 100 4100 ± 400 hexanoyl-CoA 50 50 ± 10 110 ± 10 250 80 ± 30 250 ± 30 lauroyl-CoA 1 0.5 ± 0.1 3.1 ± 0.2 1.5 0.4 ± 0.1 3.7 ± 0.5 I287A lauroyl-CoA 0.025 0.06 ± 0.02 0.53 ± 0.08 0.1 0.03 ± 0.02 0.23 ± 0.06 L276A/V277A/F295A lauroyl-CoA 5 10 ± 2 12 ± 1 10 3 ± 1 5.6 ± 0.8

* Data are mean ± s.e. (n ≥ 3) as determined from non-linear curve-fitting. Error in the Ki and Ki´ parameters were obtained from propagation of error from the individual kinetic terms.

Table 3.3. Kinetic constants for the product inhibition of tdTer. Inhibition of wild-type and mutant tdTers by hexanoyl-CoA and lauroyl-CoA.

Figure 3.14. Substrate inhibition of tdTer. Substrate inhibition was determined by characterizing tdTer activity with respect to NADH with addition of hexenoyl-CoA and dodecenoyl-CoA. (A) Double reciprocal plot with hexenoyl-CoA. (B) Double reciprocal plot with dodecenoyl-CoA. (C) Kinetic constants were determined by non-linear curve fitting and indicate that there is no significant change of Vmax or KM, NADH within error.

64

Figure 3.15. Product inhibition of tdTer. Product inhibition was determined by characterizing tdTer activity with respect to crotonyl-CoA with addition of acyl-CoA products. (A) Double reciprocal plot of wild-type tdTer with hexanoyl-CoA. (B) Double reciprocal plot of wild-type tdTer with lauroyl-CoA. (C) Kinetic constants were determined by non-linear curve fitting to a mixed inhibition model.

65

3.4. Conclusions

Enoyl-ACP (CoA) reductases catalyze a key step in the biosynthesis of reduced hydrocarbons and are important for driving the pathway equilibrium forward towards chain elongation. The diversity of isozymes available for this reaction is interesting with regard to the evolution of different FAS systems as well as the engineering of pathways to produce advanced biofuels. In this work, we have biochemically and structurally characterized a member of the Ter family of reductases, which is proposed to participate in CoA- rather than ACP-dependent pathways [21]. Although the Ters are distinct from the FabI, FabK, and FabL enoyl-ACP reductase isozymes, they are highly related to the FabV family based on their close phylogeny as well as their structural similarity. One interesting difference between the tdTer structure and the FabV structures from X. orzyae and Y. pestis is the difference in the ACP binding surface, which may lead to differences in acyl carrier specificity. However, it would not be surprising if Ters are competent to reduce both CoA- and ACP-linked substrates.

Initial characterization of tdTer indicates that Y240 is the catalytic tyrosine and that K249 is likely the active site lysine shared by FabVs and FabIs. Further biochemical studies show that tdTer prefers NADH over NADPH and uses an ordered bi-bi mechanism initiated by binding of the redox cofactor. Although tdTer had been previously reported to prefer C4 substrates [13], we observe that the catalytic efficiency is higher on both C6 and C12 substrates. The strong product inhibition of wild-type and mutant Ters implies that the hydrocarbon tail of the substrate is important for binding and recognition. Preliminary mutagenesis experiments reveal that the substrate binding loop appears to be involved in determining chain length specificity through changes either in the minor or major portal. We have identified two mutants, I287A and I276A/V277A/F295A, which demonstrate higher preference for C6 over C4 substrates compared to wild-type. In this regard, the high impact of the I287A mutation, which is located near the front of the major portal, on interaction with both C6 and C12 acyl-CoAs could be consistent with the binding mode observed in FabI structures [54].

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54. D. A. Rozwarski, C. Vilchèze, M. Sugantino, R. Bittman and J. C. Sacchettini, Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate, J. Biol. Chem. 1999, 274, 15582-15589.

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Chapter 4: Analysis of pathway flux utilizing co-localization via a protein scaffold

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4.1. Introduction

Living systems have developed a variety of methods for the control of flux through metabolic networks. One common theme involves colocalization of enzymes in a biosynthetic pathway [1-3]. Natural systems have developed several methods of colocalization in order to increase flux [4-6] and reduce the concentration of toxic intermediates [7-9]. Some of the means of colocalization seen in living systems include formation of organelles or protein capsids [10], localization of proteins to a membrane [11-13], multi protein systems with high affinity for partner proteins [5], and fusions enzymes [14, 15]. In synthetic systems, involving enzymes taken from several different organisms to create a synthetic pathway, a loss of native protein-protein interactions is typically observed. Steps that can be catalyzed by enzymes closely coupled in space under native conditions now see intermediates and enzymes independently diffusing in the cell. As a result, colocalization can be capable of increasing flux in these engineered systems and several different approaches to engineer protein co-localization have been taken, including fusion proteins [16], fusion of interaction domains [17], protein scaffolds [8, 18], DNA/RNA scaffolds [19-21], and compartamentalization [22]. In one example, the use of a protein scaffold for colocalization has been shown to facilitate metabolic flux through the mevalonate pathway to prevent accumulation of a toxic intermediate [8] and the glucaric acid pathway by increasing local effective concentrations of a key precursor [18] (Figure 4.1).

The previously described n-butanol biosynthetic pathway, based on the Clostridium acetobutylicum ABE fermentation system, has been demonstrated to depend on a kinetically controlled reduction in the biosynthetic pathway [23]. The reliance on this kinetic trap suggests that the pathway could significantly benefit from one of the colocalization strategies described above in order to facilitate flux through the controlling step. To explore this possibility, the enzymes catalyzing the reactions around the kinetic trap have been fused to affinity tags for use with a previously characterized protein scaffold in order to examine the viability of engineered colocalization for increasing n-butanol titers [8].

Figure 4.1. The design of the protein scaffold system. A single GBD binding domain in linked to x SH3 binding domains and y PDZ binding domains (A). The ratios of the binding domains (B).

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Materials and Methods. Terrific Broth (TB), LB Broth Miller (LB), LB Agar Miller, sulfuric acid, glacial acetic acid, potassium chloride, dimethyl sulfoxide (DMSO), and glycerol were purchased from EMD Biosciences (Darmstadt, Germany). Isopropyl-β-D-1-thiogalactopyranoside (IPTG), D-glucose, dithiothreitol (DTT), Tris-HCl, phenylmethanesulfonyl fluoride (PMSF), carbenicillin (Cb), streptomycin sulfate, tetracycline hydrochloride (Tc), magnesium chloride hexahydrate, sodium phosphate monobasic monohydrate, calcium chloride, and sodium chloride were purchased from Fisher Scientific (Pittsburgh, PA). Spectinomycin dihydrochloride pentahydrate (Sp), chloramphenicol (Cm), kanamycin sulfate (Km), L-arabinose, acetyl-CoA, butyryl-CoA, crotonyl-CoA, acetaldehyde, butyraldehyde, N,N,N′,N′-tetramethylethylenediamine (TEMED), and NADH were purchased from Sigma-Aldrich (St. Louis, MO). Anhydrotetracycline hydrochloride (aTc) and imidazole were purchased from Acros (Geel, Belgium). Acrylamide/bis-acrylamide solution (30%, 37.5:1), electrophoresis grade sodium dodecyl sulfate (SDS), and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). PageRuler Plus Prestained Protein Ladder was purchased from Fermentas (Glen Burnie, MD). Deoxynucleotides (dNTPs), T4 DNA ligase, and Platinum Taq High-Fidelity DNA polymerase (Pt Taq HF) were purchased from Invitrogen (Carlsbad, CA). Antarctic phosphatase, T4 DNA ligase, Phusion polymerase, and all restriction enzymes were purchased from New England Biolabs (Ipswich, MA). DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick Gel Extraction Kit, and QIAquick PCR Purification Kit (QIAGEN; Valencia, CA) as appropriate. pCDFDuet-1 was purchased from Novagen (San Diego, CA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and resuspended at a stock concentration of 100 μM in 10 mM Tris-Cl, pH 8.5. Following plasmid construction, all gene inserts were sequenced using appropriate sequencing primers by Quintara Biosciences (Berkeley, CA). All absorbance readings were taken on a DU-800 spectrometer (Beckman-Coulter; Fullerton, CA) or a SpectraMax M2 plate reader (Molecular Devices; Toronto, Canada).

Bacterial strains. E. coli BL21(de3) was used for protein overexpression and purification. E. coli DH1 was used for n-butanol production studies. E. coli DH10B-T1R was used for plasmid construction and DNA isolation.

Plasmid construction. All plasmids were constructed using standard molecular biology techniques or the one-step, isothermal in vitro recombination system described by Gibson et al (Gibson cloning) [24]. PCR amplifications were carried out with either Pt Taq HF or Phusion polymerase using olignucleotides listed in (Table 4.1). A complete list of plasmids used in this study can be found in (Table 4.2).

pBT33-phaA.hbd-SH3_crt. The SH3_crt gene was constructed using splicing by overlap extension (SOE) PCR. The crt gene and was amplified using the crt SOE F7 and crt R5 primers and the trc promoter was amplified using the pTrc99a F12 and crt SOE R7 primers. The two PCR products were gel purified and amplified using pTrc99a F12 and crt R5. The resulting PCR product was inserted into the XmaI-EagI restriction sites of pBT33-phaA.hbd-crt using Gibson cloning.

pBT33-phaA.hbd-crt_PDZ. The trc promoter and the crt gene were amplified using the pTrc99a F12 and crt R6 primers and inserted at the XmaI-EagI restriction sites of pBT33-phaA.hbd-crt using Gibson cloning.

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pCDFDuet-tetR.GBD_SH3_PDZ. The pTet operon and GBD_SH3_PDZ scaffold gene were amplified using the pTet Scaff F1 and pTet Scaff R2 primers and inserted into the XbaI-KpnI restriction sites of pCDFDuet-1.

Other scaffold plasmids Each scaffold gene with 1, 2, or 4 SH3 binding domains and 1, 2, or 4 PDZ binding domains was digested from the corresponding pTET vector using BglII and BamHI-HF, gel purified, and inserted into the BglII-BamHI restriction sites of pCDFDuet-tetR.GBD_SH3_PDZ.

pCDFDuet.P(Tet). pCDFDuet-tetR.GBD_SH3_PDZ was cut with BglII and XmaI to remove the scaffold gene. The vector was ligated using Gibson cloning using the BglII into Xma linker F1 and BglII into Xma linker R1 oligonucleotides.

pCWOri-ter_PDZ.adhE2. The ter gene was amplified using the ter R105 and TdTER F1 primers and inserted into the NdeI-EcoRI restriction sites of pCWOri-ter.adhE2.

pCWOri-ter.SH3_adhE2. The adhE2 gene was amplified using the adhE2 F25 and adhE2 R1 primers and inserted into the EcoRI-KpnI restriction sites of pCWOri-ter.adhE2.

pCWOri-ter_PDZ.SH3_adhE2. The ter gene was amplified using the ter R105 and TdTER F1 primers and inserted into the NdeI-EcoRI restriction sites of pCWOri-ter.SH3_adhE2.

Name Sequence

adhE2 F1 ATGAATTCAAGAAGGAGATATACCATGAAAGTCACGAACCAGAAGGAACTGAAGCAGAAACTGAA

CGAACTGCGCG

adhE2 F25 ATGAATTCAAGAAGGAGATATACCATGCCACCGCCAGCTCTGCCACCGAAACGTCGTCGTGGTTC

TGGTTCTGGCAGCGGCAGCATGAAAGTCACGAACCAGAAGGAACTGA

adhE2 F26 GAGAATCTCTACTTCCAGGGTACCGGCGCCATGAAAGTCACGAACCAGAAGGAACTGAAG

adhE2 R1 ATCAAGCTTGGTACCTTAAAAAGATTTGATATAAATGTCTTTCAGCTCAGAGATCAGCGGGTAACG

CGGG

adhE2 R19 TTTGACAGCTTATCATCGATAAGCTTGGTACCTTAGACCAGGCTCTCTTTGACACCACCGGAACC

GCTACCGGAACCAGAGCCAAAAGATTTGATATAAATGTCTTTCAGCTCAGAGATCAGCGGGTAACGCGGG

adhE2 R20 ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAAAAAGATTTGATATAAATGTCTTTCAGCTCAGA

GATCAGCGGGTAACG

BglII into Xma linker F1 CCGGGTATGGCATAGATCTGCTGCACTTCGTC

BglII into Xma linker R1 CCGGGACGAAGTGCAGCAGATCTATGCCATAC

crt R5 CAGGTCGACTCTAGAGGATCCCCGGGTTAACGATTTTTGAAGCCTTCGATTTTACGTTTTTCGATG

AAAGCGGTCATGGC

crt R6 CAGGTCGACTCTAGAGGATCCCCGGGTTAGACCAGGCTCTCTTTGACACCACCGGAACCGCTAC

CGGAACCAGAGCCACGATTTTTGAAGCCTTCGATTTTACGTTTTTCGATGAAAGCGGTCATGGC

crt SOE F7 GCCGTCGCGGCTCCGGTAGCGGTAGCGGCAGCGGTATGGAACTGAACAACGTGATCCTGG

crt SOE R7 ACCGCTACCGGAGCCGCGACGGCGTTTCGGCGGCAGCGCCGGCGGCGGCATGGTCTGTTTCCT

GTGTGAAATTGTTATCCGC

pTet Scaff F1 GATTACACCTAGGACTAGTGATCCGTTTCCATTTAGGTGGGTA

pTet Scaff R2* GATTACAGGTACCGTTCACCGACAAACAACAGATAAAACGAAAGG

pTrc99a F12 ACTTCTGCGCTCGGCCCTTCCGGCCGACTGCACGG

SH3 F1 GAGAATCTCTACTTCCAGGGTACCGGCGCCATGCCACCGCCAGCTCTGCCACCG

TdTER F1 GAGATATACATATGATCGTCAAGCCAATGGTGCGC

TdTER F104 GGATCCATCGATGCTTAGGAGGTCATATGCCACCGCCAGCTCTGCCACCGAAACGTCGTCGTGG

TTCTGGTTCTGGCAGCGGCAGCGGCATCGTCAAGCCAATGGTGCGC

TdTER R106 ACTTTCATGGTATATCTCCTTCTTGAATTCTTAAATACGATCGAAACGTTCAACTTCTGC

ter R105 TTCTTGAATTCTTAGACCAGGCTCTCTTTGACACCACCGGAACCGCTACCGGAACCAGAGCCAAT

ACGATCGAAACGTTCAACTTCTGCC

Table 4.1. Oligonucleotides used for cloning in this study.

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pCWOri-SH3_ter.adhE2. The ter gene was amplified using the TdTER F104 and TdTER R106 primers and inserted into the NdeI-EcoRI restriction sites of pCWOri-ter.adhE2 using Gibson cloning.

pCWOri-ter.adhE2_PDZ. The adhE2 gene was amplified using the adhE2 F1 and adhE2 R19 primers and inserted into the EcoRI-KpnI restriction sites of pCWOri-ter.adhE2 using Gibson cloning.

pCWOri-SH3_ter.adhE2_PDZ. The adhE2 gene was amplified using the adhE2 F1 and adhE2 R19 primers and the ter gene was amplified using the TdTER F104 and TdTER R106 primers. The two genes were inserted into the NdeI-KpnI restriction sites of pCWOri-ter.adhE2 using Gibson cloning.

pET23a-HisTev_adhE2. The adhE2 gene was amplified using the adhE2 F26 and adhE2 R20 primers and inserted into the EcoRI-KpnI restriction sites of pCWOri-ter.adhE2 using Gibson cloning.

pET23a-HisTev_SH3_adhE2. The adhE2 gene was amplified using the SH3 F1 and adhE2 R20 primers and inserted into the EcoRI-KpnI restriction sites of pCWOri-ter.adhE2 using Gibson cloning.

Cell transformation. E. coli were transformed via electroporation using a MicroPulser Electroporator (Bio-Rad; Hercules, CA). For strains containing four plasmids, three plasmids were transformed into KCM (KCl, CaCl2, MgCl2) chemically competent DH1 E. coli cells which had previously been transformed with the pBBR2-aceE.F.lpd plasmid. All transformed bacteria were plated on LB agar containing appropriate antibiotics and 2% (w/v) glucose. Antibiotics were used at concentrations of 50 μg mL-1 for carbenicillin, kanamycin, or chloramphenicol or

Plasmid Description Source

pBT33-phaA.hbd-crt phaA, hbd (Ara), crt (Trc), araC, Cmr, p15a [23]

pCWOri-ter.adhE2 ter, adhE2 (double Tac), lacIq, Cbr, ColE1 [23]

pTET-tetR.GBD1_SH3X_PDZY GBD1_SH3X_PDZY (Tet; X = 1, 2, or 4, Y = 1, 2, or 4), TetR, Apr, ColE1

[8]

pCDF.tet-GBD1_SH3X_PDZY GBD1_SH3X_PDZY (Tet; X = 1, 2, or 4, Y = 1, 2, or 4), TetR, Smr, CloDF13

This Study

pBBR2-aceE.F.lpd aceE.aceF.lpd (lac), lacIq, Kmr, pBBR1 [23]

pBT33-phaA.hbd-SH3_crt. phaA, hbd (Ara), SH3_crt (Trc), araC, Cmr, p15a This Study

pBT33-phaA.hbd-crt_PDZ phaA, hbd (Ara), crt_PDZ (Trc), araC, Cmr, p15a This Study

pCDFDuet.P(Tet) (Tet), TetR, Smr, CloDF13 This Study

pCWOri-ter_PDZ.adhE2 ter_PDZ, adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter.SH3_adhE2 ter, SH3_adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter_PDZ.SH3_adhE2 ter_PDZ, SH3_adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-SH3_ter.adhE2 SH3_ter, adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter.adhE2_PDZ ter, adhE2_PDZ (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-SH3_ter.adhE2_PDZ SH3_ter, adhE2_PDZ (double Tac), lacIq, Cbr, ColE1 This Study

pET23a-HisTev_adhE2 HisTev_adhE2 (T7lac), lacIq, Cbr, ColE1 This Study

pET23a-HisTev_SH3_adhE2 HisTev_SH3_adhE2 (T7lac), lacIq, Cbr, ColE1 This Study

Table 4.2. The complete list of plasmids used in this study.

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100 μg mL-1 spectinomycin for strains with a single resistance marker and all solid media. In liquid media cultures with multiple resistance markers carbenicillin was used at 50 μg mL-1 , spectinomycin at 100 μg mL-1, and kanamycin and chlormaphenicol were used at 25 μg mL-1.

In vivo production of n-butanol in flasks. Freshly transformed E. coli colonies were inoculated into 5 mL of TB with 1.5% (w/v) glucose replacing the standard glycerol supplement and appropriate antibiotics (production media) in a 50 mL test tube and grown for 12-16 h at 37 °C in a rotary shaker (200 rpm). The cultures were diluted to an OD600 of 0.05 in 50 mL of production media in a 250 mL baffled flask. Cultures were grown at 37 °C in a rotary shaker (200 rpm) to an OD600 of 0.35-0.45 and induced with IPTG (1.0 mM), L-arabinose (0.2% (w/v)), and varying amounts of Tc/aTc if used. Immediately after induction, flasks were sealed with Parafilm M (Pechiney Plastic Packaging) and reduced to 30 °C. Flasks were unsealed for 10-20 min every 24 h and resealed with Parafilm M. An additional 1% (w/v) glucose was added after 24 h. Growths were continued for 72 h.

High throughput screen for in vivo production of n-butanol. Freshly transformed E. coli colonies were inoculated into 1 mL of production media in a 96-well plate with 2 mL total well volume (deep-well plate) (Corning, NY) and grown for 12-16 h at 37 °C in a rotary shaker (200 rpm). The cultures were diluted to an OD600 of 0.05 in 0.8 mL of production media in deep-well plate and sealed with sterile AeraSeal breathable film (Excel Scientific Inc., Victorville CA). Cultures were grown at 37 °C in a rotary shaker (200 rpm) for 2.5 h and induced with IPTG (1.0 mM), L-arabinose (0.2% (w/v)), and varying amounts of Tc/aTc if used. Immediately after induction, plates were sealed with Titer Tops (Diversified Biotech, MA) and growth temperature reduced to 30 °C. Plates were unsealed for 10-20 min every 24 h and resealed with Titer Tops. An additional 1% (w/v) glucose was added after 24 h. Growths were continued for 72 h.

Extraction and quantification of n-butanol and ethanol. n-Butanol quantification for cultures grown in flasks was performed as previously described [23]. In brief, 2 mL samples were removed from cell culture and cleared of biomass via centrifugation (20,817 × g for 2 min). For high throughput screens, 200 μL samples were removed from cell culture and cleared of biomass via centrifugation (3,060 × g for 20 min at 4 °C). Each cleared medium sample was then mixed in a 9:1 ratio with an aqueous solution containing the isobutanol internal standard (10,000 mg L-1). Samples were analyzed on a Trace GC Ultra (Thermo Scientific) using an HP-5MS column (0.25 μM film thickness, J & W Scientific). The oven program was as follows: 75°C for 3 min, ramp to 300 °C at 35 °C min-1. 300 °C for 1 min. n-Butanol and ethanol were quantified by flame ionization detection (FID) (flow: 350 mL min-1 air, 35 mL min-1 H2, and 30 mL min-1 helium). Samples found to have <500 mg L-1 n-butanol were quantified re-quantified: cleared media samples were mixed in a 1:1 ratio with toluene containing the isobutanol internal standard (100 mg L-1) using a Digital Vortex Mixer (Fisher) for 5 min at 2,000. The organic layer was analyzed using a DSQII single-quadrupole mass spectrometer (Thermo Scientific) using single-ion monitoring (m/z 41, 42, 43, and 56) concurrent with full scan mode (m/z 35-80), using the same GC parameters as the FID analysis. n-Butanol levels were quantified relative to a standard curve of 2, 4, 8, 16, 31, 63, 125, 250 mg L-1 n-butanol for MS detection or 125, 250, 500, 1,000, 2,000, 4,000, 8,000 mg L-1 n-butanol for FID detection. Ethanol levels were quantified relative to a standard curve of 0.031, 0.063, 0.125, 0.250, 0.500, 1.000, 2.000 % (w/v) ethanol for the FID, ethanol was not quantified by MS.

Purification of AdhE2 and SH3_AdhE2. BL21(de3) E. coli freshly transformed with the appropriate overexpression plasmids (pET23a-HisTev_adhE2 or pET23a-HisTev_SH3_adhE2)

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were inoculated into TB containing carbenicillin (50 μg/mL) and grown overnight at 37 °C in a rotary shaker (200 rpm). The cultures were back-diluted to an OD600 of 0.05 in 1 L of TB containing carbenicillin. Cultures were grown at 37 °C to an OD600 of 0.6-0.8 followed by induction with IPTG (1 mM). The cultures were grown at 30 °C for 4 h following induction and then spun down at 12,300 × g for 7 at 4 °C. The cell pellets were harvested and stored at -80 °C.

Cell pellets were thawed and resuspended at 5 mL/g cell paste using wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, 1 mM TCEP, pH 8.0 at 4 °C) and homogenized before lysis by passage through a French Pressure cell (Thermo Scientific) at 14,000 psi. Soluble and insoluble lysate fractions were separated by centrifugation (15,316 × g for 30 min at 4 °C). DNA was precipitated from the soluble lysate by addition of streptomycin sulfate (20%) over 10 min at 4 °C to a final concentration of 1%. Precipitated DNA was removed via centrifugation (15,316× g for 20 min at 4 °C). The supernatant was run through a Ni-NTA Agarose (Qiagen) column (7 mL) at a flow rate of ≤1 mL/min. The column was washed with 20 column volumes of wash buffer before elution with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, 1 mM TCEP, pH 8.0 at 4 °C). Fractions were pooled based on protein content as measured by absorbance at 280 nm and concentrated by centrifugation through an Amicon Ultracel 30K centrifugal filter (Millipore, Billerica MA). Concentrated protein samples were then treated with TEV protease (QB3, Berkeley CA) and placed in Spectra/Por Biotech Cellulose Ester dialysis membranes (Spectrum Laboratories, Inc.). Membranes were gently stirred overnight at 4 °C in dialysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, pH 8.0 at 4 °C).

The TEV-cleaved protein samples were then passed over the Ni-NTA Agarose column to remove TEV protease and His10 sequence. The flow through was run over a Sephadex G-25 column (Sigma-Aldrich, bead size 50-150 μm, 200 mL) in 150 mM sodium chloride, 100 mM Tris, 1 mM EDTA, pH 8.0 at 4 °C. Protein-containing fractions were pooled and concentrated by centrifugation through an Amicon Ultracel 30K centrifugal filter to ~1 mg/mL using the extinction coefficients calculated with the ExPASy Peptide Properties Calculator (ε280 = 63315 M-1 cm-1). Glycerol (60%) was added to a final concentration of 10% and aliquots of protein were flash-frozen in liquid N2 and stored at -80 °C. SDS-PAGE analysis of each purification step can be found in Figure 4.7.

Kinetic analysis of AdhE2 and SH3_AdhE2. Activity of AdhE2 and SH3_AdhE2 in the forward direction was measured by monitoring the oxidation of NADH at 340 nm at 30 °C. The assay mixture contained 100 mM Tris-HCl (pH 7.5), 1 mM DTT, 100 μM NADH, and 5.16 μg AdhE2 or 4.65 μg SH3_AdhE2. For AdhE2 analysis, substrates were added in concentrations of 12.5, 25, 50, 100, 250, 375, 500, and 1000 µM acetyl-CoA; 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 250, and 500 µM butyryl-CoA; 1.25, 2.5, 5, 7.5, 12.5, and 25 mM acetaldehyde; and 125, 250, 312.5, 625, 1250, 1875, and 2500 µM butyraldehyde. For SH3_AdhE2 analysis, substrates were added in concentration of 6.25, 12.5, 25, 50, 100, 125, 200, 250, 500, and 1000 µM acetyl-CoA; 3.13, 6.25, 12.5, 25, 50, 100, 125, and 250 µM butyryl-CoA; 0.25, 0.625, 1.25, 2.5, 5, 7.5, 12.5, and 25 mM acetaldehyde; and 62.5, 125, 250, 625, 1250, 2500, 5000 µM butyraldehyde. Kinetic parameters (kcat and KM) were determined by fitting the data using Origin 6.0 (OriginLab Corporation; Northampton MA) to the equation: V0 = (Vmax [S])/(KM + [S]), where V0 is the initial rate and [S] is the substrate concentration.

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4.3. Results and discussion

Design of the scaffolded butanol pathway. A set of nine scaffolds that had been reported by the Keasling lab were subcloned for compatibility with the butanol production plasmids, using expression of from a tetR promoter which has been shown to be titratable in E. coli [25]. The nine sets of scaffolds contain three types of binding domains: GTPase binding domain (GBD) [26], SRC Homology 3 domain (SH3) [27], and PSD95/DlgA/Zo-1 domain (PDZ) [28], which provides the opportunity to colocalize up to three different enzymes. Each of these scaffolds bears a single GBD domain and either one, two, or four copies of the SH3 and/or PDZ domain to allow for different stoichometries of bound enzymes (Figure 4.1). The scaffolding systems are annotated as “x/y/z scaffold” with the number of GBD, SH3, and PDZ binding domains indicated as x, y, and z respectively. We decided to initate our studies by examining of pairs of enzymes in the synthetic butanol pathway. As a result of previous work regarding the demonstration of enoyl-CoA reduction as a kinetic trap [23], it was decided to examine the enzyme pairs of Crt/Ter and Ter/AdhE2.

Screening and optimizing conditions for butanol production with scaffolds in 96-well plates. In order to establish growth conditions that could be carried at medium throughput with strong correlations to growth under standard shake flask conditions several methods of growth were tested including 5 mL cultures in 50 mL culture tubes, and various volume cultures in deep well 96-well plates (2 mL volume). While the 5 mL cultures showed the strongest correlation with 50 mL cultures in baffled flasks, the growth in plates with non-breathable films was determined to be adequately correlated with shake-flask production and significantly higher throughput than culture tubes and so was used to proceed (Figure 4.2). Breathable films for sealing the plates were also examined. While production was increased, the large variability observed between wells prevented these films from being used for plasmid comparisons (Figure 4.3). As a result, it was decided to proceed with non-breathable sealing films with a deep well 96-well plate and removing the seal for 20 to 30 min daily to allow for some oxygenation of the culture, analogous to the previously describe protocol in 250 mL shake flasks [23].

Following the establishment of a medium throughput system to predict butanol titers in shake flasks, the scaffolding systems was tested in order to optimize expression conditions. In order to differentiate between the effects of colocalization and fusion constructs, all scaffolding systems were tested with sequences to bind the SH3 and PDZ domain interchanged. The SH3 binding ligand can be functionally expressed on the N or C-terminus of the enzymes [27], but was fused to the N-terminus of the enzyme unless otherwise noted. The PDZ binding ligand has been shown to only function when fused to the C-terminus [29]. It was found that in both cases the SH3 tag significantly reduced n-butanol production titers when fused to a variety of different enzymes (Figure 4.4). In contrast, the PDZ domain, displayed less significant reductions in n-butanol titers regardless of the enzyme to which it was fused (Figure 4.4). The expression of both the PDZ and SH3 fusions results in titers consistent with the reduction in titer as a result of the SH3 fusion. The reduction of production with SH3 is consistent with results from the Dueber lab [30]. The reduction in n-butanol titer was most significant in Ter fusions, which is consistent with previous results establishing Ter activity as essential for efficiency production of n-butanol.

The analysis of the effects of colocalization is further complicated by the reduction in n-butanol titer with introduction of the scaffold expressing plasmid. There is a significant reduced in n-butanol production in cells maintaining a scaffold expression vector, even when not induced

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Figure 4.2. Variation in n-butanol production due to culture vessel. The n-butanol producing pathway was expressed both with and without heterologous expression of the pyruvate dehydrogenase complex. The same starter cultures were used to inoculate production cultures in 250 mL baffled flasks (50 mL of media), 50 mL culture tubes (5 mL of media), or deep well 96-well plates (1, 0.8, or 0.5 mL of media). All cultures were grown microaerobically for three days (n=3).

Figure 4.3. Variation in n-butanol production due to culture seal. The production level of the n-butanol synthetic pathway using the same starter cultures was used to inoculate production cultures in 250 mL baffled flasks (50 mL of media) sealed with Parafilm M or deep well 96-well plates (0.8 mL of media) sealed with either a gas permeable seal or a non-permeable seal. All cultures were grown for three days with daily aeration for 20-30 min (n=3).

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Figure 4.4. The relative amount of n-butanol production in cells expressing enzymes fused to binding ligands. The cultures either contain the PDZ fusion and replacing the plasmid expressing the SH3 fusion with the analogous plasmid expressing the untagged protein (red), the SH3 fusion and replacing the plasmid expressing the PDZ fusion with the analogous plasmid expressing the untagged protein (blue), or contain both fusions (gray). The expected production of both fusions calculated as the product of each individual fusion is also displayed (black). All data are compared to DH1 pBT33-Bu2 pCWOri-ter.adhE2 plasmids. All cultures were grown under microaerobic conditions in 250 mL shake flasks for 3 d (n=3). (Crt_PDZ + SH3_Ter, DH1 pBT33-phaA.hbd-SH3_PDZ pCWOri-SH3_ter.adhE2; SH3_Crt + Ter_PDZ, DH1 pBT33-phaA.hbd-SH3_crt pCWOri-ter_PDZ.adhE2; Ter_PDZ + SH3_AdhE2, DH1 pBT33-Bu2 pCWOri-ter_PDZ.SH3_adhE2).

Figure 4.5. n-Butanol production levels using the Crt_PDZ and/or SH3_Ter fusion enzymes and titrated expression of the protein scaffold. The cultures either contain the plasmids replacing the PDZ and SH3 fusions with the analogous plasmids expressing the untagged protein and the empty plasmid replacing the analogous plasmid for expression of the scaffold protein (red), the PDZ fusion and replacing the plasmid expressing the SH3 fusion with the analogous plasmid expressing the untagged protein and the empty plasmid replacing the analogous plasmid for expression of the scaffold protein (gray), the SH3 fusion and replacing the plasmid expressing the PDZ fusion with the analogous plasmid expressing the untagged protein and the empty plasmid replacing the analogous plasmid for expression of the scaffold protein (blue), or contain both fusions and the plasmid for expression of the GBD1_SH34_PDZ4 scaffold protein (black). Cultures were induced with anhydrotetracycline at 0, 27, or 54 nM. All cultures were grown under microaerobic conditions in 250 mL shake flasks for 3 d (n=3). (Crt+Ter+Empty (red), DH1 pBT33-Bu2 pCWOri-ter.adhE2 pCDF.Tet-0; Crt_PDZ+Ter+Empty (gray), DH1 pBT33-phaA.hbd-crt_PDZ pCWOri-ter.adhE2 pCDF.Tet-0; Crt+SH3_Ter+Empty (blue), DH1 pBT33-Bu2 pCWOri-SH3_ter.adhE2 pCDF.Tet-0; Crt_PDZ+SH3_Ter+1/4/4 Scaffold (black), DH1 pBT33-phaA.hbd-crt_PDZ pCWOri-SH3_ter.adhE2 pCDF.tet-GBD1_SH34_PDZ4).

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(Figure 4.5). This is effect is not seen in the empty plasmid control (Figure 4.5), suggesting that the effect is due to leaky expression from the Tet promoter in the DH1 system rather than plasmid maintenance; however, the transcript level for the scaffold expression plasmid has not been analyzed.

Testing the effect of scaffolding on butanol production. Since the enoyl-CoA reduction was known to facilitate high-flux butanol production, it was decided to colocalize Ter with the preceding enzyme, Crt, which has a catalytic efficiency near the diffusion limit [23], but catalyzes a reaction with an unfavorable equilibrium constant, Keq ~ 0.1 [31]. In order to test the ability of the scaffold increase flux through the crotonyl-CoA node, both pairs of crt/ter fusions were tested, crt_PDZ/SH3_ter and SH3_crt/ter_PDZ. The set of nine scaffolding architectures were tested using induction levels of 27 or 54 nM anhydrotetracycline (aTc) in the medium-throughput 96-well plate system. Analysis in the medium-throughput format indicated that the 1/4/4 scaffold architecture using the SH3_crt/ter_PDZ tagging scheme resulted in the highest titers. This system was then repeated in the standard 50 mL culture format. Compared to the butanol production system, the scaffold results in titer increases of 1.9 and 1.2-fold, at 27 nM or 54 nM aTc respectively (Figure 4.5). Due to the significant reductions as a result of making the fusion enzymes and carrying the scaffold expression plasmid, the increased titers are still significantly below the non-scaffolded system. The increased production at intermediate levels of scaffold induction is consistent with the results seen in other labs [8]. The reduction in titers at higher induction levels may be a result of increased load on cellular protein expression or aggregation of the scaffold at higher expression levels, but the causes have not been analyzed. The production levels seen with expressed scaffold and the SH3_Crt + Ter_PDZ system have yet to yield reproducible results, although based on the control production levels they are expected to yield higher titers than the Crt_PDZ + SH3_Ter system.

Analysis of the butyryl-CoA node using tagging of Ter and AdhE2 was limited to strains expressing the Ter_PDZ and SH3_AdhE2 fusion enzymes because strains expressing the AdhE2_PDZ fusion enzyme did not show detectable levels of n-butanol production (data not shown). Analysis of the scaffolding systems using the medium-throughput system showed the 1/4/4 scaffold system had the strongest enhancement, similar to that seen in the crt/ter co-localizations. The system was again repeated on the 50 mL culture format. The reductions in butanol titers due to tagging the Ter and AdhE2 enzymes did not result in as substantial a reduction in titer as was seen in the crt/ter systems, and as a result the colocalization of the enzymes with scaffold expression resulted in n-butanol titers similar to those seen in the untagged system.

Interestingly, pathway selectivity also appeared to be altered by the scaffold as the ethanol production was observed to decrease significantly (Figure 4.6). The increased specificity for butanol production rather than ethanol production may point to a method of reducing the off pathway reaction of AdhE2 with acetyl-CoA and could funnel the carbon through the n-butanol pathway. While the recovery of n-butanol titers to the original system was seen several times, there was significant variability in the titers that has not yet been attributed to a specific variable. As a result, it is not possible to fully examine the effects of colocalization in the in vivo system. As a control, we tested the in vitro selectivity of a purified SH3_AdhE2 fusion as a means to address whether the decrease in ethanol production could be derived from the enzyme itself rather than colocalization. The fusion enzyme was generated with a His10 affinity tag with a Tev

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Figure 4.6. The ethanol and n-butanol production levels for the biosynthetic system using the Ter_PDZ and/or SH3_AdhE2 fusion enzymes. The cultures contain the Ter_PDZ fusion and/or SH3_AdhE2 or are replaced by the analogous plasmid expressing the untagged protein. The cultures also also contain a plasmid expressing the GBD1_SH34_PDZ4 or the analogous plasmid without a protein coding sequence.The production of n-butanol (red) and ethanol (gray) were quantified. All cultures were grown under microaerobic conditions in 250 mL shake flasks for 3 d (n=3). (Ter AdhE2 Empty, DH1 pBT33-Bu2 pCWOri-ter.adhE2 pCDF.P(Tet)-0; Ter_PDZ AdhE2 Empty, DH1 pBT33-Bu2 pCWOri-ter_PDZ.adhE2 pCDF.P(Tet)-0; Ter SH3_AdhE2 Empty, DH1 pBT33-Bu2 pCWOri-ter.SH3_adhE2 pCDF.P(Tet)-0; Ter_PDZ SH3_AdhE2 Empty, DH1 pBT33-Bu2 pCWOri-ter_PDZ.SH3_adhE2 pCDF.P(Tet)-0; Ter_PDZ SH3_AdhE2 1/4/4 Scaffold, DH1 pBT33-Bu2 pCWOri-ter_PDZ.SH3_adhE2 pCDF.P(Tet)-GBD1_SH34_PDZ4).

AdhE2 kcat (s-1) KM (µM) kcat/KM (M

-1 s-1) Acetyl-CoA 2.31 ± 0.06 110 ± 10 (2.1 ± 0.08) × 104 Butyryl-CoA 0.87 ± 0.06 9 ± 3 (1.0 ± 0.1) × 105 Acetaldehyde 0.78 ± 0.06 6000 ± 200 (1.3 ± 0.1) × 102 Butyraldehyde 0.28 ± 0.03 700 ± 200 (3.9 ± 0.5) × 102

SH3_AdhE2 kcat (s-1) KM (µM) kcat/KM (M

-1 s-1) Acetyl-CoA 1.60 ± 0.05 94 ± 9 (1.70 ± 0.07) × 104 Butyryl-CoA 0.55 ± 0.06 5 ± 3 (1.2 ± 0.1) × 105 Acetaldehyde 0.65 ± 0.06 4000 ± 1000 (1.5 ± 0.2) × 102 Butyraldehyde 0.32 ± 0.03 600 ± 200 (5.2 ± 0.7) × 102

Table 4.3. Michaelis-Menten kinetic parameters for AdhE2 and SH3_AdhE2. Enzymes were purified from BL21(de3) pET23a-His10Tev_AdhE2 or BL21(de3) pET23a-His10Tev_SH3_AdhE2. All errors are standard deviations (n=3) with the kcat/KM error propagated from measured variables.

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Figure 4.7. SDS-PAGE analysis of adhE2 and SH3_adhE2 purifications.The purification for adhE2 (A) and SH3_adhE2 (B). From left to right on both gels, the lanes represent: PageRuler Plus Prestained Protein Ladder (lane 1,) soluble fraction (2), insoluble fraction (3), flow-through from the first Ni-NTA Agarose column (4), final fraction from wash of the Ni-NTA Agarose column (5), pre TEV cleavage (6), post TEV cleavage (7), eluent of second Ni-NTA Agarose column (8), eluant of G25 column (9), final purified protein (10), and PageRuler Plus Prestained Protein Ladder (11).

cleavage site to yield the full length protein with the addition of GA fused to the to the N-terminus of the enzyme (Figure 4.7). The analysis of the steady-state Michaelis-Menten kinetic parameters did not show any change in kcat or KM with respect to the C2 and C4 substrates that explain the significant difference in ethanol titer (Table 4.3). While neither the KM or kcat of the enzymes were perturbed in a way that could directly explain the significant decrease in ethanol production, it is possible that the SH3 fusion tag could cause other perturbations in vivo. For example, AdhE2 has also been observed to form spirosomes in the cell [32], and the disruption of these larger formations due to SH3 fusion tag could alter enzyme selectivity.

4.4. Conclusions

While there are several intriguing results suggesting an enhancement in n-butanol being induced by the expression of the protein scaffold, several significant barriers remain to using this method for enhancement of overall n-butanol production or study of flux through the pathway. The significant decrease in titer observed when expressing enzymes with SH3 ligand fusion suggests that this domain is not conducive for a high flux pathway. Modifying the scaffold to make use of the GBD binding domain may allow for avoiding use of the SH3 fusion. Additional studies may also allow for the modification of the SH3 ligand in order to avoid a significant

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reduction in titer. In addition, the decrease in titer observed in cells containing a vector from the pCDF.Tet-GBD1_SH3X_PDZY series, regardless of inducer concentration, suggests that the protein scaffolds must be expressed in an alternative vector system. The variability observed in the scaffold system is also of great concern and additional work to identify the sources of variability would be required to allow for the use of the scaffolded n-butanol pathway as a model system.

4.5. References

This work was performed in collaboration with the following person: Mr. Timothy B. Roth.

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Chapter 5: Design and optimization of a malonyl-CoA dependent pathway for production of n-butanol

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5.1. Introduction

The distribution of flux through central metabolism is regulated at several levels, including the transcriptional [1-3], translational [4-6], and protein levels [7-9]. One of the tightly regulated metabolic nodes that has drawn extensive study is the conversion of acetyl-CoA to malonyl-CoA, two of the fundamental building blocks in central metabolism (Figure 5.1) [10,11]. Malonyl-CoA has be a metabolic intermediate of intense interest because of its role in fatty acid production (Figure 5.1). The formation of malonyl-CoA is subject to many layers of regulation, including feedback inhibition acylated acyl carrier proteins (acyl-ACPs) [12], the presence of oxygen [11], the energy charge [10], and the availability of carbon and nitrogen sources [10]. Despite a variety of efforts to engineer the intracellular concentration of malonyl-CoA [13-15], the availability of this building block has continued to be a challenge in generating high-flux and industrially-relevant biosynthetic pathways for microbial production of small molecule targets. For example, the availability of malonyl-CoA is believed to be a key limiting factor in a variety of polyketide, flavonoid, and fatty acid based pathways that have been proposed for the synthesis of pharmaceutical and fuel targets [14-17]. This problem is particularly important for the production of fuels, which should ideally be produced under anaerobic conditions to achieve

Figure 5.1. The fate of carbon from glucose through central metabolism. Malonyl-CoA is generated by the carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC). Malonyl-CoA is transacylated to form malonyl-ACP which provides the 2 carbon extension unit for fatty acid production. Acetyl-CoA is used for amino acid biosynthesis and energy production.

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Figure 5.2. Design of a malonyl-CoA dependent pathway for production of n-butanol. (A) Malonyl-CoA is generated by acetyl-CoA carboxylase (ACC) hydrolyzing one ATP per malonyl-CoA generated. The enzyme NphT7 can utilize malonyl-CoA and acetyl-CoA to form acetoacetyl-CoA. (B) The replacement of the phaA gene with nphT7 in the previously described n-butanol producing pathway allows for the use of malonyl-CoA as a starting unit for n-butanol production. This pathway can report on the availability of malonyl-CoA by analyzing the n-butanol titer.

higher efficiency, as production of malonyl-CoA derived targets is down regulated under these conditions [18].

We have approached this question through the design of a malonyl-CoA-dependent version of our synthetic n-butanol pathway, which can then be used as an in vivo platform to probe the malonyl-CoA pool. Towards this goal, we have taken advantage of the recent discovery of an acetoacetyl-CoA synthase from Streptomyces sp. CL190, NphT7, which catalyzes the formation of acetoacetyl-CoA from acetyl-CoA and malonyl-CoA (Figure 5.2A) [19]. Using NphT7, we can replace the PhaA catalyzed thiolase step, which depends on the condensation of two acetyl-CoA molecules, with a step that requires malonyl-CoA (Figure 5.2B). In this way, the availability of malonyl-CoA compared to acetyl-CoA can be analyzed in vivo utilizing the n-butanol titer as a read-out. In addition to the analysis of intracellular concentrations, we can further take advantage of a selection method developed in the laboratory to select for strains with higher n-butanol production under anaerobic conditions. By applying this selection to the

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NphT7-dependent pathway, we may be able to identify strains with higher malonyl-CoA availability or higher flux through the NphT7 catalyzed reaction. Beyond the practical value of these strains, the analysis and characterization of the strains could allow for the identification of new genetic factors that control the homeostasis of the malonyl-CoA pool.


Materials. Terrific Broth (TB), LB Broth Miller (LB), LB Agar Miller, sulfuric acid, glacial acetic acid, potassium chloride, dimethyl sulfoxide (DMSO), and glycerol were purchased from EMD Biosciences (Darmstadt, Germany). Isopropyl-β-D-1-thiogalactopyranoside (IPTG), D-glucose, dithiothreitol (DTT), Tris-HCl, phenylmethanesulfonyl fluoride (PMSF), carbenicillin (Cb), streptomycin sulfate, tetracycline hydrochloride (Tc), magnesium chloride hexahydrate, sodium phosphate monobasic monohydrate, calcium chloride, and sodium chloride were purchased from Fisher Scientific (Pittsburgh, PA). Spectinomycin dihydrochloride pentahydrate (Sp), chloramphenicol (Cm), kanamycin sulfate (Km), L-arabinose, acetyl-CoA, butyryl-CoA, crotonyl-CoA, acetaldehyde, butyraldehyde, N,N,N′,N′-tetramethylethylenediamine (TEMED), and NADH were purchased from Sigma-Aldrich (St. Louis, MO). Anhydrotetracycline hydrochloride (aTc) and imidazole were purchased from Acros (Geel, Belgium). Acrylamide/bis-acrylamide solution (30%, 37.5:1), electrophoresis grade sodium dodecyl sulfate (SDS), and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). PageRuler Plus Prestained Protein Ladder was purchased from Fermentas (Glen Burnie, MD). Deoxynucleotides (dNTPs), T4 DNA ligase, and Platinum Taq High-Fidelity DNA polymerase (Pt Taq HF) were purchased from Invitrogen (Carlsbad, CA). Antarctic phosphatase, T4 DNA ligase, Phusion polymerase, and all restriction enzymes were purchased from New England Biolabs (Ipswich, MA). DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick Gel Extraction Kit, and QIAquick PCR Purification Kit (QIAGEN; Valencia, CA) as appropriate. pCDFDuet-1 was purchased from Novagen (San Diego, CA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and resuspended at a stock concentration of 100 μM in 10 mM Tris-Cl, pH 8.5. Following plasmid construction, all gene inserts were sequenced using appropriate sequencing primers by Quintara Biosciences (Berkeley, CA). All absorbance readings were taken on a DU-800 spectrometer (Beckman-Coulter; Fullerton, CA) or a SpectraMax M2 plate reader (Molecular Devices; Toronto, Canada).

Bacterial strains. E. coli DH10B-T1R was used for plasmid construction and DNA isolation. MegaX electrocompetent cells (Invitrogen) were used for transformation of libraries. Homologous recombination of plasmids was carried out in the strain SIMD90 [20]. Production strains were derived from strain DH1 (Table 5.1).

Strain construction. All knockout strains were constructed with the λ-red recombinase system [21] (Table 5.1). The pKD3 template plasmid was used for gene disruption with subsequent recycling of the CmR marker using the pCP20 plasmid. All knockout strains were verified using PCR amplification (Table 5.2).

Synthetic gene construction. The synthetic genes encoding NphT7 from Streptomyces sp. CL190, AccA3 from Mycobacterium tuberculosis, AccD4 from M. tuberculosis, AccD6 from M. tuberculosis, AccE from M. tuberculosis, AccBC from Corynebacterium glutamicum, and DtsR1 from C. glutamicum were optimized for E. coli class II codon usage and synthesized using PCR assembly. Gene2Oligo (http://berry.engin.umich.edu/gene2oligo) was used to convert the gene

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sequence into primer sets using default optimization settings (Appendix 1). To assemble the synthetic gene, each primer was added at a final concentration of 1 μM to the first PCR reaction (50 μL) containing 1 × Pt Taq HF buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4), MgSO4 (1.5 mM), dNTPs (250 μM each), and Pt Taq HF (5 U). The following thermocycler program was used for the first assembly reaction: 95oC for 5 min; 95oC for 30 s; 55oC for 2 min; 72oC for 10 s; 40 cycles of 95oC for 15 s, 55oC for 30 s, 72oC for 20 s plus 3 s/cycle; these cycles were followed by a final incuabation at 72 oC for 5 min. The second assembly reaction (50 μL) contained 16 μL of the unpurified first PCR reaction with standard reagents for Pt Taq HF. The thermocycler program for the second PCR was: 95oC for 30 s; 55oC for 2 min; 72oC for 10 s; 40 cycles of 95oC for 15 s, 55oC for 30 s, 72oC for 80 s; these cycles were followed by a final incubation at 72oC for 5 min. The second PCR reaction (16 μL) was transferred again into fresh reagents and run using the same program. Following gene construction, the DNA smear at the

Table 5.1. Plasmids and strains used in this study.


pBT33-Bu2 phaA.hbd (Ara), crt (Trc), araC, Cmr, p15a [22]



pBT33-Bu6 nphT7.hbd (Ara), crt (Trc), araC, Cmr, p15a This Study

pTT33-Bu6 nphT7.hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pTT33-(100)Bu6 nphT7.hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pUC19-nphT7 nphT7 (lac), lacIq, Cbr, pUC19 This Study

pCDF2.tac2-gg0 (Tac, Tac), aadH, Spr, pCDF2 This Study

pCDF2.tac2-nphT7 nphT7 (Tac, Tac), aadH, Spr, pCDF2 This Study

pTT33-(100K)hbd-crt hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pCWOri-ter.adh.aldh46 ter.adh.aldh46 (Tac, Tac), lacIq, Cbr, ColE1 This Study

pCDF.tet-accD6.accE.accA3 accD6.accE.accA3 (Tet), aadH, Spr, pCDF2 This Study


pBBR2-matB matB (lac), lacIq, Kmr, pBBR1 This Study

pCDF.tet-acs acs (Tet), aadH, Spr, pCDF2 This Study

pCDF.tet-accBC.dtsR1 accBC.dtsR1 (Tet), aadH, Spr, pCDF2 This Study

pCDF.tet-acs.ACC acs.accBC.dtsR1 (Tet), aadH, Spr, pCDF2 This Study

Strain Description Source

DH1 endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK- mK

+) λ- ATCC

MC1.16 DH1 ∆adhE::FRT Jeff Hanson

MC1.24 DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT Miao Wen,

Jeff Hanson

MC1.29 DH1 ∆adhE::FRT ∆atoB::FRT This Study

MC1.36 DH1 ∆adhE::FRT ∆atoB::FRT ∆fabF::FRT This Study

MC1.40 DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT ∆atoB::FRT

This Study

SIMD90 W3110 galKtyr145UAG∆lacU169 [λ cI857 (int-cIII:bet)] mutS:cat [20]

CY242 MG1655 pyrC, fabB15 [23]

CY244 MG1655 fabF1, fabB15 [24]

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Name Sequence AccA3 F101 cacagcagcggccatatcgaaggtcgtcatATGGCAAGCCACGCAGGTAGCCGTATCGCCC AccA3 F102 GCCGCTAAaaactttctaaggaggtcccacATGGCAAGCCACGCAGGTAGCCGTATCGCC AccA3 R101 tcgggctttgttagcagccggatcctcgagTTATTTAATCTCCGCCAGGACCGTGCCTTG AccA3 R103 tgagatgagtttttgttcgggcccaagcttagatctTTATTTAATCTCCGCCAGGACCGTGCCTTG AccBC F100 gaaaagatctgggctgccgaagccac AccBC R100 gtcctccttatagttgtcggtatcgctaccttacttaatctccagcagaacgacacctttattaacac AccD4 F101 cacagcagcggccatatcgaaggtcgtcatATGACGGTTACGGAGCCGGTTCTGCACACG AccD4 F103 tttcgtcttcacgctagcaaagaggagaaAacacaattttattcttaggaggtcccttATGACGGTTACGGAGCCGGTTCTGCACACG AccD4 R101 tcgggctttgttagcagccggatcctcgagTTAGACCGGGATCAGACCATGTTTACGACC AccD4 R102 agtgctcctccttattaattctcgtctaagaaTTAGACCGGGATCAGACCATGTTTACGACC AccD6 F101 cacagcagcggccatatcgaaggtcgtcatATGACGATTATGGCACCGGAAGCAGTCGGC

AccD6 F103 tttcgtcttcacgctagcaaagaggagaaAtaaaacaaaaatacaggtaaggagtacacggagaATGACGATTATGGCACCGGAAGCAGT

AccD6 R101 tcgggctttgttagcagccggatcctcgagTTACAGCGGGATGTTTTTATGACGACCACG AccD6 R102 agtgctcctccttattaattctcgtctaagaaTTACAGCGGGATGTTTTTATGACGACCACG AccE F101 cacagcagcggccatatcgaaggtcgtcatATGGGTACGTGTCCGTGCGAGAGCAGCGAG AccE F103 ttcttagacgagaattaataaggaggagcactATGGGTACGTGTCCGTGCGAGAGCAGCG AccE R101 tcgggctttgttagcagccggatcctcgagTTAGCGGCGCATGTGGGTCATTTCTTGCAA AccE R102 CTTGCCATgtgggacctccttagaaagtttTTAGCGGCGCATGTGGGTCATTTCTTGCAA ackA KF2 ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCAgtgtaggctggagctgcttc Acs F3 ctttcgtcttcacgctagcaaagaggagaaaagatctATGagccaaattcacaaacacac Acs R3 ccttctattagccgtggcttcggcagccctTTAcgatggcatcgcgatagcc ADH GR1 catgtttgacagcttatcatcgataagcttttaaaaagatttgatataaatgtctttcag adhE F1 GCTTTGCAAAAATTTGATTTGGATCACGTAATCAGTACCC adhE KF3 ATGGCTGTTACTAATGTCGCTGAACTTAACGCACTCGTAGAGCGTgtgtaggctggagctgcttc adhE KO F1 gctttgcaaaaatttgatttggatcacgtaatcagtaccc adhE KO R1 catgagcagaaagcgtcaggcagtgttgtatccac adhE KR3 TTAAGCGGATTTTTTCGCTTTTTTCTCAGCTTTAGCCGGAGCAGCcatatgaatatcctccttag adhE R1 CATGAGCAGAAAGCGTCAGGCAGTGTTGTATCCAC aldh46 GF3 tgagggtattaactacgaggc aldh46.ADH GF1 gttctggccggctaatctagacggcaccagaggaggtaaaaatgctgtggttcaaagtcc aldh46.ADH GR1 actttgaaccacagcatttttacctcctctggtgccgtctagattagccggccagaacac BT33-NphT7 100K F2 ATCGGGCTTTTTCACAACAGGAGGATTTATCatgaccgacgttcgttttcgtatcattg DtsR1 F101 GTCGTTCTGCTGGAGATTAAGTAAGGTAGCGATACCGACAACTATAAG DtsR1 R100 cgagtcaggatccttacagcggcatattaccatgcttgc fabB KO F2 attgtgcattcgaaacttactctatgtgcgacttacagaggtattgaATGgtgtaggctggagctgcttc fabB KO R2 gtaacgtcggatgcgacgctggcgcgtctactccgacctactgcgaaTTAcatatgaatatcctccttag FabF KF1 gtcccactagaatcattttttccctccctggaggacaaacGtgtaggctggagctgcttc FabF KR1 gcccgcaagcggaccttttataagggtggaaaatgacaacCatatgaatatcctccttag frdBC KF1 ATGGCTGAGATGAAAAACCTGAAAATTGAGGTGGTGCGCTATAACgtgtaggctggagctgcttc frdBC KO F1 atggctgagatgaaaaacctgaaaattgaggtggtgcgctataacgtgtaggctggagctgcttc frdBC KO R1 ttaccagtacagggcaacaaacaggattacgatggtggcaaccaccatatgaatatcctccttag frdBC KR1 TTACCAGTACAGGGCAACAAACAGGATTACGATGGTGGCAACCACcatatgaatatcctccttag

HBD F101 agcactggctgtcgaacgcaaataaCGACTACGGGTAATTCGGAACGACAGTCGCCCCAAAatgaaaaaggtttgcgttattggtgcgg

hbd GR1 atcaggctgaaaatcttctctcatccgccaaaacagccctcgagttacttggagtaatcg ldhA KF2 ATGAACTCGCCGTTTTATAGCACAAAACAGTACGACAAGAAGTACgtgtaggctggagctgcttc ldhA KR4 TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTcatatgaatatcctccttag NphT7 F100 cttgatcgtCATatgaccgacgttcgttttcgtatcattggcac NphT7 gg F1 cgacgttgtaaaacgacggccagtgaattcggtctctaATGaccgacgttcgttttcgtatcattggc nphT7 gg F2 cgacgttgtaaaacgacggccagtgaattcggtctctaATG NphT7 gg R1 tgcctgcaggtcgactctagaggatccccgggtctctaagcttaTTAccactcgatcagcgcgaag nphT7 gg R2 CGACTCTAGAGGATCCCCGGGTCTCTAAGCTTATTA NphT7 R100 AACGCAAACCTTTTTCATTATATATCTCCTTGAATTCttaCCACTCGATCAGCGCGAAGCTC pBBR2-matB F2b CACCTTAAGTCCATCATCCTAAGGAGGTTTTATatgtcctctctcttcccggccctctcc pBBR2-matB R2b tacgactcactatagggcgaattgGAGCTCtcagtcacggttcagcgcccgcttcatgat pCDF2gg-0 F1 catcaccatcatcaccacagccaggatccggtctctgagctctcatagggtaccgagacc pCDF2gg-0 R1 gcagcagcggtttctttaccagactcgagggtctcggtaccctatgagagctcagagacc poxB KF3 ATGAAACAAACGGTTGCAGCTTATATCGCCAAAACACTCGAATCGGCAGGgtgtaggctggagctgcttc poxB KR3 TTACCTTAGCCAGTTTGTTTTCGCCAGTTCGATCACTTCATCACCGCGTCcatatgaatatcctccttag ptA KR2 TGTGCAGACTGAATCGCAGTCAGCGCGATGGTGTAGACGAcatatgaatatcctccttag pta R2 ttgttggtttggattcagtgattgcggacatagcgcaaatattcccttgcacaaaacaa pTT33-hbd F1 ttcacacgagctcaaggagatatacatatgGaattcaaggagatatataatgaaaaagg TT33-NphT7 100K R2 GATAAATCCTCCTGTTGTGAAAAAGCCCGATgagctcgtgtgaaattgttatccgctcac

Table 5.2. Oligonucleotides used for cloning and gene disruption.

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appropriate size was gel purified and used as a template for the rescue PCR (50 μL) with Pt Taq HF and rescue primers under standard conditions. The resulting rescue product was either inserted directly into the appropriate vector or first cloned into pCR2.1-TOPO using a TOPO TA Cloning Kit from Invitrogen.

Plasmid construction. All plasmids were constructed by ligation using standard molecular biology techniques or the one-step, isothermal in vitro recombination system described by Gibson et al [25]. PCR amplifications were carried out with either Pt Taq HF or Phusion polymerase (oligonucleotides, Table 5.2). All constructs were verified by sequencing (oligonucleotides, Table 5.3) (Quintara Biosciences (Albany, CA)).

pBT33-Bu6. The nphT7.hbd fragment was constructed by SOE PCR by first amplifying nphT7 gene from gene synthesis mixture using the NphT7 F100/NphT7 R100 primers and hbd gene from pBT33-Bu2 using the HBD F101/hbd GR1. The two products were gel purified and both PCR products were amplified using NphT7 F100 and hbd GR1. The resulting nphT7.hbd operon was inserted into the NdeI-XhoI restriction sites of pBT33-Bu2.

pTT33-Bu6. The nphT7.hbd fragment was constructed by SOE PCR by first amplifying nphT7 gene from pBT33-Bu6 using the NphT7 F100/NphT7 R100 primers and hbd gene from pBT33-Bu6 using the HBD F101/hbd GR1. The two products were gel purified and both PCR products were amplified using NphT7 F100 and hbd GR1. The resulting nphT7.hbd operon was inserted into the NdeI-XhoI restriction sites of pTT33-Bu2.

pTT33-(100)Bu6. The pTT33-Bu6 plasmid was digested with NdeI. The digested vector was incubated with the BT33-NphT7 100K F2 and TT33-NphT7 100K R2 primers using the Gibson isothermal cloning master mix.

pUC19-nphT7. The nphT7 gene was amplified from pBT33-Bu6 using NphT7 gg F1 and NphT7 gg R1 primers. The resulting PCR product was inserted into pUC19 using Gibson cloning cloning at the EcoRI-KpnI restriction sites.

pCDF2.tac2-gg0. The pCDF2.tac2-0 plasmid was digested with BamHI and KpnI. The digested vector was incubated with the pCDF2gg-0 F1 and pCDF2gg-0 R1 primers using the Gibson isothermal cloning master mix.

pCDF2.tac2-nphT7. The nphT7 gene was amplified from pBT33-Bu6 using the nphT7 gg F2 and nphT7 gg R2 primers. The PCR product was ligated into the pCDF2.tac2-gg0 vector using the Golden Gate cloning protocol, cycling for 50 cycles [26].

pTT33-(100K)hbd-crt. The hbd gene was amplified from pBT33-Bu2 using the pTT33-hbd F1 and hbd GR1 primers. The resulting PCR product was inserted into the NdeI-XhoI restriction sites of the pTT33-Bu6 plasmid.

pCWOri-ter.aldh46.adh. The aldh46 gene was amplified from pCDF3-ter.aldh46 using the aldh46 GF3 and aldh46.ADH GR1 primers. The adh gene fragment was amplified from the pCWOri-strep_ADH plasmid using the aldh46.ADH GF1 and ADH GR1 primers. The resulting PCR products were inserted into the EcoRI-HindIII restriction sites of the pCWOri-ter.adhE2 plasmid.

pBBR2-matB. The matB gene was amplified from S. coelicolor genomic DNA using the pBBR2-matB F2b and pBBR2-matB R2b primers and inserted into the KpnI-SacI restriction sites of pBBR1-MCS2 [27].

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pCDF.tet-accD6.accE.accA3. The accD6 gene was synthesized and rescue PCR was performed using the AccD6 F101 and AccD6 R101 primers, the PCR product was inserted into the NdeI-BamHI restriction sites of pET16b to make pET16b-accD6. The accD6 gene was amplified from pET16b-accD6 using the AccD6 F103 and AccD6 R102 primers. The accE gene was synthesized and rescue PCR was performed using the AccE F101 and AccE R101 primers, the PCR product was inserted into the NdeI-BamHI restriction sites of pET16b to make pET16b-accE. The accE gene was amplified from pET16b-accE using the AccE F103 and AccE R102 primers. The accA3 gene was synthesized and rescue PCR was performed using the AccA3 F101 and AccA3 R101 primers, the PCR product was inserted into the NdeI-BamHI restriction sites of pET16b to make pET16b-accA3. The accA3 gene was amplified from pET16b-accA3 using the AccA3 F102 and AccA3 R103 primers. The accD6, ace, and accA3 genes were inserted into the BglII restriction site of pCDF.tet-0 plasmid using Gibson cloning.

pCDF.tet-accD4.accE.accA3. The accD4 gene was synthesized and rescue PCR was performed using the AccD4 F101 and AccD4 R101 primers, the PCR product was inserted into the NdeI-BamHI restriction sites of pET16b to make pET16b-accD4. The accD4 gene was amplified from pET16b-accD4 using the AccD4 F103 and AccD4 R102 primers. The accE gene was amplified from pET16b-accE using the AccE F103 and AccE R102 primers. The accA3 gene was amplified from pET16b-accA3 using the AccA3 F102 and AccA3 R103 primers. The accD4, ace, and accA3 genes were inserted into the BglII restriction site of pCDF.tet-0 plasmid using Gibson cloning.

pCDF.tet-accBC.dtsR1. The accBC gene was synthesized and rescued using the AccBC F100 and AccBC R100 primers and inserted into the NdeI-BamHI restriction sites of pET16b to make pET16b-accBC. The accBC gene was amplified using the AccBC F100 and AccBC R100 primers, the dtsR1 gene was amplified using the DtsF101 F101 and DtsR1 R100 primers. The PCR products were cloned into the BglII restriction site of pCDF.tet-0 plasmid using Gibson cloning.

pCDF.tet-acs. The acs gene was amplified from DH1 E. coli genomic DNA using the Acs F3 and Acs R4 primers and inserted into the BglII restriction site of pCDF.ter-0 plasmid using Gibson cloning.

pCDF.tet-acs.ACC. The acs gene was amplified from pCDF.tet-acs using the Acs F3 and Acs R3 primers and inserted into the BglII restriction site of pCDF.ter-accBC.dtsR1 plasmid using Gibson cloning.

Transformation of E. coli. E. coli were transformed via electroporation using a MicroPulser Electroporator (Bio-Rad). For strains containing four plasmids, three plasmids were transformed into cells which had previously been transformed with the one plasmid. All transformed bacteria were plated on LB agar containing appropriate antibiotics. For strains in liquid media with a single resistance marker and all solid medias, antibiotics were used at concentrations of 50 μg/mL for carbenicillin, kanamycin, or chloramphenicol or 100 μg/mL spectinomycin. For strains in liquid media with multiple resistance markers carbenicillin was used at 50 μg/mL, spectinomycin at 100 μg/mL, and kanamycin and chlormaphenicol were used 25 μg/mL.

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Name Sequence aadA SF1 CGAAGGATGTCGCTGCCGACTGGGCAATGGAG ackA F2 TTCAAAACATTTTGTCTTCCATACCCACTATCAGGTATCCTTTAGCAGCCTGAAGGCCT adhE KV F1 ATGGCTGTTACTAATGTCGCTGAACTTAACGCACTCGTAGAGCGTGTGTAGGCTGGAGCTGCTTC adhE KV R1 TTAAGCGGATTTTTTCGCTTTTTTCTCAGCTTTAGCCGGAGCAGCCATATGAATATCCTCCTTAG atoB SF2 CCTTTCGTGATACCCGCAGCCCAGGCGCTGG atoB SR2 GCGCTATCAGGTGTATATCAACTACTATGGTGGGCGC FabB SF2 TTGGAAAAATAGACATCGTCAAAATCTCGGGAAACAGGTGTACCCTCAGC FabB SR2 TGCAGCGCAAGGCGAGGAGTATCCCCGTCTCATCTCTCTGG FabF SF1 GTCTGCGTGGTTATGAGTAATAATTAGTGCAAAATGATTTGCG FabF SR1 CGGGCTTAGCGCCAACGTAATCCCC frdB SF1 GTATGGCGCACTCCGCAATGGCACGTAAAGAG frdBC KV F1 GTATGGCGCACTCCGCAATGGCACGTAAAGAG frdBC KV R1 CGTCAGAACGCTTTGGATTTGGATTAATCATCTCAGGCTCC frdC SR1 CGTCAGAACGCTTTGGATTTGGATTAATCATCTCAGGCTCC ldhA KV F1 CAGTAATAACAGCGCGAGAACGGCTTTATATTTACCCAGC ldhA KV R1 CTGGTCACGGGCTTACCGTTTACGCTTTCCAGCAC matB SF1 GGACCGGCGAGGACGTGC matB SF2 ACCGAGGACGGCTTCTTCCGC nphT7 SF1 GGTTACGCACTGGTCATTGGTGC nphT7 SR1 CCCGCCGGCACACGAAT poxB KV F1 AGTGGTTTCCGGTGAATATACGGTGAGCAGCAC poxB KV R1 GTTCGCAGTGACTGAGCAGAGCGACCAGGT

Table 5.3. Oligonucleotides used for sequencing. Some oligonucleotides used for cloning were also used for sequencing

NphT7 ePCR library. A library nphT7 mutants generated by error-prone PCR (ePCR) was

generated using four ePCR reactions using the primer . Three ePCR products were generated using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s protocol using a 1 min extension time. The three ePCR reactions were begun with 750, 250, and 1 ng of initial target in the ePCR reaction in order to generate mutations at low (0 - 4.5 mutations/kb), medium (4.5 – 9 mutations/kb), and high (9 – 16 mutations/kb) rates, respectively. A fourth ePCR reaction using: 1 µM primers, 1 × Taq Reaction Buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl, and 2 mM MgCl2), 200 µM dATP, 200 µM dGTP, 1 mM dCTP, 1 mM dTTP, 5.5 mM MgCl2, 20 ng template DNA, and 5 U Taq Polymerase in 50 µL reaction [28]. The following thermocycler program was used for the first assembly reaction: 95ºC for 5 min; 95ºC for 30 s; 55ºC for 30 sec; 72ºC for 60 s for 40 cycles; these cycles were followed by a final incubation at 72ºC for 5 min. Each ePCR product was incubated with 50 U DpnI (New England Biolabs) for 2 h at 37ºC followed by PCR Purification (QIAGEN).

Each ePCR product was introduced into the pCDF2.tac2-gg0 plasmid using Golden Gate cloning [26] to generate pCDF2.tac2-nphT7(M1) using the thermocycler program: 50 cycles of

Mutazyme

High Mutation Rate Mutazyme

Medium Mutation Rate Mutazyme

Low Mutation Rate Unequal dNTP

High MgCl2

Colonies Sequenced 6 3 5 6

Non-mutated Colonies 1 0 0 0

Average Mutations/kb 5.3 3.4 3.7 5.1

Total frame shifts 3 0 2 0

Maximum mutations 7 5 5 9

Minimum mutations 1 1 1 3

Table 5.4. Analysis of mutation frequency for nphT7 libraries generated by ePCR.

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37°C for 2 min, 15°C for 5 min; followed by 50°C for 15 min and 80°C for 10 min. For each ligation 2 µL of ligation mix was transformed into 40 µL of MegaX Electrocompetent Cells (Invitrogen) according to the manufacturer’s protocol. Following recovery 1% of the recovery mixture was spread on selecting solid media for sequence analysis (Table 5.4), the rest of the culture was diluted 1:50 in selecting media and grown for 12-16 h at 37°C in a rotary shaker shaker (200 rpm). The plasmid DNA was isolated and pooled at equimolar concentrations. The plasmid pool was transformed into the strain MC1.40 containing pTT33-(100K)hbd-crt and pCWOri-ter.adh.aldh46 and grown under anaerobic selection.

NphT7 saturation mutagenesis library. The Phyre 2.0 algorithm [29] was used to build a structural model of NphT7. The putative binding pocket of NphT7 was selected based on the binding of malonyl-CoA to FabH, which is a structural homolog [30]. A total of 21 residues lining the hypothetical binding site were selected for saturation mutagenesis (V31, S84, V114, D142, Y144, L158, F159, I194, V196, F217, M219, V228, L232, A258, G260, V261, G288, G315, F316, G317, G318). These residues were further separated into seven groups based on the tertiary structure of the NphT7 model (1: S84, V114, D142, Y144; 2: I194, V196, F217, M219;

Group 1: S84, V114, D142, and Y144

CGGTtattgcggtcgcaacgNNKaccccggaccgtccgcagccgccgacggcggcctacgtgcaacatcatCTGGgcgcaaccggcaccgcggca ATAACGCCAGCGTTGCnnmTGGGGCCTGGCAGGCGTCGGCGGCTGCCGCCGGATGCACGTTGTAGTAgaccCGCGTTGGCCGTGGCGCCGT tttgatgttaacgctNNKtgcagcgGcacggtttttgctctgtccagcgtggCGGGcacgctggtgtatcgtggcggttacgcactggtcattgg AAACTACAATTGCGAnnmACGTCGCCGTGCCAAAAACGAGACAGGTCGCACCgcccGTGCGACCACATAGCACCGCCAATGCGTGACCAGTAACC tgccNNKctgNNKtcccgtattctgaatccg ACGGnnmGACnnmAGGGCATAAGACTTAGGCcgcc

Group 2: I194, V196, F217, and M219

TTGGtggtctgaccgacctgNNKcgtNNKccggcgggtggcagccgccaaccgctggACACggatggcttggacgcgggtctgcaatacNNKgct ACCAGACTGGCTGGACnnmGCAnnmGGCCGCCCACCGTCGGCGGTTGGCGACCtgtgCCTACCGAACCTGCGCCCAGACGTTATGnnmCGA NNKgacggtcgcgaggtgc nnmCTGCCAGCGCTCCACGcagc

Group 3: L158 and F159

TCTGaatccggcggaccgcaagaccgttgttNNKNNKggtgacggcgcgggtgcgatggt TTAGGCCGCCTGGCGTTCTGGCAACAAnnmnnmCCACTGCCGCGCCCACGCTACCAcgac

Group 4: V228 and L232

GACGgtcgcgaggtgcgtcgttttNNKaccgaacacNNKccgcaactgattaaaggtttc CAGCGCTCCACGCAGCAAAAnnmTGGCTTGTGnnmGGCGTTGACTAATTTCCAAAGaacg

Group 5: G315, F316, G317, and G318

GTGAactggtcctgctggcgNNKNNKNNKNNKggcatggcagcgagcttcgcgctgatcgagtggtaatag TGACCAGGACGACCGCnnmnnmnnmnnmCCGTACCGTCGCTCGAAGCGCGACTAGCTCACCATTATCcgaa

Group 6: V31, G260, and V261

AATGaccgacgttcgttttcgtatcattggcacgggtgcgtacgtgccggagcgtattgtgtccaacgacga TGGCTGCAAGCAAAAGCATAGTAACCGTGCCCACGCATGCACGGCCTCGCATAACACAGGTTGCTGCTCCAC

Group 7: A258 and G288

TTTGtgccgcaccaaNNKaacggtgtcatgctggacgaggtctttggtgaactgcacctgCCGCgtgcgaccatgcaccgtaccgtcgaaaccta ACGGCGTGGTTnnmTTGCCACAGTACGACCTGCTCCAGAAACCACTTGACGTGGACggcgCACGCTGGTACGTGGCATGGCAGCTTTGGAT cggcaatacgNNKgcggccagcat GCCGTTATGCnnmCGCCGGTCGTAaggc

Figure 5.3. Oligonucleotides used to perform saturation mutagenesis in the NphT7 enzyme using the ligation based method. Oligonucleotides were designed to insert the resulting PCR product into the pCDF2.tactac-GoldenGate vector via the Golden Gate method.

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3: L158, F159; 4: V228, L232; 5: G315, F316, G317, G318; 6: V31, G260, V261; 7: A258, G288). Saturation mutagenesis was initially carried out in each of these seven groups independently using either ligation based method or homologous recombination.

Ligation based method. PCR products were generated to allow for insertion into the pCDF2.tactac-GoldenGate vector via the Golden Gate method [26]. For each residue or string of residues to be mutagenized, pairs of homologous oligonucleotides were designed with randomized NNK codons at the appropriate mutagenesis sites (Table 5.2). All oligonucleotides contained at least 25 bases of homology on the 5'- and 3'-ends followed by a 4-base 5'-sticky end designed for ligation by Golden Gate (Figure 5.3). Sections of the nphT7 not targeted for saturation mutagenesis were amplified using standard conditions with appropriate primers (Table X). The Golden Gate ligation was ligated using the thermocycler program: 50 cycles of 37 °C for 2 min, 15 °C for 5 min; followed by 50 °C for 15 min and 80 °C for 10 min. The ligation was transformed in batches of 2 µL of ligation mix in 40 µL of MegaX Electrocompetent Cells (Invitrogen) according to the manufacturer’s protocol. The target vector was not observed in any ligation.

Homologous recombination method. Freshly transformed colonies of the pCDF2.tactac-nphT7 plasmid in strain SIMD90 were inoculated in low-salt LB (tryptone, 10 g/L; NaCl, 5 g/L; yeast extract, 5 g/L) and grown for 12-16 h at 37°C in a rotary shaker at 200 rpm. The culture was then diluted to OD600 = 0.05 and the growth was continued until the culture reached an OD600 of 0.3 to 0.4. The culture was then transferred into a 50 mL conical vial and incubated for 15 min at 42°C in a water bath with frequent mixing. The conical vial was then submerged in an ice water bath for 30 min with frequent mixing. The culture was spun at 6,500 g for 8 min at 4°C. The media was removed via aspiration and the culture was resuspended in sterile double deionized water (sddH2O, 50 mL) at 4°C. The culture was again spun at 6,500 g for 8 min at 4°C and the sddH2O removed. The culture was washed once more with sddH2O (2 mL) by centrifuging at 20,817 g for 20 s at 4°C. Following the final wash, the cells were resuspended with sddH2O (100 µL) and used for 3 × 40 µL transformations with 100 µM oligonucleotide (2 µL, Table 5.5). The rate of mutation has not been analyzed for any library generated.

Name Sequence ResiduenphT7 V31 F gtgtccaacgacgaggtgggtgcgccggctggtNNKgatgatgactggattacccgtaagaccggcatt V31 nphT7 S84 F ccggagcaactgacggttattgcggtcgcaacgNNKaccccggaccgtccgcagccgccgacggcggcc S84 nphT7 V114 F gcaaccggcaccgcggcatttgatgttaacgctNNKtgcagcggcacggtttttgctctgtccagcgtg V114 nphT7 D142 F tatcgtggcggttacgcactggtcattggtgccNNKctgtattcccgtattctgaatccggcggaccgc D142 nphT7 Y144 F ggcggttacgcactggtcattggtgccgatctgNNKtcccgtattctgaatccggcggaccgcaagacc Y144 nphT7 L158 F attctgaatccggcggaccgcaagaccgttgttNNKtttggtgacggcgcgggtgcgatggtgctgggt L158 nphT7 F159 F ctgaatccggcggaccgcaagaccgttgttctgNNKggtgacggcgcgggtgcgatggtgctgggtccg F159 nphT7 I194 F gccctgcacacgtttggtggtctgaccgacctgNNKcgtgtgccggcgggtggcagccgccaaccgctg I194 npht7 V196 F cacacgtttggtggtctgaccgacctgattcgtNNKccggcgggtggcagccgccaaccgctggacacg V196 nphT7 F217 F gacacggatggcttggacgcgggtctgcaatacNNKgctatggacggtcgcgaggtgcgtcgttttgtt F217 nphT7 M219 F gatggcttggacgcgggtctgcaatacttcgctNNKgacggtcgcgaggtgcgtcgttttgttaccgaa M219 nphT7 V228 F ttcgctatggacggtcgcgaggtgcgtcgttttNNKaccgaacacttgccgcaactgattaaaggtttc V228 nphT7 L232 F ggtcgcgaggtgcgtcgttttgttaccgaacacNNKccgcaactgattaaaggtttcttgcacgaggcg L232 nphT7 A258 F gcggcagatattagccattttgtgccgcaccaaNNKaacggtgtcatgctggacgaggtctttggtgaa A258 nphT7 G260 F gatattagccattttgtgccgcaccaagcgaacNNKgtcatgctggacgaggtctttggtgaactgcac G260 nphT7 V261 F attagccattttgtgccgcaccaagcgaacggtNNKatgctggacgaggtctttggtgaactgcacctg V261 nphT7 G288 F atgcaccgtaccgtcgaaacctacggcaatacgNNKgcggccagcattccgattacgatggatgcagca G288 nphT7 G315 F agcttccgtccgggtgaactggtcctgctggcgNNKtttggtggtggcatggcagcgagcttcgcgctg G315 nphT7 F316 F ttccgtccgggtgaactggtcctgctggcgggtNNKggtggtggcatggcagcgagcttcgcgctgatc F316 nphT7 G317 F cgtccgggtgaactggtcctgctggcgggttttNNKggtggcatggcagcgagcttcgcgctgatcgag G317 nphT7 G318 F ccgggtgaactggtcctgctggcgggttttggtNNKggcatggcagcgagcttcgcgctgatcgagtgg G318

Table 5.5. Oligonucleotides used to perform saturation mutagenesis in the NphT7 enzyme using the homologous recombination method.

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Mutagenized host library. A freshly transformed colony was inoculated into LB Broth with appropriate antibiotics and grown for at 37°C for 12 to 16 h at 200 rpm. The culture was diluted into LB with appropriate antibiotics (50 mL) and grown to OD600 ~ 0.3. Cultures were washed twice with phosphate buffered saline (PBS, 50 mL) and concentrated into PBS (2 mL). Ethylmethanesulfonoate (EMS; 45 µL; final concentration, 2.25%) was added to the culture, which was incubated at 37 °C for 45 min at 200 rpm. Following EMS incubation, the culture was washed twice with PBS (2 mL) and once with TB (5 mL) followed by resuspension in production media (5 mL). This culture was grown overnight at 37°C and for n-butanol production as described below.

Selection. Following generation of library starter culture, cultures were grown anaerobically at 37°C in a rotary shaker (200 rpm) in TB production media (TB with 2.5% (w/v) glucose replacing the standard glycerol supplement and appropriate antibiotics, 50 mL) in a 250 mL-screw top anaerobic baffled flask (Chemglass). When the cultures reached OD600 of 0.35-0.45, they were induced by addition of IPTG (1.0 mM). Immediately after induction, flasks were screwed closed and the head space was deaerated by flowing Ar (~1.5 atm) into the flask for ≥ 5 min using a needle (21 G). The flasks were then returned to the shaker with the growth temperature reduced to 30°C. The OD600 of the cultures was monitored daily. If the OD600 was ≥ 0.8 or the OD600 ≥ 0.4 and had not increased for the previous 48 h period, cultures were diluted to OD600 = 0.1 in anaerobic production media (50 mL). Prior to dilution, the media was sonicated and the head space was deaerated by flowing Ar into the flask for ≥3 min. The culture was then introduced into the deaerated flask via syringe (1 - 50 mL) while flowing Ar. Following transfer, the head space was deaerated again with Ar for an additional ≥3 min. Following five dilutions, aliquots of mutagenized culture (5 × 1 mL) were frozen as15% glycerol stocks. The remaining culture was then frozen for sequencing.

n-Butanol production. Freshly-transformed single E. coli colonies were inoculated into the appropriate anaerobic or microaerobic production media (5 mL) in a 50 mL test tube and grown for 12-16 h at 37°C in a rotary shaker (200 rpm).

Anaerobic production. The cultures were diluted to OD600 = 0.05 in anaerobic production media (50 mL) in a 250 mL screw top baffled flask. Cultures were grown at 37°C in a rotary shaker (200 rpm) until the OD600 reached 0.35-0.45 before inducing (IPTG, 1.0 mM; L-arabinose, 0.2% (w/v)). Immediately after induction, flasks were screwed closed and the head space was deaerated by flowing Ar (~1.5 atm) into the flask for ≥5 min using a needle (21 G). The flasks were then returned to the shaker with the growth temperature reduced to 30°C. Samples for 1 day (2 mL) were removed with a syringe (21 G). Growths were continued for 72 h.

Microaerobic production. The cultures were diluted to OD600 = 0.05 in TB production media (50 mL) in a 250 mL baffled flask. Cultures were grown at 37 °C in a rotary shaker (200 rpm) until the OD600 reached 0.35-0.45 and before inducing (IPTG, 1.0 mM; L-arabinose; 0.2% (w/v)). Immediately after induction, flasks were sealed with Parafilm M (Pechiney Plastic Packaging) and the growth temperature was reduced to 30°C. Flasks were opened to air for 10-20 min every 24 h and resealed with Parafilm M afterwards before returning to the shaker. An additional aliquot of glucose (1% (w/v)) was added after 24 h. Growths were continued for 72 h.

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MatB growths. Cultures were supplemented with malonate from a stock solution of filter-sterilized sodium malonate, pH 7.0 (1 M) to the appropriate final concentration (0, 2, 10, or 50 mM) at inoculation.

n-Butanol extraction and quantitation. The extraction and quantitation of n-butanol was performed as previously described [22]. Samples (2 mL) were removed from cell culture and cleared of biomass by centrifugation at 20,817 g for 10 min using an Eppendorf 5417R centrifuge (Hamburg, Germany). The cleared media was then mixed in a 9:1 ratio with an aqueous solution containing the isobutanol internal standard (10,000 mg/L). These samples were then analyzed on a Trace GC Ultra (Thermo Scientific; Waltham, MA) using an HP-5MS column (0.25 mm 30 m, 0.25 μM film thickness, J & W Scientific). The oven program was as follows: 75C for 3 min, ramp to 300C at 45C/min, 300C for 1 min. n-Butanol was quantified by flame ionization detection (FID) (flow: 350 ml/min air, 35 ml/min H2, and 30 ml/min He). Samples containing n-butanol levels below 500 mg/L were re-quantified after extraction of the cleared media sample or standard (500 µL) with toluene (500 µL) containing the isobutanol internal standard (100 mg/L) using a Digital Vortex Mixer (Fisher) for 5 min set at 2000. The organic layer was then quantified using the same GC parameters with a DSQII single-quadrupole mass spectrometer (Thermo Scientific; Waltham, MA) using single ion monitoring (m/z 41 and 56) concurrent with full scan mode (m/z 35-80). Samples were quantified relative to a standard curve of 2, 4, 8, 16, 31, 63, 125, 250, 500 mg/L n-butanol for MS detection or 125, 250, 500, 1000, 2000, 4000, 8000 mg/L n-butanol for FID detection. Standard curves were prepared freshly during each run and normalized for injection volume using the internal isobutanol standard (100 or 1000 mg/L for MS and FID, respectively).

Enzyme assays. Cell cultures were prepared as described for n-butanol production studies and were harvested 20 h after induction by centrifugation at 15,316 g for 20 min at 4C (Sorvall Legend RT+; Thermo Scientific; Waltham, MA) and stored at -80C after removal of the supernatant. Frozen cell pellets were thawed and resuspended at 5 mL/g cell paste in 100 mM Tris-HCl, 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, pH 7.5. The cell suspension was homogenized and lysed by passage through a French Pressure Cell (Thermo Scientific) at 14,000 psi. The crude lysate was centrifuged at 15,316 g for 30 min at 4C to remove cell debris and insoluble protein. The supernatant was used for enzyme assays as described below. Protein concentration of samples was measured using Protein Assay Reagent (Bio-Rad Laboratories; Hercules, CA) relative to a standard curve of bovine serum albumin (New England Biolabs; Ipswich, MA; 50-900 μg/mL; ε280 = 0.66 ml/mg/cm). Spectrophotometric assays for PhaA, PhaB, Ccr, Ter, and AdhE2 activity were carried out on a SpectraMax M2 plate reader (Molecular Devices; Sunnyvale, CA). The pathlength was calculated using the water constant protocol in order to convert readings to a 1 cm-pathlength. Absorbance measurements for Crt assays were collected with DU-800 spectrophotometer (Beckman-Coulter; Brea, CA). All assays were carried out at 30C except for Ter assays, which were performed at 25C.

Acetoacetyl-CoA synthetase. Synthetase activity was assayed in the forward direction by monitoring NADPH oxidation at 340 nm at 30ºC in a coupled assay with His6-PhaB [31,32]. The assay mixture contained acetyl-CoA (1 mM), malonyl-CoA (1 mM), NADPH (100 μM), and His6-PhaB (7.5 U/mL) in 100 mM Tris-HCl, 4.5 mM MgCl2, 1.5 mM DTT, pH 7.5. The reaction was initiated by addition of malonyl-CoA.

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Acetoacetyl-CoA thiolase. Thiolase activity was assayed in the forward direction by monitoring NADPH oxidation at 340 nm at 30ºC in a coupled assay with His6-PhaB [31,32]. The assay mixture contained acetyl-CoA (1 mM), NADPH (100 μM), and His6-PhaB (7.5 U/mL) in 100 mM Tris-HCl, 4.5 mM MgCl2, 1.5 mM DTT, pH 7.5. The reaction was initiated by addition of acetyl-CoA.

Acetoacetyl-CoA reductase. The reduction of acetoacetyl-CoA was assayed by monitoring the oxidation of NAD(P)H at 340 nm at 30ºC [33]. The assay mixture contained acetoacetyl-CoA (100 μM) and NAD(P)H (100 μM ) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of acetoacetyl-CoA.

3-Hydroxybutyryl-CoA dehydratase (crotonase). Dehydratase activity was measured in the reverse direction by monitoring hydration of the double bond at 263 nm at 30ºC [34]. The assay mixture contained crotonyl-CoA (30 μM) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of crotonyl-CoA.

Trans-enoyl reductase. Crotonyl-CoA reduction activity was assayed in the forward direction by monitoring the oxidation of NAD(P)H at 340 nm at 30ºC [35]. The assay mixture contained crotonyl-CoA (50 μM) and NAD(P)H (100 μM) in 100 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of crotonyl-CoA.

5.3. Results and discussion

Design of a malonyl-CoA dependent pathway for production of n-butanol. To initiate these studies, a host strain was first designed to both to limit the background production of acetoacetyl-CoA as well as to reduce its breakdown via E. coli native enzymes. There are two known acetoacetyl-CoA thiolases in E. coli, atoB [36] and yqeF [37], that were targeted for deletion as they are both capable of operating in the biosynthetic and degradative direction. As such, the deletion of these genes should reduce the production of n-butanol from native pathways independent of NphT7 and also should prevent the creation of a futile cycle through degradation of acetoacetyl-CoA back to two acetyl-CoA with loss of ATP (Figure 5.4). While the introduction of the atoB gene deletion in the both the E. coli DH1 adhE::FRT (MC1.16) and DH1 DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT (MC1.24) strain backgrounds was achieved, the double deletion of atoB and yqeF in both backgrounds was not easily accomplished. As such, MC1.16 atoB (MC1.29) and MC1.24 atoB (MC1.40) strains were used for all of the experiments presented in this chapter. However, no thiolase activity above background was observed in cell lysate activity in DH1 or MC1.29 cultures despite the presence of genomically encoded acetoacetyl-CoA thiolase enzymes (data not shown). As a note, the MC1.16 atoByqeF and MC1.24 atoB yqeF double thiolase knockout strains have since been generated for future experiments.

We set out to construct an NphT7-dependent version of our previously reported n-butanol pathway by replacing the gene encoding phaA with a codon-optimized nphT7 gene. The plasmids constructed were analogous to those previously used to analyze changes in expression of a phaA operon on n-butanol titer [22]. A series of three promoters were used to express the nphT7.hbd operon varying from relatively low to high expression utilizing the arabinose, T5, and trc promoters. The plasmids for both the NphT7-dependent and PhaA-dependent pathways were then transformed into MC1.29 in order to directly compare their n-butanol productivity in the the same strain background. We observed that the replacement of PhaA with NphT7 (Figure 5.5; 2)

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Figure 5.4. Design of an E. coli knockout strain for studying n-butanol production with NphT7. The E. coli genome encodes 2 acetoacetyl-CoA thiolases, AtoB and YqeF. In order to analyze a strain using the nphT7 based n-butanol synthetic pathway there should be no thiolase activity in the cell. The atoB was successfully deleted from the strains analyzed in this study. Deletion of the yqeF gene was not easily achieved, as a result, all strains used in this study contain the genomically encoded yqeF.

reduces n-butanol titers by over 10-fold compared to the standard pathway (Figure 5.5; 1), indicating that the use of NphT7 for acetoacetyl-CoA production has become a bottleneck.

To further address this question, we attempted to increase the expression of nphT7 through the use of stronger promoters as well as to titrate nphT7 expression levels through the design of ribosome binding site (RBS) variants using the Salis RBS calculator [38,39]. These experiments demonstrate that n-butanol titers appear to be directly connected to the expression of nphT7 as the use of stronger promoters (Figure 5.5; 3 and 4) increased n-butanol production while the use of weaker RBS strengths decreased yields (Figure 5.5; 5 - 7). A comparison of the activity of NphT7 in cell lysates shows that the stronger promoters do lead to higher functional expression (Figure 5.5) with corresponding changes in activity for the Hbd enzyme which is expressed from the same operon but no significant change of activities for the other enzyme expressed from same plasmid (Crt) (Table 5.6).

While increasing the intracellular activity of NphT7 by an order of magnitude leads to a ~4-fold increase of n-butanol titers, assays of cell lysate activity of PhaA in the standard pathway showed that their overall activities were equivalent (Figure 5.5). Although it is possible that there are other changes to in vivo activity that cannot be observed by in vitro cell lysate based assays, these data suggest that malonyl-CoA availability could be a limiting factor to high n-butanol flux. Indeed, the intracellular concentration of malonyl-CoA (15 to 57 µM [14]) is similar to the Km of NphT7 (28 µM) [19], which would result in sensitivity to changes in malonyl-CoA concentration. In contrast, the reported values for intracellular concentration of acetyl-CoA in E. coli (0.7 to 1.4 mM) [14,40] is above the Km(acetyl-CoA) for PhaA (0.3 mM) [41] and NphT7 (68 µM) [19], respectively.

Increasing intracellular malonyl-CoA availability. Several approaches have been reported to increase malonyl-CoA concentration in vivo. The Cronan laboratory has reported that the use strains lacking functional fatty acid synthases results in increased malonyl-CoA

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Figure 5.5. Promoter and RBS optimization of n-butanol production using an nphT7 based pathway. The nphT7 pathways are titrated with promoters of varying strength with the same rbs (Ara < T5 < Trc), rbs of varying strength with the Ara promoter, and the T7 promoter using the 100K rbs. All cultures use strain MC1.29 grown microaerobically over 3 d. All strains contain the pCWOri-ter.adhE2 plasmid. Each strain also contains the additional plasmid noted in the table. The promoter variants and the phaA pathway are labeled with the specific activity (µmol/min/mg) of the acetoacetyl-CoA producing step as measured in cell lysate. All data are n=3.

Plasmid Hbd S.A. (µmol/min/mg) Crt S.A. (µmol/min/mg)

pBT33-Bu2 2.8 ± 0.4 2300 ± 400

pBT33-Bu6 3.0 ± 0.6 2800 ± 200

pT5T33-Bu6 9.3 ± 0.9 2700 ± 400

pTT33-Bu6 20 ± 3 2800 ± 200

Table 5.6. Specific activity of other pathway enzymes. All cultures use strain MC1.29 grown microaerobically over 3 d. All strains contain the pCWOri-ter.adhE2 plasmid. Each strain also contains the additional plasmid noted in the table. In vitro activity is calculated as the specific activity (µmol/min/mg) as measured in cell lysate. All data are n=3.

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Figure 5.6. Examining the effect of ACS and ACCase overexpression on n-butanol production using an nphT7 based pathway. (A) Effect of mutations in fatty acid biosynthetic genes. The n-butanol production after 3 days under microaerobic conditions was tested using the pCWOri-ter.adhE2 plasmid in addition to either the pBT33-Bu2 (phaA containing) plasmid or the pTT33-Bu6 (nphT7 containing) plasmid. The production was performed in either the DH1 (red), CY242 (black), or CY244 (blue) strains. The DH1 and CY242 growths were performed at 30 °C while the CY244 growth was performed at 42 °C. (B) The cultures expressing the nphT7 pathway contain the pBBR2 plasmid expressing an enzyme proposed to increase intracellular malonyl-CoA. All growths were carried out in strain MC1.29 at 30 °C under microaerobic conditions for 3 days. The strains contained plasmids pBT33-Bu2 and pCWOri-ter.adhE2 (1) or pTT33-Bu6 and pCWOri-ter.adhE2 (strains 2 – 7). In addition the strains contained the plasmids pBBR2-0 (strains 1 and 2), pBBR2-acs (strain 3), pBBR2-accD4.accE.accA3 (strain 4), pBBR2-accD6.accE.accA3 (strain 5), pBBR2-accBC.dtsR1 (strain 6), pBBR2-acs.ACC (strain 7). All data are n=3.

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concentrations [23,24]. In addition, the Zhao laboratory has shown to expression of acetyl-CoA carboxylases (ACCase) can increase the intracellular malonyl-CoA concentration, especially when expressed concurrently with acetyl-CoA synthases (ACS). Based on these reports, we have tried several of these strategies to improve n-butanol production using the NphT7 pathway. Using E. coli strains CY242 (∆fabB) and CY244 (fabF(ts)) from the Cronan lab we were able to observe an increase or no change in n-butanol production when changing from a PhaA based pathway to an NphT7, in contrast to the decrease seen in the DH1 strain (Figure 5.6A). However, the overall production remains significantly lower than in the DH1 host background. The ability to see higher flux with NphT7 based pathway in an MG1655 derived strain suggests that engineering of DH1 metabolism may yield malonyl-CoA n-butanol titers competitive with the PhaA based pathway.

In addition to introduction of genomic modifications to the native E. coli fatty acid synthesis system, we have overexpressed genes involved in acetyl-CoA and malonyl-CoA generation. Towards this goal, plasmids for co-expression of the ACCases from Mycobacterium tuberculosis (accA3 and accE with either accD4 or accD6) and Corynebacterium glutamicum (accBC and dtsR1) were constructed, as theses ACCases have been previously used to increase flux to malonyl-CoA based pathways is E. coli [14,42,43] and can be used to engineer increases in E. coli malonyl-CoA levels. While the co-expression of the ACS from E. coli (Figure 5.6B; 3), ACCase complexes from M. tuberculosis (Figure 5.6B; 4 and 5) and C. glutamicum (Figure 5.6B; 6), as well as C. glutamicum accCB and dtsR1 with E. coli ACS (Figure 5.6B; 7) result in elevated n-butanol titers, but the titers still remained significantly below the acetyl-CoA based pathway (Figure 5.6B; 1).

We next turned our attention using feeding experiments to further explore the relationship between intracellular malonyl-CoA concentration and n-butanol production via the NphT7 pathway. To facilitate these experiments, a malonyl-CoA ligase (MatB) from Streptomyces coelicolor was co-expressed with the NphT7 based n-butanol pathway. MatB has been shown to be competent convert malonate to malonyl-CoA in E. coli, elevating the intracellular concentration [44]. We expressed the matB gene with either the PhaA based pathway or one of two variants of the NphT7 pathway; however, error among samples expressing the NphT7 pathway prevent us from drawing any conclusions (Figure 5.7).

Efforts at selecting for E. coli host mutations. In conjunction with rational design approaches to increasing malonyl-CoA availability, we decided to take advantage of an n-butanol selection method developed by other members of our group. Since the intracellular concentration of malonyl-CoA is tightly controlled by multiple factors, we decided that one approach towards increasing n-butanol titers could be to improve NphT7 through directed evolution. In order to identify improved NphT7 variants, we have implemented a redox based selection system developed by Matthew Davis in collaboration with Dr. Miao Wen. The selection system uses the strain, MC1.40, derived from DH1 with the lactate, acetate, succinate, and ethanol fermentation pathways knocked out (DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT) in order to link n-butanol production to redox balance and therefore cell growth under anaerobic conditions.

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Figure 5.7. Examining the effect of malonic acid feeding with MatB expression using an nphT7 based pathway. The strains contained the plasmids pCWOri-ter.adhE2, pBBR2-matB, and either pBT33-Bu2 (red), pTT33-(100K)Bu6 (gray), or pCDF2.tac2-nphT7 and pTT33-(100K)hbd-crt (blue). At induction of n-butanol pathway the cultures were also fed 0, 2, 10, or 50 mM malonic acid. All data are n=3.

Figure 5.8. Production of n-butanol using the nphT7 based pathway in cultures mutagenized the EMS. Strain MC1.40 containing the plasmids pTT33-Bu6 and pCWOri-ter.aldh46.adh were exposed to 2.25% EMS for 15, 30, 45, or 60 min. Cultures were analyzed every 24 h and diluted to OD600 = 0.1 when at least one culture showed an OD600 > 1.0 or the cultures had maintained a near constant OD600 for 48 h.The cultures were selected under anaerobic conditions over 5 dilutions followed by a 3 day growth after which n-butanol titer was assayed. n-Butanol titers are noted above bar with standard deviation. All data are n=3.

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Towards this goal, we decided to use a chemical mutagen, ethyl methanesulfonate (EMS), to introduce chromosomal mutations into the host. As the number of mutations is dependent on the EMS concentration and incubation time, we used a 2.25 % EMS incubation for 45 min in order to generate on the order of 75 to150 mutations in the E. coli genome [45]. Because of the difficulty in obtaining a large number of transformants given the number of plasmids required to express the n-butanol pathway, we decided to introduce EMS to the culture after transformation. Although plasmid-borne mutations would also be possible, we were interested in any mutations that could help to increase n-butanol titer even within the synthetic pathway itself. While the initial n-butanol titers for this pathway appear to be too low for significant improvement given the diversity of our libraries and the large drop in production related to the MC1.40 selection strain, we were able to select for hosts with increased n-butanol titers after five rounds of culture dilution (Figure 5.8).

However, the extremely low n-butanol production in the starting strain results in very slow growth and does not appear to provide an adequate platform for evolution. Therefore, we decided to reevaluate the plasmids used to the express the NphT7 basd n-butanol synthetic pathway to attempt to increase the initial titer and therefore also increase the growth rate of the initial cultures. Several different systems were tested and optimized, while all showed low n-butanol production compared to the PhaA based pathway, alternative plasmids were identified that resulted in increased titers under selecting conditions (Figure 5.9).

Efforts at directed evolution of nphT7. We also decided to explore the possibility of evolving the NphT7 enzyme using the selection system. We used two major approaches to generating nphT7 libraries, involving either error-prone PCR (ePCR) or saturation mutagenesis. Since ePCR is typically the simplest approach to generate diversity, our first libraries were produced using this method. Four variations of ePCR were used to generate clones with varying mutation rates. Each library was independently ligated into the pCDF2.tac2 vector using the cycling Golden Gate ligation method [26] and their mutation rates were independently analyzed (Table 5.4).

Figure 5.9. Optimization of production of n-butanol under selecting growth conditions. The production of the MC1.29 strain under microaerobic conditions for 3 days containing pBT33-Bu6 and pCWOri-ter.aldh46.adh plasmid (strain 1). The production of the C1.40 strain under anaerobic conditions for 3 days containing the pCWOri-ter.aldh46.adh plasmid and pBT33-Bu6 (2), pTT33-Bu6 (3), pT7T33-Bu6 (4), pTT33-(100K)Bu6 (5), pT5T33-(100K)Bu6, or pCDF2.tac2-nphT7 and pTT33-hbd-crt (7). n-Butanol titers are noted above bar with standard deviation. All data are n=3.

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Figure 5.10. Growth of cultures expressing ePCR generated nphT7 mutants in the n-butanol producing pathway. Strain MC1.40 containing the plasmids pCDF2.tac2-nphT7 (mutagenized with ePCR), pTT33-hbd-crt, and pCWOri-ter.aldh46.adh. The cultures contain an ePCR generated nphT7 gene using two independent ligations/transformations each in triplicate (triplicates represented as red and black). Cultures were analyzed every 24 h and diluted to OD600 = 0.1 when at least one culture showed an OD600 > 1.0 or the cultures had maintained a near constant OD600 for 48 h.

The four ePCR generated libraries were pooled together and transformed into the selection strain of MC1.40 containing the plasmids pTT33-(100K)hbd.crt and pCWOri-ter.aldh46.adh for a library size of ~1.3 × 108. After five rounds of selection, the growth rate of the culture increased significantly (Figure 5.10), however, it was found that the phaA gene could be amplified via PCR from all selected cultures which suggests that a phaA expressing strain had contaminated the culture and was selected.

As saturation mutagenesis of key residues has often been found to be more successful in identifying improved mutants, we also decided to use the approach developed by the Reetz laboratory for iterative mutagenesis of grouped amino acid residues [46]. We first generated a homology model of NphT7 using Phyre 2.0 [29]and located the putative active site based on the malonyl-CoA-bound structure of the E. coli FabH [30] (Figure 5.11A). Using this structural model, we chose seventeen different sites within the hypothetical binding pocket for malonyl-CoA that were divided into seven groups (Figure 5.11B-D). Attempts to generate libraries with saturation mutagenesis at specific residues have thus far resulted in plasmids that have been shown to not contain the full length nphT7 gene upon sequencing. Modified cloning

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Figure 5.11. Selecting residues in NphT7 for saturation mutagenesis. (A) The alignment of FabH from E. coli (gray) crystallized with malonyl-CoA bound with the Phyre 2.0 prediction of the NphT7 structure (slate). The location of the residues selected for saturation mutagenesis: (B) group 1 (black), group 2 (red), (C) group 3 (black), group 4 (red), group 7 (blue), (D) group 5 (black), group 6 (red).

Mutagenesis target Method Status

Genome EMS

Variable exposure time

Attempted once. Cultures exposed for 0, 15, 30, 45, and 60 min were selected in triplicate. After 5 dilutions the cultures that had been exposed to EMS for 30, 45, and 60 min produced more n-butanol than the control

over a three day growth (Figure X).

Genome EMS

45 min exposure time

Attempted twice. In both trials, the culture became saturated with a strain that grew rapidly but produced

no n-butanol.

nphT7 epPCR

Attempted once. With selection the culture grew at increased an increased rate and the final sample

showed significant n-butanol production increase. It appears the culture was contaminated by a strain

expressing the phaA gene (Figure X).

nphT7 Ligation saturation

mutagenesis

Attempted 3 times. Each attempt resulted in all sequenced clones lacking a full length nphT7 gene. Alternative cloning methods and/or an alternative

vector backbone must be developed to allow for ligation based saturation mutagenesis.

nphT7 Recombination saturation

mutagenesis Attempted once. Sequencing has not been completed

to determine the rate of mutation.

Table 5.7. Summary of selection efforts for optimizing an nphT7 dependent pathway. The status of methods in use or under development for generating libraries for use with the selection of strains with increased n-butanol production.

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techniques and the use of homologous recombination are currently being explored to generate these libraries. Neither of these techniques has yielded a library to date.

At this time, only a single selection of cultures mutated with EMS has successfully evolved cultures that yield detectable levels on n-butanol (Figure 5.9). All other attempts at evolving strains treated with EMS have resulted in selection of faster growing strains in which no n-butanol can be detected (Table 5.7). Further characterization of the selected strains following evolution must be undertaken. Selection of cultures containing a mutagenized nphT7 gene have been selected for cultures growing rapidly and producing n-butanol; however, these cultures appear to be contaminated with strains that contain the phaA gene, and therefore use a non-malonyl-CoA dependent pathway that likely is the cause of the enhanced n-butanol production (Figure 5.10). Work the generate saturation mutagenesis libraries of nphT7 have yet to yield plasmids that can be utilized for selections (Table 5.7).

5.4. Conclusions

The production of n-butanol in the NphT7 based pathway is limited by flux through the NphT7 node. While increasing concentration of the enzyme can increase production, the activity level is competent for n-butanol titers competitive with the PhaA based pathway. The overexpression of ACC and ACS enzymes suggest that low intracellular levels of malonyl-CoA are limiting flux to n-butanol. The redox based selection method developed within the lab appears capable of selecting for strains with elevated n-butanol production. Unfortunately, the low levels of production observed, most significantly under anaerobic conditions, limit the ability of the selection to identify mutants yielding high flux through the NphT7 based pathway.

5.5. References

Portions of this work were performed in collaboration with the following persons: Strains and methods for genetic selections for n-butanol production were developed by Mr. Matthew Davis and Dr. Miao Wen.

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Appendix 1: Oligonucleotides maps and primer sequences for gene synthesis

113

Table A1.1. Oligonucleotides used for the synthesis of the accA3 gene. Name Sequence AccA3 R1 TGCGTGGCTTGCCAT AccA3 F1 ATGGCAAGCCACGCAGGTAGCCGTATCGCCCG AccA3 R2 GCCACCAGGACTTTGCTAATGCGGGCGATACGGCTACC AccA3 F2 CATTAGCAAAGTCCTGGTGGCCAACCGTGGTGAGATCGC AccA3 R3 GCACGGATGACACGAACTGCGATCTCACCACGGTTG AccA3 F3 AGTTCGTGTCATCCGTGCCGCACGTGATGCAGGT AccA3 R4 CGCCACAGACGGCAAACCTGCATCACGTGCG AccA3 F4 TTGCCGTCTGTGGCGGTCTACGCAGAGCCGGA AccA3 R5 TGCGGGGATTCGGCATCCGGCTCTGCGTAGAC AccA3 F5 TGCCGAATCCCCGCACGTCCGTCTGGCCGA AccA3 R6 CCCAGTGCAAAAGCCTCGTCGGCCAGACGGACG AccA3 F6 CGAGGCTTTTGCACTGGGTGGTCAGACGAGCGCG AccA3 R7 CGCAAAGTCCAGGTAGCTTTCCGCGCTCGTCTGACCA AccA3 F7 GAAAGCTACCTGGACTTTGCGAAAATTTTGGACGCCGCG AccA3 R8 TGGCACCGCTCTTTGCCGCGGCGTCCAAAATTTT AccA3 F8 GCAAAGAGCGGTGCCAACGCGATTCATCCGGGT AccA3 R9 TTTCGGCCAGAAAGCCATAACCCGGATGAATCGCGT AccA3 F9 TATGGCTTTCTGGCCGAAAACGCGGACTTTGCACA AccA3 R10 CGGCGTCAATAACCGCTTGTGCAAAGTCCGCGT AccA3 F10 AGCGGTTATTGACGCCGGTCTGATTTGGATCGGCCC AccA3 R11 GGATGCTTTGCGGGCTCGGGCCGATCCAAATCAGAC AccA3 F11 GAGCCCGCAAAGCATCCGCGACCTGGGTGACAAAG AccA3 R12 CGATGTGACGTGCCGTGACTTTGTCACCCAGGTCGC AccA3 F12 TCACGGCACGTCACATCGCGGCGCGTGCCCAAG AccA3 R13 CCGGCACCAGCGGCGCTTGGGCACGCGCCG AccA3 F13 CGCCGCTGGTGCCGGGTACGCCGGACCCGG AccA3 R14 GACTTCGTCTGCACCTTTCACCGGGTCCGGCGTAC AccA3 F14 TGAAAGGTGCAGACGAAGTCGTTGCGTTCGCAGAAGAGTA AccA3 R15 GCGATCGGCAGACCGTACTCTTCTGCGAACGCAAC AccA3 F15 CGGTCTGCCGATCGCTATCAAGGCAGCCCACGG AccA3 R16 CCTTTACCGCCGCCGCCGTGGGCTGCCTTGATA AccA3 F16 CGGCGGCGGTAAAGGTATGAAAGTTGCGCGTACCA AccA3 R17 GCTCCGGGATTTCGTCGATGGTACGCGCAACTTTCATA AccA3 F17 TCGACGAAATCCCGGAGCTGTACGAAAGCGCAGTGC AccA3 R18 CCGCCGTCGCCTCACGCACTGCGCTTTCGTACA AccA3 F18 GTGAGGCGACGGCGGCCTTCGGTCGTGGCGA AccA3 R19 CCAAATAACGTTCAACGTAGCATTCGCCACGACCGAAGG AccA3 F19 ATGCTACGTTGAACGTTATTTGGATAAGCCGCGCCACG AccA3 R20 TCACCTGGGCCTCCACGTGGCGCGGCTTAT AccA3 F20 TGGAGGCCCAGGTGATTGCCGACCAGCACG AccA3 R21 CGGCCACCACAACGTTACCGTGCTGGTCGGCAA AccA3 F21 GTAACGTTGTGGTGGCCGGTACGCGCGACTGCTC AccA3 R22 TGATAGCGACGCTGCAAGGAGCAGTCGCGCGTAC AccA3 F22 CTTGCAGCGTCGCTATCAGAAACTGGTCGAAGAGGCA AccA3 R23 CAGGAACGGGGCCGGTGCCTCTTCGACCAGTTTC AccA3 F23 CCGGCCCCGTTCCTGACCGACTTTCAACGTAAGGAG AccA3 R24 TCGCGCTGTCGTGGATCTCCTTACGTTGAAAGTCGGT AccA3 F24 ATCCACGACAGCGCGAAACGCATTTGCAAGGAGGC AccA3 R25 CCGCACCGTGGTAATGCGCCTCCTTGCAAATGCGTT AccA3 F25 GCATTACCACGGTGCGGGCACCGTCGAGTATCTGGT AccA3 R26 AATCAGGCCATCTTGACCCACCAGATACTCGACGGTGC AccA3 F26 GGGTCAAGATGGCCTGATTTCTTTTCTGGAAGTGAACACGC AccA3 R27 GGGTGTTCAACTTGCAGGCGCGTGTTCACTTCCAGAAAAGA AccA3 F27 GCCTGCAAGTTGAACACCCGGTCACGGAAGAAACGGC AccA3 R28 GCAGCACCAGATCAATACCCGCCGTTTCTTCCGTGACC AccA3 F28 GGGTATTGATCTGGTGCTGCAACAATTCCGCATTGCGAAC AccA3 R29 GTGATGTCCAGTTTTTCGCCGTTCGCAATGCGGAATTGTT AccA3 F29 GGCGAAAAACTGGACATCACCGAGGACCCGACCCC AccA3 R30 ATCGCGTGACCACGCGGGGTCGGGTCCTCG AccA3 F30 GCGTGGTCACGCGATCGAGTTCCGCATTAATGGTGA AccA3 R31 AAGTTACGACCCGCATCCTCACCATTAATGCGGAACTCG

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AccA3 F31 GGATGCGGGTCGTAACTTTCTGCCGGCTCCGGG AccA3 R32 GATGAAACTTCGTCACCGGACCCGGAGCCGGCAGA AccA3 F32 TCCGGTGACGAAGTTTCATCCGCCGAGCGGTCCGG AccA3 R33 CGCTATCCACACGAACGCCCGGACCGCTCGGCG AccA3 F33 GCGTTCGTGTGGATAGCGGTGTTGAGACGGGCAGC AccA3 R34 TATCAAACTGGCCACCAATGACGCTGCCCGTCTCAACAC AccA3 F34 GTCATTGGTGGCCAGTTTGATAGCATGTTGGCGAAACTGATT AccA3 R35 ACGATCTGCACCGTGAACAATCAGTTTCGCCAACATGC AccA3 F35 GTTCACGGTGCAGATCGTGCGGAGGCGCTGGCG AccA3 R36 CGCACGACGCGCACGCGCCAGCGCCTCCGC AccA3 F36 CGTGCGCGTCGTGCGCTGAACGAGTTCGGTGTGGA AccA3 R37 GGAATCACGGTAGCCAAACCTTCCACACCGAACTCGTTCAG AccA3 F37 AGGTTTGGCTACCGTGATTCCGTTCCATCGCGCAGTTGT AccA3 R38 TAAAAGCCGGGTCGCTCACAACTGCGCGATGGAAC AccA3 F38 GAGCGACCCGGCTTTTATTGGTGATGCTAATGGCTTCT AccA3 R39 AGCGCGTGTGAACGGAGAAGCCATTAGCATCACCAA AccA3 F39 CCGTTCACACGCGCTGGATCGAGACCGAGTGGAA AccA3 R40 GTGAACGGCTCGATGGTATTGTTCCACTCGGTCTCGATCC AccA3 F40 CAATACCATCGAGCCGTTCACCGATGGTGAGCCGCTG AccA3 R41 GGACGCGCATCCTCGTCCAGCGGCTCACCATCG AccA3 F41 GACGAGGATGCGCGTCCGCGTCAAAAAGTCGTGGTCGA AccA3 R42 ACGCGACGACCGTCGATTTCGACCACGACTTTTTGACGC AccA3 F42 AATCGACGGTCGTCGCGTCGAGGTGAGCCTGCCGG AccA3 R43 CTCAGCGCCAGATCCGCCGGCAGGCTCACCTCG AccA3 F43 CGGATCTGGCGCTGAGCAACGGTGGTGGCTGT AccA3 R44 GAATAACACCGACCGGGTCACAGCCACCACCGTTG AccA3 F44 GACCCGGTCGGTGTTATTCGCCGTAAGCCGAAGCC AccA3 R45 AGCACCGCGCTTACGCGGCTTCGGCTTACGGC AccA3 F45 GCGTAAGCGCGGTGCTCACACCGGCGCGGCG AccA3 R46 GGCGTCGCCGCTGGCCGCCGCGCCGGTGTG AccA3 F46 GCCAGCGGCGACGCCGTGACGGCACCGATGCA AccA3 R47 CAAATTTCACAACGGTGCCCTGCATCGGTGCCGTCAC AccA3 F47 GGGCACCGTTGTGAAATTTGCAGTTGAGGAAGGCCAAGA AccA3 R48 TCGCCGGCCACGACCTCTTGGCCTTCCTCAACTG AccA3 F48 GGTCGTGGCCGGCGACCTGGTGGTCGTCCTGGA AccA3 R49 CGGGTTCTCCATCTTCATTGCCTCCAGGACGACCACCAGG AccA3 F49 GGCAATGAAGATGGAGAACCCGGTTACCGCGCACAAAGACG AccA3 R50 CCAGACCCGTGATGGTGCCGTCTTTGTGCGCGGTAAC AccA3 F50 GCACCATCACGGGTCTGGCGGTGGAGGCGGGTGC AccA3 R51 GTGCCTTGGGTAATCGCCGCACCCGCCTCCACCG AccA3 F51 GGCGATTACCCAAGGCACGGTCCTGGCGGAGATTAAATAA AccA3 R52 tcacgtctacgttagagcttctaTTATTTAATCTCCGCCAGGACCAccA3 F52 tagaagctctaacgtagacgtga

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Table A1.2. Oligonucleotides used for the synthesis of the accBC gene. Name Sequence accBC R1 GTGGCTTCGGCAGCCC accBC F1 GGGCTGCCGAAGCCACGGCTAATAGAAGGAAGGATAAGGC accBC R2 GCGGGTTTCAACGCTCATGCCTTATCCTTCCTTCTATTAGCC accBC F2 ATGAGCGTTGAAACCCGCAAGATTACGAAAGTTCTGGTGGC accBC R3 GGCGATTTCACCACGGTTTGCCACCAGAACTTTCGTAATCTT accBC F3 AAACCGTGGTGAAATCGCCATTCGTGTTTTCCGTGCGG accBC R4 CGATGCCTTCGTCACGTGCCGCACGGAAAACACGAAT accBC F4 CACGTGACGAAGGCATCGGCAGCGTCGCAGTTTACG accBC R5 TCCGCATCCGGCTCGGCGTAAACTGCGACGCTGC accBC F5 CCGAGCCGGATGCGGATGCGCCGTTCGTGAGC accBC R6 GAACGCCTCATCGGCATAGCTCACGAACGGCGCA accBC F6 TATGCCGATGAGGCGTTCGCGTTGGGTGGTCAGAC accBC R7 CCAGATAGCTCTCTGCGCTCGTCTGACCACCCAACGC accBC F7 GAGCGCAGAGAGCTATCTGGTTATCGACAAAATTATCGATGCGG accBC R8 CGCGCCGCTCTTACGCGCCGCATCGATAATTTTGTCGATAA accBC F8 CGCGTAAGAGCGGCGCGGATGCGATCCATCCGGGC accBC R9 GCATTTTCGGCCAAAAAACCGTAGCCCGGATGGATCGCATC accBC F9 TACGGTTTTTTGGCCGAAAATGCAGACTTTGCGGAAGCAGTCA accBC R10 CGATCCAAATCAGACCTTCGTTAATGACTGCTTCCGCAAAGTCT accBC F10 TTAACGAAGGTCTGATTTGGATCGGCCCGAGCCCGGAATC accBC R11 ACCCAGGCTGCGGATGGATTCCGGGCTCGGGC accBC F11 CATCCGCAGCCTGGGTGACAAAGTCACCGCGCG accBC R12 GCGGTATCTGCGATGTGACGCGCGGTGACTTTGTC accBC F12 TCACATCGCAGATACCGCAAAAGCCCCGATGGCTC accBC R13 GGCTCCTTGGTGCCCGGAGCCATCGGGGCTTTT accBC F13 CGGGCACCAAGGAGCCGGTTAAGGATGCGGCGG accBC R14 CGCAAACGCAACGACCTCCGCCGCATCCTTAACC accBC F14 AGGTCGTTGCGTTTGCGGAGGAGTTCGGCCTGC accBC R15 CGCTGCTTTAATTGCGATCGGCAGGCCGAACTCCTC accBC F15 CGATCGCAATTAAAGCAGCGTTTGGCGGCGGTGGTC accBC R16 GTACGCGACCTTCATACCACGACCACCGCCGCCAAA accBC F16 GTGGTATGAAGGTCGCGTACAAGATGGAAGAAGTCGCAGATT accBC R17 GGGTAGCGCTTTCGAACAAATCTGCGACTTCTTCCATCTT accBC F17 TGTTCGAAAGCGCTACCCGTGAAGCGACCGCGGC accBC R18 ACATTCACCACGACCGAACGCCGCGGTCGCTTCAC accBC F18 GTTCGGTCGTGGTGAATGTTTCGTGGAGCGCTATCTGGATA accBC R19 TTCAACGTGGCGAGCCTTATCCAGATAGCGCTCCACGAA accBC F19 AGGCTCGCCACGTTGAAGCTCAGGTCATTGCGGAC accBC R20 CCACGACAACATTGCCATGTTTGTCCGCAATGACCTGAGC accBC F20 AAACATGGCAATGTTGTCGTGGCGGGCACGCGCGACTG accBC R21 ACGGCGTTGCAGGCTACAGTCGCGCGTGCCCG accBC F21 TAGCCTGCAACGCCGTTTCCAGAAACTGGTCGAGGA accBC R22 AAACGGTGCCGGTGCTTCCTCGACCAGTTTCTGGAA accBC F22 AGCACCGGCACCGTTTCTGACGGACGACCAACG accBC R23 GCTATGCAGGCGCTCGCGTTGGTCGTCCGTCAG accBC F23 CGAGCGCCTGCATAGCTCTGCAAAAGCAATTTGCAAGG accBC R24 CGCCATAGTAACCGGCCTCCTTGCAAATTGCTTTTGCAGA accBC F24 AGGCCGGTTACTATGGCGCAGGCACCGTTGAGTATCTG accBC R25 AGACCGTCGCTACCGACCAGATACTCAACGGTGCCTG accBC F25 GTCGGTAGCGACGGTCTGATCTCCTTTCTGGAGGTCAAC accBC R26 CCACTTGCAAGCGGGTGTTGACCTCCAGAAAGGAGATC accBC F26 ACCCGCTTGCAAGTGGAGCACCCGGTTACCGA accBC R27 CAATGCCGGTGGTCTCCTCGGTAACCGGGTGCT accBC F27 GGAGACCACCGGCATTGACCTGGTGCGTGAGATGT accBC R28 CATGACCTTCGGCAATACGAAACATCTCACGCACCAGGT accBC F28 TTCGTATTGCCGAAGGTCATGAGCTGAGCATCAAGGAGGAC accBC R29 GACCACGCGGTGCCGGGTCCTCCTTGATGCTCAGCT accBC F29 CCGGCACCGCGTGGTCACGCATTCGAGTTCCGTATC accBC R30 CCCGCATCTTCGCCATTGATACGGAACTCGAATGCGT accBC F30 AATGGCGAAGATGCGGGCAGCAACTTCATGCCGGC

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accBC R31 TGGTGATCTTGCCCGGTGCCGGCATGAAGTTGCTG accBC F31 ACCGGGCAAGATCACCAGCTACCGCGAGCCGCAG accBC R32 TACGCACGCCCGGACCCTGCGGCTCGCGGTAGC accBC F32 GGTCCGGGCGTGCGTATGGACAGCGGCGTGGT accBC R33 CTAATCTCGCTGCCCTCGACCACGCCGCTGTCCA accBC F33 CGAGGGCAGCGAGATTAGCGGTCAGTTCGACTCTATGC accBC R34 CCCAAACAATCAGTTTTGCCAGCATAGAGTCGAACTGACCG accBC F34 TGGCAAAACTGATTGTTTGGGGCGATACCCGTGAGCAG accBC R35 ACGAGAACGTTGCAGTGCCTGCTCACGGGTATCGC accBC F35 GCACTGCAACGTTCTCGTCGTGCGCTGGCGGAATA accBC R36 CGGCATACCTTCCACGACATATTCCGCCAGCGCACG accBC F36 TGTCGTGGAAGGTATGCCGACCGTTATTCCGTTCCACC accBC R37 GCCGGGTTTTCGACAATATGTTGGTGGAACGGAATAACGGT accBC F37 AACATATTGTCGAAAACCCGGCGTTCGTCGGTAATGATGAGGG accBC R38 TCCACTTCGTGTAGATTTCGAAACCCTCATCATTACCGACGAAC accBC F38 TTTCGAAATCTACACGAAGTGGATTGAGGAGGTGTGGGATAACC accBC R39 ACGTACGGGGCGATCGGGTTATCCCACACCTCCTCAA accBC F39 CGATCGCCCCGTACGTTGATGCGTCCGAGTTGGA accBC R40 GGGGTTTTATCCTCGTCTTCATCCAACTCGGACGCATCA accBC F40 TGAAGACGAGGATAAAACCCCGGCCCAAAAAGTTGTCGTC accBC R41 ACACGGCGACCGTTAATTTCGACGACAACTTTTTGGGCC accBC F41 GAAATTAACGGTCGCCGTGTTGAAGTGGCCCTGCCG accBC R42 CCCAATGCCAAGTCGCCCGGCAGGGCCACTTCA accBC F42 GGCGACTTGGCATTGGGTGGTACGGCAGGTCCG accBC R43 GGCGCTTCTTCGCTTTTTTTTTCGGACCTGCCGTACCA accBC F43 AAAAAAAAAGCGAAGAAGCGCCGCGCAGGTGGCGCTAA accBC R44 GCCGCTCACGCCTGCTTTAGCGCCACCTGCGC accBC F44 AGCAGGCGTGAGCGGCGATGCGGTCGCAGCTCC accBC R45 CTTAATAACCGTGCCTTGCATCGGAGCTGCGACCGCATC accBC F45 GATGCAAGGCACGGTTATTAAGGTCAATGTTGAAGAAGGTGCAG accBC R46 GGTGTCACCTTCGTTGACTTCTGCACCTTCTTCAACATTGAC accBC F46 AAGTCAACGAAGGTGACACCGTTGTGGTTCTGGAGGCA accBC R47 TCACCGGATTCTCCATTTTCATTGCCTCCAGAACCACAAC accBC F47 ATGAAAATGGAGAATCCGGTGAAGGCCCACAAAAGCGG accBC R48 TCAGACCCGTCACGGTGCCGCTTTTGTGGGCCT accBC F48 CACCGTGACGGGTCTGACGGTTGCGGCGGGTGA accBC R49 AGAACGACACCTTTATTAACACCCTCACCCGCCGCAACCG accBC F49 GGGTGTTAATAAAGGTGTCGTTCTGCTGGAGATTAAGTAAgccgaaccBC R50 cccatggggctgaggctcggcTTACTTAATCTCCAGC accBC F50 gcctcagccccatggg

117

Table A1.3. Oligonucleotides used for the synthesis of the accD4 gene. Name Sequence AccD4 R1 GTCATtcccgcctttccag AccD4 F1 ctggaaaggcgggaATGACGGTTACGGAGCCGGTT AccD4 R2 CGCCGTCGTGTGCAGAACCGGCTCCGTAACC AccD4 F2 CTGCACACGACGGCGGAAAAGCTGGCTGAGCTG AccD4 R3 TTCCAGGCGCTCACGCAGCTCAGCCAGCTTTTC AccD4 F3 CGTGAGCGCCTGGAACTGGCTAAAGAGCCGGG AccD4 R4 CGCAGCTTTCTCGCCACCCGGCTCTTTAGCCAG AccD4 F4 TGGCGAGAAAGCTGCGGCGAAGCGCGATAAGAAA AccD4 R5 GCGCGGACGGGATACCTTTCTTATCGCGCTTCGC AccD4 F5 GGTATCCCGTCCGCGCGTGCACGTATCTATGAGCTGG AccD4 R6 CATAAAGCTACCCGGATCGACCAGCTCATAGATACGTGCAC AccD4 F6 TCGATCCGGGTAGCTTTATGGAGATTGGTGCGCTGTG AccD4 R7 TCACCCGGCGTGCGGCACAGCGCACCAATCTC AccD4 F7 CCGCACGCCGGGTGACCCGAACGCCTTGTACG AccD4 R8 TCACCACGCCGTCACCGTACAAGGCGTTCGGG AccD4 F8 GTGACGGCGTGGTGACCGGCCACGGTCTGA AccD4 R9 CCACCGGACGACCATTAATCAGACCGTGGCCGG AccD4 F9 TTAATGGTCGTCCGGTGGGCGTTTTTAGCCATGACCA AccD4 R10 GCCACCGAACACGGTCTGGTCATGGCTAAAAACGC AccD4 F10 GACCGTGTTCGGTGGCACGGTGGGTGAAATGTTTG AccD4 R11 CAGACGTGCAACTTTACGACCAAACATTTCACCCACCGT AccD4 F11 GTCGTAAAGTTGCACGTCTGATGGAGTGGTGTGCGAT AccD4 R12 AATCGGACAGCCGACCATCGCACACCACTCCAT AccD4 F12 GGTCGGCTGTCCGATTGTCGGCATCAACGACTCT AccD4 R13 TGAATACGGGCACCACCAGAGTCGTTGATGCCGAC AccD4 F13 GGTGGTGCCCGTATTCAGGATGCAGTGACCAGCT AccD4 R14 CGGCGTACCATGCCAAGCTGGTCACTGCATCC AccD4 F14 TGGCATGGTACGCCGAGCTGGGCCGTCGTC AccD4 R15 CCGCTCAGCAGTTCGTGACGACGGCCCAGCT AccD4 F15 ACGAACTGCTGAGCGGCCTGGTCCCGCAAATCT AccD4 R16 CACACTTACCCAGAATGATGGAGATTTGCGGGACCAGG AccD4 F16 CCATCATTCTGGGTAAGTGTGCAGGTGGCGCGGTTTA AccD4 R17 CGGTTTGGATCGGGCTGTAAACCGCGCCACCTG AccD4 F17 CAGCCCGATCCAAACCGATCTGGTCGTTGCCGT AccD4 R18 ATAGCCCTGATCGCGGACGGCAACGACCAGAT AccD4 F18 CCGCGATCAGGGCTATATGTTCGTCACCGGCC AccD4 R19 GTAACATCCTTGATAACGTCCGGGCCGGTGACGAACAT AccD4 F19 CGGACGTTATCAAGGATGTTACGGGCGAGGACGTTAGC AccD4 R20 CCACCCAGCTCATCCAGGCTAACGTCCTCGCCC AccD4 F20 CTGGATGAGCTGGGTGGTGCCGACCACCAGGC AccD4 R21 CTGATGAATGTTACCGTAGCTGGCCTGGTGGTCGGCA AccD4 F21 CAGCTACGGTAACATTCATCAGGTGGTCGAAAGCGAAGC AccD4 R22 ACATACTGGTAGGCTGCCGCTTCGCTTTCGACCAC AccD4 F22 GGCAGCCTACCAGTATGTGCGCGACTTCCTGAGC AccD4 R23 AGCAATTAGACGGCAGGAAGCTCAGGAAGTCGCGC AccD4 F23 TTCCTGCCGTCTAATTGCTTCGATAAACCGCCGGTG AccD4 R24 CCAGGCCCGGATTGACCACCGGCGGTTTATCGA AccD4 F24 GTCAATCCGGGCCTGGAGCCGGAAATTACGGGT AccD4 R25 TGTCCAGCTCCAAATCGTGACCCGTAATTTCCGGCT AccD4 F25 CACGATTTGGAGCTGGACAGCATCGTGCCGGATAGC AccD4 R26 TGCATGTCATACGCCATGTTATCGCTATCCGGCACGATGC AccD4 F26 GATAACATGGCGTATGACATGCACGAGGTTCTGCTGCGT AccD4 R27 AGAAAATCACCATCGTCAAAGATACGCAGCAGAACCTCG AccD4 F27 ATCTTTGACGATGGTGATTTTCTGGATGTCGCGGCACA AccD4 R28 GATTGCTTGGCCTGCCTGTGCCGCGACATCC AccD4 F28 GGCAGGCCAAGCAATCATTACGGGTTACGCCCG AccD4 R29 CCGTACGACCATCGACACGGGCGTAACCCGTAAT AccD4 F29 TGTCGATGGTCGTACGGTTGGCGTTGTTGCGAAT AccD4 R30 GCTCATGTGCATCGGTTGATTCGCAACAACGCCAA AccD4 F30 CAACCGATGCACATGAGCGGCGCGATTGACAATGAG AccD4 R31 AGCCTTGTCGCTCGCCTCATTGTCAATCGCGCC

118

AccD4 F31 GCGAGCGACAAGGCTGCACGTTTCATTCGCTTCA AccD4 R32 GGAATATCGAACGCATCGCTGAAGCGAATGAAACGTGC AccD4 F32 GCGATGCGTTCGATATTCCGTTGGTGTTCGTCGTCGA AccD4 R33 CAGGAAACCCGGCGTATCGACGACGAACACCAAC AccD4 F33 TACGCCGGGTTTCCTGCCGGGCGTCGAGCAAG AccD4 R34 GCGCTTGATGATACCATTCTTTTCTTGCTCGACGCCCGG AccD4 F34 AAAAGAATGGTATCATCAAGCGCGGCGGTCGCTTCCTG AccD4 R35 GCTTCCACGACCGCATACAGGAAGCGACCGCC AccD4 F35 TATGCGGTCGTGGAAGCTGATGTGCCGAAAGTTACGA AccD4 R36 CCATAGCTCTTGCGAATCGTAATCGTAACTTTCGGCACATCA AccD4 F36 TTACGATTCGCAAGAGCTATGGCGGCGCGTACGCTGT AccD4 R37 GTTGCTTGCTGCCCATCACAGCGTACGCGCCG AccD4 F37 GATGGGCAGCAAGCAACTGACCGCCGATCTGAAT AccD4 R38 CGGTCGGCCACGCGAAATTCAGATCGGCGGTCA AccD4 F38 TTCGCGTGGCCGACCGCACGCATCGCAGTGATTG AccD4 R39 GGCAGCGCCATCTGCACCAATCACTGCGATGCGTG AccD4 F39 GTGCAGATGGCGCTGCCCAATTGCTGATGAAACGCTTCC AccD4 R40 GGGGCATTCGGGTCCGGGAAGCGTTTCATCAGCAATTG AccD4 F40 CGGACCCGAATGCCCCGGAGGCGCAGGCGATC AccD4 R41 AATTCTCAACGAAGGACTTACGGATCGCCTGCGCCTCC AccD4 F41 CGTAAGTCCTTCGTTGAGAATTACAATCTGAATATGGCTATTCCGAccD4 R42 GTTCCGCTGCGATCCACGGAATAGCCATATTCAGATTGT AccD4 F42 TGGATCGCAGCGGAACGCGGTTTTATCGATGCCG AccD4 R43 GTTTCATGCGGGTCAATGACGGCATCGATAAAACCGC AccD4 F43 TCATTGACCCGCATGAAACGCGCTTGCTGCTGCG AccD4 R44 GCAGCAGGTGCATAGATTTGCGCAGCAGCAAGCGC AccD4 F44 CAAATCTATGCACCTGCTGCGCGACAAACAACTGTGGT AccD4 R45 TACGACCGACACGCCACCACAGTTGTTTGTCGC AccD4 F45 GGCGTGTCGGTCGTAAACATGGTCTGATCCCGG AccD4 R46 ccgcgtaatggggTTAGACCGGGATCAGACCATGTT AccD4 F46 TCTAAccccattacgcgg

119

Table A1.4. Oligonucleotides used for the synthesis of the accD5 gene. Name Sequence AccD5 R1 GGTCATgatcgactgtggga AccD5 F1 tcccacagtcgatcATGACCAGCGTGACCGATCGTAGC AccD5 R2 ACGTTCAGCAGAGTGAGCGCTACGATCGGTCACGCT AccD5 F2 GCTCACTCTGCTGAACGTTCTACCGAACACACCATTGA AccD5 R3 CGCGGTGGTGTGGATGTCAATGGTGTGTTCGGTAGA AccD5 F3 CATCCACACCACCGCGGGTAAGCTGGCGGAGC AccD5 R4 TCTCTTCACGACGTTTATGCAGCTCCGCCAGCTTACC AccD5 F4 TGCATAAACGTCGTGAAGAGAGCCTGCATCCGGTCGG AccD5 R5 CCTTCTCAACGGCATCTTCACCGACCGGATGCAGGC AccD5 F5 TGAAGATGCCGTTGAGAAGGTCCACGCGAAAGGTAAGC AccD5 R6 CGCTCACGAGCCGTCAGCTTACCTTTCGCGTGGA AccD5 F6 TGACGGCTCGTGAGCGTATCTACGCGCTGTTGGAC AccD5 R7 CAGTTCAACGAAGCTGTCTTCGTCCAACAGCGCGTAGATA AccD5 F7 GAAGACAGCTTCGTTGAACTGGACGCGCTGGCGAAAC AccD5 R8 AGATTGAAGTTCGTGCTACGATGTTTCGCCAGCGCGTC AccD5 F8 ATCGTAGCACGAACTTCAATCTGGGTGAGAAACGTCCGCT AccD5 R9 TGACCACACCGTCGCCCAGCGGACGTTTCTCACCC AccD5 F9 GGGCGACGGTGTGGTCACGGGCTACGGCACCATC AccD5 R10 CACACGTCACGACCGTCGATGGTGCCGTAGCCCG AccD5 F10 GACGGTCGTGACGTGTGCATCTTTTCCCAAGACGCCA AccD5 R11 GCTGCCACCAAACACGGTGGCGTCTTGGGAAAAGATG AccD5 F11 CCGTGTTTGGTGGCAGCCTGGGTGAAGTTTATGGTGAAAA AccD5 R12 AACTCTTGGACCTTGACGATCTTTTCACCATAAACTTCACCCAG AccD5 F12 GATCGTCAAGGTCCAAGAGTTGGCAATTAAAACGGGTCGTCC AccD5 R13 CCATCGTTGATACCGATCAGCGGACGACCCGTTTTAATTGCC AccD5 F13 GCTGATCGGTATCAACGATGGCGCTGGTGCTCGTATTCA AccD5 R14 CAGGCTAACCACACCTTCTTGAATACGAGCACCAGCG AccD5 F14 AGAAGGTGTGGTTAGCCTGGGCCTGTACAGCCGTATC AccD5 R15 GCTCGCCAGAATATTATTACGAAAGATACGGCTGTACAGGCC AccD5 F15 TTTCGTAATAATATTCTGGCGAGCGGTGTTATTCCGCAAATCTCTCAccD5 R16 GCCGCGCCCATAATCAGAGAGATTTGCGGAATAACACC AccD5 F16 TGATTATGGGCGCGGCTGCCGGTGGTCACGTC AccD5 R17 GGTCAGTGCCGGGCTATAGACGTGACCACCGGCA AccD5 F17 TATAGCCCGGCACTGACCGACTTCGTTATCATGGTTGACC AccD5 R18 GGTAATAAACATCTGGGAGGTCTGGTCAACCATGATAACGAAGTC AccD5 F18 AGACCTCCCAGATGTTTATTACCGGCCCGGATGTTATCAAGA AccD5 R19 TCCTCACCCGTCACCGTCTTGATAACATCCGGGCC AccD5 F19 CGGTGACGGGTGAGGAGGTCACGATGGAAGAGCT AccD5 R20 CGTATGCGCACCGCCCAGCTCTTCCATCGTGACC AccD5 F20 GGGCGGTGCGCATACGCACATGGCGAAGTCCGG AccD5 R21 GCTGCGTAATGTGCCGTGCCGGACTTCGCCATGTG AccD5 F21 CACGGCACATTACGCAGCAAGCGGTGAGCAAGATGC AccD5 R22 AGTTCACGGACGTAATCGAAGGCATCTTGCTCACCGCTT AccD5 F22 CTTCGATTACGTCCGTGAACTGCTGAGCTACTTGCCGC AccD5 R23 GGCATCCGTGGAATTATTCGGCGGCAAGTAGCTCAGC AccD5 F23 CGAATAATTCCACGGATGCCCCGCGCTATCAGGCAG AccD5 R24 GGACCCGTCGGAGCCGCTGCCTGATAGCGCGG AccD5 F24 CGGCTCCGACGGGTCCGATTGAAGAGAATCTGACGGACG AccD5 R25 GCGTATCCAGTTCCAGATCCTCGTCCGTCAGATTCTCTTCAATC AccD5 F25 AGGATCTGGAACTGGATACGCTGATCCCGGATAGCCCG AccD5 R26 GCATGTCGTACGGCTGATTCGGGCTATCCGGGATCA AccD5 F26 AATCAGCCGTACGACATGCACGAGGTTATTACCCGTTTGC AccD5 R27 TCCAGGAACTCATCGTCCAGCAAACGGGTAATAACCTCGT AccD5 F27 TGGACGATGAGTTCCTGGAGATTCAAGCCGGTTACGCA AccD5 R28 AGCCCACCACAATGTTCTGTGCGTAACCGGCTTGAATC AccD5 F28 CAGAACATTGTGGTGGGCTTTGGCCGCATCGATGG AccD5 R29 AATGCCCACCGGGCGGCCATCGATGCGGCCAA AccD5 F29 CCGCCCGGTGGGCATTGTCGCCAACCAACCGA AccD5 R30 GACAGCCTGCAAAATGGGTCGGTTGGTTGGCGAC AccD5 F30 CCCATTTTGCAGGCTGTCTGGACATTAACGCCAGCG AccD5 R31 CGAAACGTGCGGCTTTTTCGCTGGCGTTAATGTCCA

120

AccD5 F31 AAAAAGCCGCACGTTTCGTTCGTACGTGTGACTGCTT AccD5 R32 CAGCATAACGATCGGGATATTGAAGCAGTCACACGTACGAA AccD5 F32 CAATATCCCGATCGTTATGCTGGTGGATGTTCCGGGTTTCC AccD5 R33 TGATCCGTACCCGGCAGGAAACCCGGAACATCCAC AccD5 F33 TGCCGGGTACGGATCAAGAGTATAATGGCATTATCCGCC AccD5 R34 CAGCAATTTCGCACCGCGGCGGATAATGCCATTATACTCT AccD5 F34 GCGGTGCGAAATTGCTGTATGCCTATGGTGAGGCAAC AccD5 R35 GATGACCGTAATTTTCGGAACCGTTGCCTCACCATAGGCATA AccD5 F35 GGTTCCGAAAATTACGGTCATCACCCGTAAAGCGTATGGTG AccD5 R36 CCCATAACGCAGTACGCGCCACCATACGCTTTACGGGT AccD5 F36 GCGCGTACTGCGTTATGGGCAGCAAGGACATGGGTTGC AccD5 R37 CCAGGCCAGGTTAACGTCGCAACCCATGTCCTTGCTG AccD5 F37 GACGTTAACCTGGCCTGGCCGACGGCGCAAATCGC AccD5 R38 CGCTGGCACCCATCACCGCGATTTGCGCCGTCGG AccD5 F38 GGTGATGGGTGCCAGCGGCGCGGTGGGCTTCGT AccD5 R39 CGGCCAGTTGTTGACGATAGACGAAGCCCACCGCGC AccD5 F39 CTATCGTCAACAACTGGCCGAAGCAGCCGCTAACGG AccD5 R40 AGACGCAACTTATCAATATCCTCACCGTTAGCGGCTGCTT AccD5 F40 TGAGGATATTGATAAGTTGCGTCTGCGCCTGCAACAGGAG AccD5 R41 CGGATTGACCAAGGTATCTTCATACTCCTGTTGCAGGCGC AccD5 F41 TATGAAGATACCTTGGTCAATCCGTACGTTGCGGCGGAAC AccD5 R42 GGCGTCAACATAGCCGCGTTCCGCCGCAACGTA AccD5 F42 GCGGCTATGTTGACGCCGTGATCCCGCCGAGCC AccD5 R43 GCCAATGTAACCACGGGTATGGCTCGGCGGGATCAC AccD5 F43 ATACCCGTGGTTACATTGGCACCGCGTTGCGCCTGT AccD5 R44 TGCGCAATCTTACGTTCCAACAGGCGCAACGCGGT AccD5 F44 TGGAACGTAAGATTGCGCAACTGCCGCCGAAAAAGC AccD5 R45 CAGCGGGACGTTACCATGCTTTTTCGGCGGCAGT AccD5 F45 ATGGTAACGTCCCGCTGTAAagggtaacgtctacgaggta AccD5 R46 tacctcgtagacgttaccctTTA

121

Table A1.5. Oligonucleotides used for the synthesis of the accD6 gene. Name Sequence AccD6 R1 TTCCGGTGCCATAATCGTCATc AccD6 F1 gATGACGATTATGGCACCGGAAGCAGTCGGCGAAAGCC AccD6 R2 GGGTCGCGCGGGTCCAGGCTTTCGCCGACTGC AccD6 F2 TGGACCCGCGCGACCCGCTGCTGCGTCTGAGCAA AccD6 R3 TCCACGCTACCATCATCAAAAAAATTGCTCAGACGCAGCAGC AccD6 F3 TTTTTTTGATGATGGTAGCGTGGAGTTGCTGCACGAACGTGA AccD6 R4 CAGGACGCCGCTACGGTCACGTTCGTGCAGCAAC AccD6 F4 CCGTAGCGGCGTCCTGGCGGCAGCAGGCACCG AccD6 R5 GGTGCGCACACCGTTGACGGTGCCTGCTGCCGC AccD6 F5 TCAACGGTGTGCGCACCATCGCGTTTTGCACGGATG AccD6 R6 CACCACCCATAACGGTGCCATCCGTGCAAAACGCGAT AccD6 F6 GCACCGTTATGGGTGGTGCAATGGGCGTTGAAGGCT AccD6 R7 CGTAGGCATTAACAATGTGGGTACAGCCTTCAACGCCCATTG AccD6 F7 GTACCCACATTGTTAATGCCTACGATACGGCGATTGAAGACCAAAG AccD6 R8 CCAGATGCCAACAATCGGGCTTTGGTCTTCAATCGCCGTAT AccD6 F8 CCCGATTGTTGGCATCTGGCACTCTGGCGGTGCACG AccD6 R9 CGAACACCTTCCGCCAGACGTGCACCGCCAGAGTG AccD6 F9 TCTGGCGGAAGGTGTTCGCGCTTTGCACGCTGTGG AccD6 R10 CGCCTCGAACACCTGGCCCACAGCGTGCAAAGCG AccD6 F10 GCCAGGTGTTCGAGGCGATGATCCGCGCTTCTGGC AccD6 R11 CACAACGGAAATCTGCGGAATATAGCCAGAAGCGCGGATCAT AccD6 F11 TATATTCCGCAGATTTCCGTTGTGGTGGGTTTCGCGGCGG AccD6 R12 CCGTAGGCCGCACCACCCGCCGCGAAACCCAC AccD6 F12 GTGGTGCGGCCTACGGTCCGGCCTTGACCGAC AccD6 R13 CGGGGCCATAACCACAACGTCGGTCAAGGCCGGA AccD6 F13 GTTGTGGTTATGGCCCCGGAAAGCCGCGTTTTTGTCACC AccD6 R14 GAACCACGTCCGGGCCGGTGACAAAAACGCGGCTTTC AccD6 F14 GGCCCGGACGTGGTTCGTAGCGTCACGGGTGAGG AccD6 R15 AGGCTCGCCATATCAACGTCCTCACCCGTGACGCTAC AccD6 F15 ACGTTGATATGGCGAGCCTGGGCGGTCCGGAAACC AccD6 R16 CAAACGCCGCTCTTTTTATGATGGGTTTCCGGACCGCCC AccD6 F16 CATCATAAAAAGAGCGGCGTTTGTCACATCGTGGCGGATGA AccD6 R17 GGTCGTATGCGTCCAGTTCGTCATCCGCCACGATGTGA AccD6 F17 CGAACTGGACGCATACGACCGTGGCCGTCGCCTGGT AccD6 R18 TGCTGGCAGAACAGACCCACCAGGCGACGGCCAC AccD6 F18 GGGTCTGTTCTGCCAGCAGGGTCACTTTGACCGCAGC AccD6 R19 CGCCCGCCTCCGCCTTGCTGCGGTCAAAGTGACCC AccD6 F19 AAGGCGGAGGCGGGCGACACCGACATCCACGCG AccD6 R20 GAGCTTTCCGGCAGCAGCGCGTGGATGTCGGTGT AccD6 F20 CTGCTGCCGGAAAGCTCCCGTCGTGCGTACGACG AccD6 R21 CCGTAACGATCGGGCGAACGTCGTACGCACGACGG AccD6 F21 TTCGCCCGATCGTTACGGCAATCCTGGATGCAGATACGC AccD6 R22 TGGCTTGAAATTCGTCGAACGGCGTATCTGCATCCAGGATTG AccD6 F22 CGTTCGACGAATTTCAAGCCAACTGGGCACCGAGCAT AccD6 R23 CGACCCAAACCGACAACCATGCTCGGTGCCCAGT AccD6 F23 GGTTGTCGGTTTGGGTCGTCTGAGCGGTCGTACCG AccD6 R24 GGATTATTCGCCAGGACACCAACGGTACGACCGCTCAGA AccD6 F24 TTGGTGTCCTGGCGAATAATCCGTTGCGTTTGGGCGGCT AccD6 R25 CGGCGCTCTCGCTATTCAGACAGCCGCCCAAACGCAAC AccD6 F25 GTCTGAATAGCGAGAGCGCCGAGAAAGCTGCTCGCTTTGTCC AccD6 R26 CAAAAGCGTCGCACAGGCGGACAAAGCGAGCAGCTTTCT AccD6 F26 GCCTGTGCGACGCTTTTGGTATCCCGCTGGTCGTTGTG AccD6 R27 GGTAGCCCGGAACATCGACCACAACGACCAGCGGGATAC AccD6 F27 GTCGATGTTCCGGGCTACCTGCCGGGTGTGGACCA AccD6 R28 CACCACACCACCCCATTCCTGGTCCACACCCGGCA AccD6 F28 GGAATGGGGTGGTGTGGTGCGCCGTGGTGCGAAATT AccD6 R29 TCGCCGAATGCGTGCAACAATTTCGCACCACGGCG AccD6 F29 GTTGCACGCATTCGGCGAGTGCACCGTTCCGCGCG AccD6 R30 CTTACGGGTAACCAGGGTCACGCGCGGAACGGTGCAC AccD6 F30 TGACCCTGGTTACCCGTAAGACCTACGGTGGCGCATAT AccD6 R31 GAGAACGGCTATTCATTGCGATATATGCGCCACCGTAGGT

122

AccD6 F31 ATCGCAATGAATAGCCGTTCTCTGAACGCGACGAAGGTCT AccD6 R32 GCATCCGGCCAGGCAAAGACCTTCGTCGCGTTCA AccD6 F32 TTGCCTGGCCGGATGCGGAGGTGGCCGTCATGG AccD6 R33 ACCGCCGCTTTGGCACCCATGACGGCCACCTCC AccD6 F33 GTGCCAAAGCGGCGGTCGGCATTCTGCACAAGAAGA AccD6 R34 GGTGCGGCTGCCAGCTTCTTCTTGTGCAGAATGCCG AccD6 F34 AGCTGGCAGCCGCACCGGAGCATGAACGTGAGGCT AccD6 R35 CGCCAGTTGATCGTGCAGAGCCTCACGTTCATGCTCC AccD6 F35 CTGCACGATCAACTGGCGGCGGAACATGAACGCATCG AccD6 R36 CAGAATCAACACCGCCCGCGATGCGTTCATGTTCCGC AccD6 F36 CGGGCGGTGTTGATTCTGCTCTGGATATTGGTGTGGTTGATG AccD6 R37 GGGTATGAGCCGGATCAATTTTTTCATCAACCACACCAATATCCAGAGAccD6 F37 AAAAAATTGATCCGGCTCATACCCGTAGCAAGCTGACCGAAGC AccD6 R38 CGGCGCTTGAGCCAGGGCTTCGGTCAGCTTGCTAC AccD6 F38 CCTGGCTCAAGCGCCGGCTCGTCGTGGTCGTCATA AccD6 R39 cggTTACAGCGGGATGTTTTTATGACGACCACGACGAGC AccD6 F39 AAAACATCCCGCTGTAAccgagggggtggtgtagccg AccD6 R40 cggctacaccaccccct

123

Table A1.6. Oligonucleotides used for the synthesis of the accE gene. Name Sequence AccE R1 CCCATcccgcgacttcctt AccE F1 aaggaagtcgcgggATGGGTACGTGTCCGTGCGAGA AccE R2 CTCGTTACGCTCGCTGCTCTCGCACGGACACGTA AccE F2 GCAGCGAGCGTAACGAGCCGGTGAGCCGCGTTA AccE R3 ACTTCGTTGGTGCCGCTAACGCGGCTCACCGG AccE F3 GCGGCACCAACGAAGTCAGCGACGGCAACGAG AccE R4 ACTTCAGCCGGATTGTTCGTCTCGTTGCCGTCGCTG AccE F4 ACGAACAATCCGGCTGAAGTCTCCGACGGTAACGAAACCAA AccE R5 GAAACTTCCGCCGGGTTGTTGGTTTCGTTACCGTCGGAG AccE F5 CAACCCGGCGGAAGTTTCTGACGGTAATGAAACCAATAACCC AccE R6 ACGGGAGACCGGGGCCGGGTTATTGGTTTCATTACCGTCA AccE F6 GGCCCCGGTCTCCCGTGTGTCTGGCACGAATGAAGT AccE R7 TGGTCTCGTTACCATCGCTAACTTCATTCGTGCCAGACAC AccE F7 TAGCGATGGTAACGAGACCAACAATCCGGCACCGGT AccE R8 TACCGCTCACACGGCTGACCGGTGCCGGATTGT AccE F8 CAGCCGTGTGAGCGGTACGAATGAGGTGTCCGACGG AccE R9 AGCCGGATTATTGGTCTCATTGCCGTCGGACACCTCATTCG AccE F9 CAATGAGACCAATAATCCGGCTCCGGTTACGGAAAAGCCG AccE R10 GGTTCGTGCGGATGCAGCGGCTTTTCCGTAACCGG AccE F10 CTGCATCCGCACGAACCGCACATTGAGATCCTGCGT AccE R11 CTGATCGGTCGGTTGGCCACGCAGGATCTCAATGTGC AccE F11 GGCCAACCGACCGATCAGGAGTTGGCCGCACTGATC AccE R12 CGCTGATGCTACCCAAAACAGCGATCAGTGCGGCCAACTC AccE F12 GCTGTTTTGGGTAGCATCAGCGGTTCTACCCCGCCGGCG AccE R13 GGGTCGGCTCCGGCTGCGCCGGCGGGGTAGAAC AccE F13 CAGCCGGAGCCGACCCGTTGGGGCCTGCCGGTC AccE R14 CCGGGTAGCGCAGTTGATCGACCGGCAGGCCCCAAC AccE F14 GATCAACTGCGCTACCCGGTGTTCTCTTGGCAGCGTATTAC AccE R15 GCATGTGGGTCATTTCTTGCAAGGTAATACGCTGCCAAGAGAACAAccE F15 CTTGCAAGAAATGACCCACATGCGCCGCTAAcccctcgc AccE R16 gcgaggggTTAGCGGC

124

Table A1.7. Oligonucleotides used for the synthesis of the tbACS4 gene. Name Sequence TbACS4 R2 AGCCACCCATgcgttca TbACS4 F2 tgaacgcATGGGTGGCTGCGTAATCTCTGTGATGGACTA TbACS4 R3 TCTACTTCGGAACGGTTATTCATATAGTCCATCACAGAGATTACGC TbACS4 F3 TATGAATAACCGTTCCGAAGTAGAAAACGAACATGTTAAAAAGTTCCGT TbACS4 R4 GCCACTTTGCCCAGCGCACGGAACTTTTTAACATGTTCGTTT TbACS4 F4 GCGCTGGGCAAAGTGGCTGTTCCGGTTCCTGGTTC TbACS4 R5 GGAGAGCAATCAGAGGTTTCAGAACCAGGAACCGGAACA TbACS4 F5 TGAAACCTCTGATTGCTCTCCAATCTACCGCCTGGTTACTG TbACS4 R6 TCGATGTCCTTACCGTCGTCAGTAACCAGGCGGTAGATT TbACS4 F6 ACGACGGTAAGGACATCGAGGAAGTTCGCCGTGAATGG TbACS4 R7 GCAGACATTCGCCTTCGTACCATTCACGGCGAACTTCC TbACS4 F7 TACGAAGGCGAATGTCTGCCGCAGCGTTTTGCGGCA TbACS4 R8 TGGCTGACGTTTGCACAGTGCCGCAAAACGCTGCG TbACS4 F8 CTGTGCAAACGTCAGCCAAAACAACGTGCACTGGCG TbACS4 R9 GCGGTCCACTGGACGATACGCCAGTGCACGTTGTTT TbACS4 F9 TATCGTCCAGTGGACCGCGTTGAGAAAGCGGTGATCAAG TbACS4 R10 CCGTACCGGTATGCAGATCCTTGATCACCGCTTTCTCAAC TbACS4 F10 GATCTGCATACCGGTACGGAAAAGATGATGAATGTTACGCACT TbACS4 R11 TAGTCCAGATATTTGGTTTCCTTGAAGTGCGTAACATTCATCATCTTTT TbACS4 F11 TCAAGGAAACCAAATATCTGGACTACGGTACGTTCTGGGACTATATC TbACS4 R12 ACCACGACCGAAGGATTCGATATAGTCCCAGAACGTACCG TbACS4 F12 GAATCCTTCGGTCGTGGTCTGGTAGAGCTGGGTATTTCTC TbACS4 R13 GGCGACACGGGAAGACGGAGAAATACCCAGCTCTACCAG TbACS4 F13 CGTCTTCCCGTGTCGCCATTTACGAAGAAACTCGTTGGGA TbACS4 R14 CGTAGATGGTAGCCAGCCATTCCCAACGAGTTTCTTCGTAAAT TbACS4 F14 ATGGCTGGCTACCATCTACGGTATCTGGAGCCAGAATATGGT TbACS4 R15 TGCATAAACAGTGGTCGCAACCATATTCTGGCTCCAGATAC TbACS4 F15 TGCGACCACTGTTTATGCAAACCTGGGCGAGGACGC TbACS4 R16 CGCAGAGCATAAGCCAGCGCGTCCTCGCCCAGGTT TbACS4 F16 GCTGGCTTATGCTCTGCGTGAAACGGGCTGCAAAGG TbACS4 R17 ACGTTTTTAGCGTTGCAGATAATACCTTTGCAGCCCGTTTCA TbACS4 F17 TATTATCTGCAACGCTAAAAACGTGTCCGTGGTTATCAAATTCATGT TbACS4 R18 TCGGGGTAATGCCTTCAGACATGAATTTGATAACCACGGAC TbACS4 F18 CTGAAGGCATTACCCCGAGCGCTCCGATCATTTATAACG TbACS4 R19 AGAAGCCGGCAGGCTACCGTTATAAATGATCGGAGCGC TbACS4 F19 GTAGCCTGCCGGCTTCTGTTGACCAGGAAGCCTGT TbACS4 R20 TCTTCCCAAGAAACCAGGTGACAGGCTTCCTGGTCAAC TbACS4 F20 CACCTGGTTTCTTGGGAAGAAGTGGTGAAGCTGGGCC TbACS4 R21 ACGGTCGCGTGCTTCACGGCCCAGCTTCACCACT TbACS4 F21 GTGAAGCACGCGACCGTCTGCCGCTGAACAATTCTG TbACS4 R22 CCAGATCGTCTGCACGACCAGAATTGTTCAGCGGCAG TbACS4 F22 GTCGTGCAGACGATCTGGCACTGATTATGTATACGAGCGGC TbACS4 R23 TTCGGATCGCCAGTGGTGCCGCTCGTATACATAATCAGTG TbACS4 F23 ACCACTGGCGATCCGAAGGGTGTTATCCACACTCATGG TbACS4 R24 CGCCGGACATCAGAGAGCCATGAGTGTGGATAACACCC TbACS4 F24 CTCTCTGATGTCCGGCGTGCATGCTCTGGACCATC TbACS4 R25 CCATCACCGCATTCAGGCGATGGTCCAGAGCATGCA TbACS4 F25 GCCTGAATGCGGTGATGGGTCCTCTGCGCGACGGC TbACS4 R26 CAGGTAGCTCAGGTAAGTCTCGCCGTCGCGCAGAGGAC TbACS4 F26 GAGACTTACCTGAGCTACCTGCCACTGGCTCACATCCTG TbACS4 R27 GGACAGGACACCCAGTTCCAGGATGTGAGCCAGTGG TbACS4 F27 GAACTGGGTGTCCTGTCCGTTTTCATTGCGCGTGGT TbACS4 R28 GCCAAAGCAGATCAGTGCACCACGCGCAATGAAAAC TbACS4 F28 GCACTGATCTGCTTTGGCTCTCCGTTCACCCTGACC TbACS4 R29 GGGCGAGCAGTCAGGTCGGTCAGGGTGAACGGAGA TbACS4 F29 GACCTGACTGCTCGCCCTCGCGGTGACCTGGCTG TbACS4 R30 CAGCAGAGACGGGTTGTATTCAGCCAGGTCACCGCGA TbACS4 F30 AATACAACCCGTCTCTGCTGATTGGTGTTCCGCGTATTTATG TbACS4 R31 TGGATTGCCTTTTTCAGGGTATCATAAATACGCGGAACACCAAT TbACS4 F31 ATACCCTGAAAAAGGCAATCCAGGCGAAACTGCCTGCGC TbACS4 R32 ACGACGTTTAAAGGTGCCAGGCGCAGGCAGTTTCGCC

125

TbACS4 F32 CTGGCACCTTTAAACGTCGTGCTTTCGACCACGCTTTTC TbACS4 R33 CGCACGCAGACGAGACTGAAAAGCGTGGTCGAAAGC TbACS4 F33 AGTCTCGTCTGCGTGCGTTCAAAGATGGTAAAGACTCTCCG TbACS4 R34 GACTTTCGCGTCCCAGTACGGAGAGTCTTTACCATCTTTGAA TbACS4 F34 TACTGGGACGCGAAAGTCTTTGCGGCGACCCGTGC TbACS4 R35 ATACATGTTTTTGCCCAGTACCGCACGGGTCGCCGCAAA TbACS4 F35 GGTACTGGGCAAAAACATGTATATGGTGCTGTCCGGCGG TbACS4 R36 CAGTAGACAGCGGGCCACCGCCGGACAGCACCAT TbACS4 F36 TGGCCCGCTGTCTACTGCCACCCAGGATTTCCTGAA TbACS4 R37 TACGAACCACGGCAACGTTCAGGAAATCCTGGGTGG TbACS4 F37 CGTTGCCGTGGTTCGTATTATCCAAGGTTGGGGTCTG TbACS4 R38 TACGCAAACGGTTTCGGTCAGACCCCAACCTTGGATAA TbACS4 F38 ACCGAAACCGTTTGCGTAGGCGGCGTTCAGCTGAC TbACS4 R39 CGCCAGTTTCAATATCACCGGTCAGCTGAACGCCGCC TbACS4 F39 CGGTGATATTGAAACTGGCGCTGTTGGTCCGCCTCTG TbACS4 R40 GCAGTTTTACCTCTTCGCTCAGCAGAGGCGGACCAACAG TbACS4 F40 CTGAGCGAAGAGGTAAAACTGCTGGATGTGGAAGGCTACAAG TbACS4 R41 TCCGGTTCGTCAGTGTGCTTGTAGCCTTCCACATCCA TbACS4 F41 CACACTGACGAACCGGACCCGCGTGGCGAGATCC TbACS4 R42 GAACGGGCCACGCAGCAGGATCTCGCCACGCGGG TbACS4 F42 TGCTGCGTGGCCCGTTCCTGTTCAAGGGTTACTACAAACAGG TbACS4 R43 TCGATAGCTTCTTTAGTCAGTTCTTCCTGTTTGTAGTAACCCTTGAACAGTbACS4 F43 AAGAACTGACTAAAGAAGCTATCGACGAAGACGGTTGGTTTCACA TbACS4 R44 CCAATAGAACCTACGTCACCAGTGTGAAACCAACCGTCTTCG TbACS4 F44 CTGGTGACGTAGGTTCTATTGGTCCGAACGGCACCCTGC TbACS4 R45 GCTTTTACACGGCCGATAATACGCAGGGTGCCGTTCGGA TbACS4 F45 GTATTATCGGCCGTGTAAAAGCACTGGCTAAAAATGTCCTGGG TbACS4 R46 TCTCCATAGCCACGTATTCGCCCAGGACATTTTTAGCCAGT TbACS4 F46 CGAATACGTGGCTATGGAGACTCTGGAATCTATGTACGCTCA TbACS4 R47 TTGGCATGCTCAGAGAGTTATGAGCGTACATAGATTCCAGAG TbACS4 F47 TAACTCTCTGAGCATGCCAAACGGCGTGTGCGTCCTG TbACS4 R48 GGGCGATCCGGATGGACCAGGACGCACACGCCGT TbACS4 F48 GTCCATCCGGATCGCCCTTACATCTGTGCCCTGGTG TbACS4 R49 CACTTTTGCTTCATCGGTCAGCACCAGGGCACAGATGTAA TbACS4 F49 CTGACCGATGAAGCAAAAGTGGTGGCATTTACTCGTGAGCA TbACS4 R50 GGGTACTTACCTTTCAGACCATGCTCACGAGTAAATGCCAC TbACS4 F50 TGGTCTGAAAGGTAAGTACCCGGAAGTACTGCAGGACCC TbACS4 R51 GCAGTAGCTTTTTTTTGGAACTCCGGGTCCTGCAGTACTTCC TbACS4 F51 GGAGTTCCAAAAAAAAGCTACTGCGTCCTTCCAGGAAACTGCG TbACS4 R52 TGACGATCAGAAGCGCGCGCAGTTTCCTGGAAGGAC TbACS4 F52 CGCGCTTCTGATCGTCAGAAATTCGAAATCGTGCGTCA TbACS4 R53 CGGACAGCAGACGAACGTGACGCACGATTTCGAATTTC TbACS4 F53 CGTTCGTCTGCTGTCCGACGAGTGGACGCCGGAA TbACS4 R54 GCGGTCAGAACACCGTTTTCCGGCGTCCACTCGT TbACS4 F54 AACGGTGTTCTGACCGCTGCGGGCAAACTGAAACG TbACS4 R55 TACTTCTCGTCGATAACACGGCGTTTCAGTTTGCCCGCA TbACS4 F55 CCGTGTTATCGACGAGAAGTACACCGACACCATTGTGTCT TbACS4 R56 gccTTAGCATTCTTCTACGAACAGAGACACAATGGTGTCGGTG TbACS4 F56 CTGTTCGTAGAAGAATGCTAAggcggcatgctgtttcactcatatt TbACS4 R57 aatatgagtgaaacagcatgcc

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Table A1.8. Oligonucleotides used for the synthesis of the aldh593 gene. Name Sequence sALDH593 R2 TATCTCtcgctctattgtcg sALDH593 F2 cgacaatagagcgaGAGATATACATATGAACAAAGATACCCT sALDH593 R3 TCTTTCGTAGTCGGGATCAGGGTATCTTTGTTCATATGTA sALDH593 F3 GATCCCGACTACGAAAGACCTGAAACTGAAAACCAAC sALDH593 R4 TCAGGTTGATGTTTTCCACGTTGGTTTTCAGTTTCAGG sALDH593 F4 GTGGAAAACATCAACCTGAAAAATTACAAAGACAATTCCTCT sALDH593 R5 TCAAAAACGCCGAAACAAGAGGAATTGTCTTTGTAATTTT sALDH593 F5 TGTTTCGGCGTTTTTGAGAACGTTGAGAACGCA sALDH593 R6 AACGGCGCTGTTGATTGCGTTCTCAACGTTC sALDH593 F6 ATCAACAGCGCCGTTCATGCGCAGAAAATTCTG sALDH593 R7 CTTTGGTATAATGCAGGGACAGAATTTTCTGCGCATG sALDH593 F7 TCCCTGCATTATACCAAAGAACAGCGTGAGAAAATCA sALDH593 R8 GCTTTACGGATTTCGGTAATGATTTTCTCACGCTGTT sALDH593 F8 TTACCGAAATCCGTAAAGCTGCTCTGGAAAACAAAGA sALDH593 R9 CATGGTCGCCAGTACTTCTTTGTTTTCCAGAGCA sALDH593 F9 AGTACTGGCGACCATGATCCTGGAAGAAACCCA sALDH593 R10 TCGTAGCGACCCATGTGGGTTTCTTCCAGGAT sALDH593 F10 CATGGGTCGCTACGAGGATAAGATTCTGAAACACG sALDH593 R11 GGTATATTTGGCAACCAGTTCGTGTTTCAGAATCTTATCC sALDH593 F11 AACTGGTTGCCAAATATACCCCTGGTACTGAAGACCT sALDH593 R12 TCCAAGCCGTGGTAGTCAGGTCTTCAGTACCAGG sALDH593 F12 GACTACCACGGCTTGGAGCGGCGACAACGGTC sALDH593 R13 ACATCTCTACTACGGTCAGACCGTTGTCGCCGC sALDH593 F13 TGACCGTAGTAGAGATGTCTCCGTACGGCGTTA sALDH593 R14 GGAGTGATCGCACCAATAACGCCGTACGGAG sALDH593 F14 TTGGTGCGATCACTCCGAGCACCAACCCAAC sALDH593 R15 TTGCAGATAACAGTCTCCGTTGGGTTGGTGCTC sALDH593 F15 GGAGACTGTTATCTGCAATTCCATCGGTATGATTGCA sALDH593 R16 CAACAGCATTGCCCGCTGCAATCATACCGATGGAA sALDH593 F16 GCGGGCAATGCTGTTGTGTTTAACGGTCACCCTG sALDH593 R17 ACACATTTCTTCGCGCCAGGGTGACCGTTAAACA sALDH593 F17 GCGCGAAGAAATGTGTTGCTTTCGCAATTGAAATG sALDH593 R18 CAGCTGATGATAGCTTTATTGATCATTTCAATTGCGAAAGCA sALDH593 F18 ATCAATAAAGCTATCATCAGCTGCGGTGGCCCAGAGAAC sALDH593 R19 GTTTTTGATAGTCGTTACCAGGTTCTCTGGGCCACCG sALDH593 F19 CTGGTAACGACTATCAAAAACCCGACGATGGAAAGC sALDH593 R20 TTGATGATGGCGTCCAGGCTTTCCATCGTCGG sALDH593 F20 CTGGACGCCATCATCAAACACCCGCTGATTAAACT sALDH593 R21 CCAGTGCCGCACAGCAGTTTAATCAGCGGGTGT sALDH593 F21 GCTGTGCGGCACTGGCGGCCCAGGTATGGT sALDH593 R22 CTGTTCAGCAGGGTTTTCACCATACCTGGGCCG sALDH593 F22 GAAAACCCTGCTGAACAGCGGTAAAAAGGCAATTGGC sALDH593 R23 GTTGCCAGCACCCGCGCCAATTGCCTTTTTACCG sALDH593 F23 GCGGGTGCTGGCAACCCGCCGGTTATTGTTG sALDH593 R24 CAATATCGGCGGTATCATCAACAATAACCGGCGG sALDH593 F24 ATGATACCGCCGATATTGAAAAAGCTGGTAAGAGCA sALDH593 R25 CAAAAGAACAGCCTTCAATAATGCTCTTACCAGCTTTTT sALDH593 F25 TTATTGAAGGCTGTTCTTTTGATAATAACCTGCCGTGC sALDH593 R26 CGAATACTTCTTTTTCAGCAATGCACGGCAGGTTATTAT sALDH593 F26 ATTGCTGAAAAAGAAGTATTCGTCTTCGAAAACGTCGC sALDH593 R27 GTTGCTGATCAGATCATCAGCGACGTTTTCGAAGA sALDH593 F27 TGATGATCTGATCAGCAACATGCTGAAAAACAACGC sALDH593 R28 TGGTCTTCGTTGATGATAACAGCGTTGTTTTTCAGCAT sALDH593 F28 TGTTATCATCAACGAAGACCAGGTATCTAAACTGATTGATCTGGsALDH593 R29 TTCGTTGTTTTTCTGCAGTACCAGATCAATCAGTTTAGATACC sALDH593 F29 TACTGCAGAAAAACAACGAAACTCAAGAATACTTCATCAACAA sALDH593 R30 TTTGCCCACCCATTTCTTGTTGATGAAGTATTCTTGAGT sALDH593 F30 GAAATGGGTGGGCAAAGACGCTAAACTGTTCTCCG sALDH593 R31 GATTCTACGTCGATTTCGTCGGAGAACAGTTTAGCGTC sALDH593 F31 ACGAAATCGACGTAGAATCCCCGTCCAACATCAAA sALDH593 R32 TAACTTCGCACACGATACATTTGATGTTGGACGGG

127

sALDH593 F32 TGTATCGTGTGCGAAGTTAATGCAAACCACCCATTC sALDH593 R33 TCATCAGCTCGGTCATAACGAATGGGTGGTTTGCAT sALDH593 F33 GTTATGACCGAGCTGATGATGCCGATTCTGCCAAT sALDH593 R34 ATGTCTTTAACACGGACGATTGGCAGAATCGGCA sALDH593 F34 CGTCCGTGTTAAAGACATCGACGAAGCGGTTAAATA sALDH593 R35 TGTTCGGCAATTTTGGTGTATTTAACCGCTTCGTCG sALDH593 F35 CACCAAAATTGCCGAACAGAACCGCAAACACTCTG sALDH593 R36 AATGTTCTTGGAATAGATGTATGCAGAGTGTTTGCGGTTC sALDH593 F36 CATACATCTATTCCAAGAACATTGACAACCTGAACCGTTT sALDH593 R37 TGTCAATTTCGCGCTCAAAACGGTTCAGGTTGTC sALDH593 F37 TGAGCGCGAAATTGACACTACCATCTTCGTCAAAAACG sALDH593 R38 CCTGCGAAGGATTTGGCGTTTTTGACGAAGATGGTAG sALDH593 F38 CCAAATCCTTCGCAGGTGTCGGTTATGAGGCTG sALDH593 R39 TGAAAGTGGTAAAGCCCTCAGCCTCATAACCGACA sALDH593 F39 AGGGCTTTACCACTTTCACTATCGCTGGCAGCAC sALDH593 R40 GTGATGCCCTCGCCGGTGCTGCCAGCGATAG sALDH593 F40 CGGCGAGGGCATCACCTCTGCGCGCAACTT sALDH593 R41 CGACGTTGACGGGTGAAGTTGCGCGCAGAG sALDH593 F41 CACCCGTCAACGTCGTTGCGTACTGGCCGG sALDH593 R42 tgcgtaatATCTCTAGATCAGCCGGCCAGTACGCAA sALDH593 F42 CTGATCTAGAGATattacgca

128

Table A1.9. Oligonucleotides used for the synthesis of the alkK gene.

Name Sequence sAlkKR1 AGCATgtggcgtaacgcta sAlkKF1 tagcgttacgccacATGCTGGGTCAGATGATGCGTAATCA sAlkKR2 CCAGGCTACCGATAACCAGTTGATTACGCATCATCTGACCC sAlkKF2 ACTGGTTATCGGTAGCCTGGTGGAGCACGCCGCTCG sAlkKR3 ACGGGCACCGTGGTAACGAGCGGCGTGCTCCA sAlkKF3 TTACCACGGTGCCCGTGAGGTGGTGTCTGTTGAAACG sAlkKR4 GGGTGACCTCACCGCTCGTTTCAACAGACACCACCTC sAlkKF4 AGCGGTGAGGTCACCCGTTCTTGCTGGAAAGAAGTTGAA sAlkKR5 AGTTTACGCGCACGCAGTTCAACTTCTTTCCAGCAAGAAC sAlkKF5 CTGCGTGCGCGTAAACTGGCGAGCGCATTGGGTA sAlkKR6 TCGGGGTCAAACCCATTTTACCCAATGCGCTCGCC sAlkKF6 AAATGGGTTTGACCCCGAGCGACCGCTGTGCGAC sAlkKR7 GCGGATATTATTCCAGGCAATGGTCGCACAGCGGTCGC sAlkKF7 CATTGCCTGGAATAATATCCGCCACCTGGAGGTCTATTACGCG sAlkKR8 ATGCCTGCGCCGCTAACCGCGTAATAGACCTCCAGGTG sAlkKF8 GTTAGCGGCGCAGGCATGGTTTGCCACACGATCAAC sAlkKR9 CTGTTCGATAAACAGACGCGGGTTGATCGTGTGGCAAACC sAlkKF9 CCGCGTCTGTTTATCGAACAGATTACGTATGTCATTAATCACGCG sAlkKR10 CCAGCAAAACCACCTTATCTTCCGCGTGATTAATGACATACGTAAT sAlkKF10 GAAGATAAGGTGGTTTTGCTGGACGACACCTTCCTGCCG sAlkKR11 GCTACCATGAATCTCTGCGATAATCGGCAGGAAGGTGTCGT sAlkKF11 ATTATCGCAGAGATTCATGGTAGCCTGCCGAAAGTGAAGGCC sAlkKR12 GTTATTGTGTGCCATCAGAACAAAGGCCTTCACTTTCGGCAG sAlkKF12 TTTGTTCTGATGGCACACAATAACTCCAATGCGAGCGCCC sAlkKR13 CGATCAGACCCGGCATTTGGGCGCTCGCATTGGA sAlkKF13 AAATGCCGGGTCTGATCGCCTACGAGGATCTGATTGGTCA sAlkKR14 GGCCAGATATAGTTATCATCACCTTGACCAATCAGATCCTCGTAGG sAlkKF14 AGGTGATGATAACTATATCTGGCCGGATGTCGATGAGAACGAGG sAlkKR15 TGTAGCACAGGCTGCTTGCCTCGTTCTCATCGACATCC sAlkKF15 CAAGCAGCCTGTGCTACACCAGCGGCACCACGGG sAlkKR16 AGTACAAAACACCTTTCGGGTTACCCGTGGTGCCGCTGG sAlkKF16 TAACCCGAAAGGTGTTTTGTACTCCCATCGTAGCACCGTG sAlkKR17 CGTGGTCATGGAGTGCAACACGGTGCTACGATGGG sAlkKF17 TTGCACTCCATGACCACGGCGATGCCGGACACCC sAlkKR18 CGTGCGCTCAGGTTCAGGGTGTCCGGCATCGC sAlkKF18 TGAACCTGAGCGCACGTGACACGATCCTGCCGGT sAlkKR19 GCATTAACATGAAACATCGGGACGACCGGCAGGATCGTGTCA sAlkKF19 CGTCCCGATGTTTCATGTTAATGCCTGGGGCACCCCGTATT sAlkKR20 ACCAACCATCGCGGCGGAATACGGGGTGCCCCAG sAlkKF20 CCGCCGCGATGGTTGGTGCCAAGCTGGTGCTGCC sAlkKR21 GTCCAGCGCCGGACCCGGCAGCACCAGCTTGGC sAlkKF21 GGGTCCGGCGCTGGACGGTGCAAGCCTGAGCA sAlkKR22 GCCTTCGCTTGCAATCAGTTTGCTCAGGCTTGCACC sAlkKF22 AACTGATTGCAAGCGAAGGCGTCAGCATCGCTCTGGG sAlkKR23 CCACACCACCGGAACGCCCAGAGCGATGCTGAC sAlkKF23 CGTTCCGGTGGTGTGGCAGGGCCTGCTGGCGG sAlkKR24 GCTACCATTACCAGCTTGCGCCGCCAGCAGGCCCTG sAlkKF24 CGCAAGCTGGTAATGGTAGCAAGTCCCAGAGCCTGACC sAlkKR25 CCACCCACAACAACGCGGGTCAGGCTCTGGGACTT sAlkKF25 CGCGTTGTTGTGGGTGGCTCCGCATGTCCGGCG sAlkKR26 TCATTGAACTCACGAATCATGGACGCCGGACATGCGGAG sAlkKF26 TCCATGATTCGTGAGTTCAATGACATCTATGGTGTTGAGGTCATCC sAlkKR27 CGGTCATACCCCACGCATGGATGACCTCAACACCATAGATG sAlkKF27 ATGCGTGGGGTATGACCGAGCTGAGCCCGTTCGG sAlkKR28 CGGGGTATTCGCGGTGCCGAACGGGCTCAGCT sAlkKF28 CACCGCGAATACCCCGCTGGCGCACCACGTTG sAlkKR29 TTCGTCCGGGCTCAGATCAACGTGGTGCGCCAG sAlkKF29 ATCTGAGCCCGGACGAAAAACTGAGCCTGCGTAAAAGCC sAlkKR30 TACGGCGGACGGCCCTGGCTTTTACGCAGGCTCAGTTT sAlkKF30 AGGGCCGTCCGCCGTACGGTGTTGAGCTGAAAATCG

129

sAlkKR31 CGGATGCCCTCATCATTCACGATTTTCAGCTCAACACCG sAlkKF31 TGAATGATGAGGGCATCCGTCTGCCGGAGGACGGT sAlkKR32 CATCAAGTTACCTTTGCTGCGACCGTCCTCCGGCAGA sAlkKF32 CGCAGCAAAGGTAACTTGATGGCGCGTGGTCACTGGG sAlkKR33 GCTGTGGAAGTAGTCCTTAATCACCCAGTGACCACGCGC sAlkKF33 TGATTAAGGACTACTTCCACAGCGATCCGGGCAGCACCC sAlkKR34 AACCAGCCGTCGGACAGGGTGCTGCCCGGATC sAlkKF34 TGTCCGACGGCTGGTTTAGCACCGGCGATGTCG sAlkKR35 CGTCGCTATCGATGGTGGCGACATCGCCGGTGCTA sAlkKF35 CCACCATCGATAGCGACGGTTTTATGACCATTTGCGATCGT sAlkKR36 GCTCTTGATGATGTCCTTCGCACGATCGCAAATGGTCATAAAAC sAlkKF36 GCGAAGGACATCATCAAGAGCGGTGGTGAGTGGATCAGC sAlkKR37 TCTCCAGCTCGACGGTGCTGATCCACTCACCACC sAlkKF37 ACCGTCGAGCTGGAGAGCATCGCCATTGCGCA sAlkKR38 ATCGACGATGTGCGGGTGCGCAATGGCGATGC sAlkKF38 CCCGCACATCGTCGATGCTGCGGTGATTGCGG sAlkKR39 TCCCATTTCTCGTGACGTGCCGCAATCACCGCAGC sAlkKF39 CACGTCACGAGAAATGGGACGAACGTCCGCTGCTG sAlkKR40 GGGCTTTTCACCGCGATCAGCAGCGGACGTTCG sAlkKF40 ATCGCGGTGAAAAGCCCGAACTCTGAGCTGACCAGC sAlkKR41 GCAAAGTAGTTACAGACTTCGCCGCTGGTCAGCTCAGAGTTC sAlkKF41 GGCGAAGTCTGTAACTACTTTGCGGACAAAGTCGCCCGT sAlkKR42 GCGTCCGGGATTTGCCAACGGGCGACTTTGTCC sAlkKF42 TGGCAAATCCCGGACGCTGCAATTTTCGTGGAGGAATTGC sAlkKR43 TACCCGTACCGTTACGCGGCAATTCCTCCACGAAAATTGCA sAlkKF43 CGCGTAACGGTACGGGTAAGATTTTGAAGAATCGTCTGCGTG sAlkKR44 GCAGCAGAATATCACCGTATTTTTCACGCAGACGATTCTTCAAAATCTsAlkKF44 AAAAATACGGTGATATTCTGCTGCGTTCTAGCAGCAGCGTCT sAlkKR45 cgacaccacgttaataTTATTCGCAGACGCTGCTGCTAGAAC sAlkKF45 GCGAATAAtattaacgtggtgtcg

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Table A1.10. Oligonucleotides used for the synthesis of the atfA gene. Name Sequence AtfA R2 CGCATgaattgctgggaag AtfA F2 cttcccagcaattcATGCGCCCACTGCACCCGATT AtfA R3 TCCAGAGACAGGAAAATAAAGTCAATCGGGTGCAGTGGG AtfA F3 GACTTTATTTTCCTGTCTCTGGAAAAACGTCAGCAGCCAA AtfA R4 AGGCCACCAACGTGCATTGGCTGCTGACGTTTT AtfA F4 TGCACGTTGGTGGCCTGTTTCTGTTCCAGATCCCG AtfA R5 AGGTGTCTGGAGCGTTATCCGGGATCTGGAACAGAAAC AtfA F5 GATAACGCTCCAGACACCTTCATCCAGGATCTGGTAAACG AtfA R6 ATAGATTTGCTGATACGGATGTCGTTTACCAGATCCTGGATGAAtfA F6 ACATCCGTATCAGCAAATCTATCCCGGTCCCGCCATTTA AtfA R7 AGACCGTTCAGTTTGTTGTTAAATGGCGGGACCGGG AtfA F7 ACAACAAACTGAACGGTCTGTTCTGGGACGAAGATGAG AtfA R8 TGGTGATCCAGATCGAACTCCTCATCTTCGTCCCAGAAC AtfA F8 GAGTTCGATCTGGATCACCACTTCCGTCACATTGCGCT AtfA R9 TACGACCCGGATGCGGCAGCGCAATGTGACGGAAG AtfA F9 GCCGCATCCGGGTCGTATTCGTGAGCTGCTGATCTA AtfA R10 AGTAGAGTGTTCCTGAGAAATGTAGATCAGCAGCTCACGAA AtfA F10 CATTTCTCAGGAACACTCTACTCTGCTGGACCGTGCGA AtfA R11 GCAGGTCCACAGTGGTTTCGCACGGTCCAGCAG AtfA F11 AACCACTGTGGACCTGCAACATTATTGAGGGCATCGA AtfA R12 CGCGAAACGGTTGCCTTCGATGCCCTCAATAATGTT AtfA F12 AGGCAACCGTTTCGCGATGTATTTCAAAATCCACCACGC AtfA R13 AGCAACACCATCGACCATCGCGTGGTGGATTTTGAAATACAT AtfA F13 GATGGTCGATGGTGTTGCTGGTATGCGTCTGATTGAAAAGT AtfA R14 TACGTCGTGGCTCAGGGACTTTTCAATCAGACGCATACC AtfA F14 CCCTGAGCCACGACGTAACCGAGAAATCCATCGTGCC AtfA R15 TTCCACGCACCACGGCGGCACGATGGATTTCTCGGT AtfA F15 GCCGTGGTGCGTGGAAGGCAAACGTGCTAAACGT AtfA R16 GGTCTTTGGTTCACGCAGACGTTTAGCACGTTTGCC AtfA F16 CTGCGTGAACCAAAGACCGGCAAGATCAAGAAAATCATGT AtfA R17 GCTGAGATTTGATGCCGGACATGATTTTCTTGATCTTGCC AtfA F17 CCGGCATCAAATCTCAGCTGCAGGCAACCCCGAC AtfA R18 TGAGACAGTTCCTGAATAACGGTCGGGGTTGCCTGCA AtfA F18 CGTTATTCAGGAACTGTCTCAGACGGTATTCAAAGACATCGG AtfA R19 GTGGTCCGGGTTACGACCGATGTCTTTGAATACCGTC AtfA F19 TCGTAACCCGGACCACGTAAGCAGCTTCCAGGC AtfA R20 TCAGGATGGAGCATGGCGCCTGGAAGCTGCTTAC AtfA F20 GCCATGCTCCATCCTGAACCAGCGTGTCTCCTC AtfA R21 GGCGAAGCGGCGAGAAGAGGAGACACGCTGGT AtfA F21 TTCTCGCCGCTTCGCCGCTCAGAGCTTCGACCT AtfA R22 GATGTTACGGAAGCGATCCAGGTCGAAGCTCTGAGC AtfA F22 GGATCGCTTCCGTAACATCGCTAAATCCCTGAATGTCACG AtfA R23 CCAGTACAACGTCGTTGATCGTGACATTCAGGGATTTAGC AtfA F23 ATCAACGACGTTGTACTGGCAGTTTGCAGCGGTGC AtfA R24 CAGGTAAGCACGCAGCGCACCGCTGCAAACTG AtfA F24 GCTGCGTGCTTACCTGATGAGCCATAACTCTCTGCC AtfA R25 GCGATCAGCGGTTTAGACGGCAGAGAGTTATGGCTCAT AtfA F25 GTCTAAACCGCTGATCGCTATGGTGCCGGCGAGC AtfA R26 GCTGTCGTCGTTACGAATGCTCGCCGGCACCATA AtfA F26 ATTCGTAACGACGACAGCGACGTCTCTAACCGTATCACC AtfA R27 CCAGGTTAGCCAGGATCATGGTGATACGGTTAGAGACGTC AtfA F27 ATGATCCTGGCTAACCTGGCTACCCACAAGGACGATCC AtfA R28 TTCCAGACGCTGCAGTGGATCGTCCTTGTGGGTAG AtfA F28 ACTGCAGCGTCTGGAAATCATCCGTCGTTCCGTA AtfA R29 GAAACGCTGTTTGCTATTCTGTACGGAACGACGGATGAT AtfA F29 CAGAATAGCAAACAGCGTTTCAAGCGCATGACGTCCG AtfA R30 GCGGAATAGTTCAGAATCTGATCGGACGTCATGCGCTT AtfA F30 ATCAGATTCTGAACTATTCCGCCGTTGTTTACGGTCCAGC AtfA R31 GAGATGATGTTCAGGCCCGCTGGACCGTAAACAACG AtfA F31 GGGCCTGAACATCATCTCCGGTATGATGCCTAAACGC AtfA R32 GAAATGACCAGATTAAATGCCTGGCGTTTAGGCATCATACCG

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AtfA F32 CAGGCATTTAATCTGGTCATTTCCAATGTTCCGGGTCCTCG AtfA R33 CGTTCCAATACAGAGGTTCACGAGGACCCGGAACATTG AtfA F33 TGAACCTCTGTATTGGAACGGCGCTAAACTGGACGC AtfA R34 GCTCGCCGGGTACAGCGCGTCCAGTTTAGCGC AtfA F34 GCTGTACCCGGCGAGCATCGTTCTGGACGGTCA AtfA R35 CTAGTCATAGTAATGTTCAGTGCTTGACCGTCCAGAACGAT AtfA F35 AGCACTGAACATTACTATGACTAGCTACCTGGACAAGCTGGA AtfA R36 CAGGCGATCAGACCAACTTCCAGCTTGTCCAGGTAG AtfA F36 AGTTGGTCTGATCGCCTGCCGCAATGCTCTGCCGC AtfA R37 GGTCAGCAGGTTCTGCATACGCGGCAGAGCATTGCGG AtfA F37 GTATGCAGAACCTGCTGACCCACCTGGAAGAGGAGATTCA AtfA R38 GATAACGCCCTCAAACAGTTGAATCTCCTCTTCCAGGTG AtfA F38 ACTGTTTGAGGGCGTTATCGCCAAACAGGAAGACATTAAAAC AtfA R39 cgacccttTTAGTTCGCGGTTTTAATGTCTTCCTGTTTGGC AtfA R40 CGCGAACTAAaagggtcg

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Table A1.11. Oligonucleotides used for the synthesis of the bdhA gene. Name Sequence bdhA R2 TCTCCTTCTTGGTACCAAGC bdhA F2 GCTTGGTACCAAGAAGGAGATATCATATGCTGTCCTTCGA bdhA R3 TACTTTGGTCGGGATAGAATAATCGAAGGACAGCATATGATA bdhA F3 TTATTCTATCCCGACCAAAGTATTCTTCGGTAAAGGCAAAAT bdhA R4 TCTTCGCCAATTACGTCGATTTTGCCTTTACCGAAGAA bdhA F4 CGACGTAATTGGCGAAGAGATCAAGAAATATGGTTCCCG bdhA R5 ACCATACACGATCAGAACGCGGGAACCATATTTCTTGATC bdhA F5 CGTTCTGATCGTGTATGGTGGCGGCAGCATTAAAC bdhA R6 GCGATCATAAATGCCGTTACGTTTAATGCTGCCGCC bdhA F6 GTAACGGCATTTATGATCGCGCTACGGCCATCCTGA bdhA R7 AACGCGATGTTGTTTTCTTTCAGGATGGCCGTAGC bdhA F7 AAGAAAACAACATCGCGTTCTACGAGCTGTCTGGC bdhA R8 CGAGGGTTAGGTTCGACGCCAGACAGCTCGTAG bdhA F8 GTCGAACCTAACCCTCGTATCACTACCGTGAAAAAGGG bdhA R9 GTTCTCACGACAGATTTCAATACCCTTTTTCACGGTAGTGATA bdhA F9 TATTGAAATCTGTCGTGAGAACAATGTCGATCTGGTTCTGG bdhA R10 GAACCGCCACCGATAGCCAGAACCAGATCGACATT bdhA F10 CTATCGGTGGCGGTTCTGCAATCGACTGCTCC bdhA R11 CCGCAGCGATGACCTTGGAGCAGTCGATTGCA bdhA F11 AAGGTCATCGCTGCGGGCGTATATTATGACGGCGA bdhA R12 TCACCATGTCCCAGGTATCGCCGTCATAATATACGC bdhA F12 TACCTGGGACATGGTGAAAGACCCGTCTAAGATCAC bdhA R13 GCAATCGGCAGAACTTTGGTGATCTTAGACGGGTCTT bdhA F13 CAAAGTTCTGCCGATTGCTTCCATCCTGACTCTGAGC bdhA R14 TTTCAGAGCCCGTAGCGCTCAGAGTCAGGATGGAA bdhA F14 GCTACGGGCTCTGAAATGGATCAGATCGCGGT bdhA R15 CGTTGGTTTCCATATTGCTAATAACCGCGATCTGATCCA bdhA F15 TATTAGCAATATGGAAACCAACGAAAAACTGGGCGTCGG bdhA R16 ACGCATGTCGTCATGACCGACGCCCAGTTTTT bdhA F16 TCATGACGACATGCGTCCGAAATTTTCTGTACTGGAC bdhA R17 CGGTGAACGTATAGGTAGGGTCCAGTACAGAAAATTTCGG bdhA F17 CCTACCTATACGTTCACCGTACCGAAAAATCAGACCGCA bdhA R18 TATCTGCGGTGCCCGCTGCGGTCTGATTTTTCGGTA bdhA F18 GCGGGCACCGCAGATATTATGAGCCACACCTTCG bdhA R19 CGCCGCTAAAGTAGGATTCGAAGGTGTGGCTCATAA bdhA F19 AATCCTACTTTAGCGGCGTAGAAGGTGCCTACGTACA bdhA R20 TTCGGCGATACCATCCTGTACGTAGGCACCTTCTA bdhA F20 GGATGGTATCGCCGAAGCCATCCTGCGTACCT bdhA R21 TAGCGATTTTACCGTATTTGATACAGGTACGCAGGATGGC bdhA F21 GTATCAAATACGGTAAAATCGCTATGGAGAAAACTGATGATTATGAAbdhA R22 TCAGGTTCGCACGGGCTTCATAATCATCAGTTTTCTCCA bdhA F22 GCCCGTGCGAACCTGATGTGGGCCTCTTCTCT bdhA R23 AGCAGACCATTGATAGCCAGAGAAGAGGCCCACA bdhA F23 GGCTATCAATGGTCTGCTGAGCCTGGGTAAAGATCG bdhA R24 GGTGGCAAGACCATTTGCGATCTTTACCCAGGCTC bdhA F24 CAAATGGTCTTGCCACCCGATGGAACATGAACTGAG bdhA R25 GGTGATGTCGTAATAAGCGCTCAGTTCATGTTCCATCG bdhA F25 CGCTTATTACGACATCACCCACGGCGTCGGTCTGG bdhA R26 TTCGGGGTCAGGATCGCCAGACCGACGCCGTG bdhA F26 CGATCCTGACCCCGAATTGGATGGAGTACATTCTGA bdhA R27 TGCAGGGTGTCGTCATTCAGAATGTACTCCATCCAA bdhA F27 ATGACGACACCCTGCACAAGTTCGTGTCTTACGGTA bdhA R28 CGATACCCCAGACGTTAATACCGTAAGACACGAACTTG bdhA F28 TTAACGTCTGGGGTATCGACAAAAACAAAGACAACTACGA bdhA R29 TGGCTTCACGTGCAATTTCGTAGTTGTCTTTGTTTTTGT bdhA F29 AATTGCACGTGAAGCCATCAAAAACACTCGCGAGTA bdhA R30 TACCCAGGCTGTTGAAGTACTCGCGAGTGTTTTTGA bdhA F30 CTTCAACAGCCTGGGTATCCCTTCTAAACTGCGTG bdhA R31 TTTACCGATGCCCACTTCACGCAGTTTAGAAGGGA bdhA F31 AAGTGGGCATCGGTAAAGATAAACTGGAACTGATGGC bdhA R32 TGCGAACCGCCTGTTTAGCCATCAGTTCCAGTTTATC

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bdhA F32 TAAACAGGCGGTTCGCAACTCTGGTGGCACCATC bdhA R33 TTGGGCGCAGAGAGCCGATGGTGCCACCAGAGT bdhA F33 GGCTCTCTGCGCCCAATCAACGCCGAGGACGT bdhA R34 GCTTTTCTTGAAAATTTCCAGAACGTCCTCGGCGTTGA bdhA F34 TCTGGAAATTTTCAAGAAAAGCTATTAAAAGCTTATCcgcaacc bdhA R35 gagctaggcaccagcgggttgcgGATAAGCTTTTAATA bdhA F35 cgctggtgcctagctc

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Table A1.12. Oligonucleotides used for the synthesis of the bdhB gene. Name Sequence sBdhB R2 CTCcgccctacggcaat sBdhB F2 attgccgtagggcgGAGCTCGGCGCGCCGAATT sBdhB R3 GGTATATCTCCTTGGATCCGAATTCGGCGCGCCGAG sBdhB F3 CGGATCCAAGGAGATATACCATGGTGGACTTCGAGTATTC sBdhB R4 AAAAGATACGGGTTGGGATAGAATACTCGAAGTCCACCAT sBdhB F4 TATCCCAACCCGTATCTTTTTCGGTAAAGATAAAATCAATGTGC sBdhB R5 TTCAGCTCACGACCCAGCACATTGATTTTATCTTTACCGA sBdhB F5 TGGGTCGTGAGCTGAAGAAATACGGCTCTAAAGTTCTG sBdhB R6 CCGCCACCATAAACGATCAGAACTTTAGAGCCGTATTTC sBdhB F6 ATCGTTTATGGTGGCGGTAGCATCAAACGTAACGGTA sBdhB R7 ACCGCCTTGTCGTAGATACCGTTACGTTTGATGCTA sBdhB F7 TCTACGACAAGGCGGTGTCTATTCTGGAAAAGAACTCC sBdhB R8 CAGCCAGTTCGTAGAATTTAATGGAGTTCTTTTCCAGAATAGAC sBdhB F8 ATTAAATTCTACGAACTGGCTGGCGTCGAACCAAACCC sBdhB R9 AACGGTCGTCACACGCGGGTTTGGTTCGACGC sBdhB F9 GCGTGTGACGACCGTTGAAAAAGGCGTAAAAATCTGCC sBdhB R10 TTCTACGCCGTTCTCACGGCAGATTTTTACGCCTTTTTC sBdhB F10 GTGAGAACGGCGTAGAAGTGGTGCTGGCCATCGG sBdhB R11 CGATTGCAGAGCCACCACCGATGGCCAGCACCAC sBdhB F11 TGGTGGCTCTGCAATCGACTGCGCGAAAGTGATT sBdhB R12 TATTCGCACGCTGCCGCAATCACTTTCGCGCAGT sBdhB F12 GCGGCAGCGTGCGAATATGACGGCAACCCGTGG sBdhB R13 GCCGTCCAGCACAATGTCCCACGGGTTGCCGTCA sBdhB F13 GACATTGTGCTGGACGGCAGCAAAATTAAGCGCGTAC sBdhB R14 AATGGAAGCAATCGGCAGTACGCGCTTAATTTTGCT sBdhB F14 TGCCGATTGCTTCCATTCTGACTATTGCTGCCACGG sBdhB R15 CCAGGTATCCATTTCGCTACCCGTGGCAGCAATAGTCAG sBdhB F15 GTAGCGAAATGGATACCTGGGCAGTAATTAACAACATGGACAC sBdhB R16 CCGCGATCAGCTTTTCGTTAGTGTCCATGTTGTTAATTACTGC sBdhB F16 TAACGAAAAGCTGATCGCGGCCCACCCGGACATGGC sBdhB R17 GGGTCCAGAATAGAGAATTTTGGTGCCATGTCCGGGTGGG sBdhB F17 ACCAAAATTCTCTATTCTGGACCCGACCTACACCTACACTGTG sBdhB R18 GCAGCAGTCTGATTAGTCGGCACAGTGTAGGTGTAGGTC sBdhB F18 CCGACTAATCAGACTGCTGCTGGCACTGCGGATATTATG sBdhB R19 TAGACTTCGAAGATGTGGGACATAATATCCGCAGTGCCA sBdhB F19 TCCCACATCTTCGAAGTCTATTTTTCTAATACCAAAACTGCGTA sBdhB R20 CATGCGGTCTTGCAGATACGCAGTTTTGGTATTAGAAAAA sBdhB F20 TCTGCAAGACCGCATGGCGGAAGCTCTGCTGC sBdhB R21 GCCGTATTTAATGCAGGTACGCAGCAGAGCTTCCGC sBdhB F21 GTACCTGCATTAAATACGGCGGTATTGCGCTGGAAAAA sBdhB R22 CCTCGTAGTCGTCCGGTTTTTCCAGCGCAATACC sBdhB F22 CCGGACGACTACGAGGCTCGCGCTAACCTGATG sBdhB R23 GCCAGAGAAGATGCCCACATCAGGTTAGCGCGAG sBdhB F23 TGGGCATCTTCTCTGGCTATCAACGGTCTGCTGAC sBdhB R24 GTTCGTGTCCTTACCGTAGGTCAGCAGACCGTTGATA sBdhB F24 CTACGGTAAGGACACGAACTGGAGCGTCCACCTGA sBdhB R25 CAGACAGTTCATGCTCCATCAGGTGGACGCTCCA sBdhB F25 TGGAGCATGAACTGTCTGCATATTACGACATCACTCACGGT sBdhB R26 GTCAGAATTGCCAGACCTACACCGTGAGTGATGTCGTAATATG sBdhB F26 GTAGGTCTGGCAATTCTGACGCCAAACTGGATGGAATATATC sBdhB R27 TTGTAAACAGTATCGTTGTTCAGGATATATTCCATCCAGTTTGGC sBdhB F27 CTGAACAACGATACTGTTTACAAGTTCGTTGAGTATGGTGTTAAC sBdhB R28 TTTGTCGATACCCCACACGTTAACACCATACTCAACGAAC sBdhB F28 GTGTGGGGTATCGACAAAGAGAAAAACCACTACGACATC sBdhB R29 TGGATCGCCTGGTGTGCGATGTCGTAGTGGTTTTTCTC sBdhB F29 GCACACCAGGCGATCCAGAAGACCCGTGATTACTTCGT sBdhB R30 GGCAGACCCAGAACGTTCACGAAGTAATCACGGGTCTTC sBdhB F30 GAACGTTCTGGGTCTGCCGTCCCGCCTGCGTGAC sBdhB R31 AGTTTTTCTTCTTCGATACCAACGTCACGCAGGCGGGAC sBdhB F31 GTTGGTATCGAAGAAGAAAAACTGGATATTATGGCAAAAGAAAGCGsBdhB R32 CCGCCGGTCAGCTTGACGCTTTCTTTTGCCATAATATCC

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sBdhB F32 TCAAGCTGACCGGCGGCACCATCGGCAACCTG sBdhB R33 AAGCGTTAACCGGGCGCAGGTTGCCGATGGTG sBdhB F33 CGCCCGGTTAACGCTTCCGAGGTACTGCAAATTTTC sBdhB R34 CGGTACCTTAAACGGATTTCTTGAAAATTTGCAGTACCTCGG sBdhB F34 AAGAAATCCGTTTAAGGTACCGAATTCAAGCTTaaatccagtaagasBdhB R35 tcttactggatttAAGCTTGAATT

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Table A1.13. Oligonucleotides used for the synthesis of the ccr gene from Streptomyces cinnamonensis. Name Sequence CCR (S. cinna) R1 AGCGTCCAGGATTTCTTTCATc CCR (S. cinna) F1 gATGAAAGAAATCCTGGACGCTATTCAGGCACAAACGGCA CCR (S. cinna) R2 CCGTACCGCTTGCGGTTGCCGTTTGTGCCTGAAT CCR (S. cinna) F2 ACCGCAAGCGGTACGGCGGCTGTTACCTCTGCT CCR (S. cinna) R3 GCAGGGCCGCAAAATCAGCAGAGGTAACAGCCG CCR (S. cinna) F3 GATTTTGCGGCCCTGCCGCTGCCGGACTCTTAC CCR (S. cinna) R4 GTGAACCGTGATCGCACGGTAAGAGTCCGGCAGCG CCR (S. cinna) F4 CGTGCGATCACGGTTCACAAGGATGAAACCGAGATGTTCGC CCR (S. cinna) R5 TATCACGGGATTCCAGACCGGCGAACATCTCGGTTTCATCCTT CCR (S. cinna) F5 CGGTCTGGAATCCCGTGATAAAGATCCTCGTAAATCTCTGCA CCR (S. cinna) R6 TGGCACGTCATCCAGGTGCAGAGATTTACGAGGATCTT CCR (S. cinna) F6 CCTGGATGACGTGCCAATTCCGGAACTGGGTCC CCR (S. cinna) R7 AACCAGCGCTTCGCCCGGACCCAGTTCCGGAAT CCR (S. cinna) F7 GGGCGAAGCGCTGGTTGCTGTGATGGCTTCCTCT CCR (S. cinna) R8 GTCCAGACGCTGTTATAATTCACAGAGGAAGCCATCACAGC CCR (S. cinna) F8 GTGAATTATAACAGCGTCTGGACTTCCATCTTCGAACCGGT CCR (S. cinna) R9 CCAGGAAGCTGAAGGTAGATACCGGTTCGAAGATGGAA CCR (S. cinna) F9 ATCTACCTTCAGCTTCCTGGAACGTTACGGTCGTCTGT CCR (S. cinna) R10 TGACGTTTGCTCAGATCGGACAGACGACCGTAACGTT CCR (S. cinna) F10 CCGATCTGAGCAAACGTCATGACCTGCCGTACCACA CCR (S. cinna) R11 GCCAGATCAGAACCGATGATGTGGTACGGCAGGTCA CCR (S. cinna) F11 TCATCGGTTCTGATCTGGCCGGTGTCGTTCTGCGTA CCR (S. cinna) R12 GTTGACACCAGGACCGGTACGCAGAACGACACCG CCR (S. cinna) F12 CCGGTCCTGGTGTCAACGCTTGGAACCCAGGTGA CCR (S. cinna) R13 ATGCGCCACCACTTCGTCACCTGGGTTCCAAGC CCR (S. cinna) F13 CGAAGTGGTGGCGCATTGCCTGAGCGTGGAAC CCR (S. cinna) R14 GCCATCGGAGGACTCCAGTTCCACGCTCAGGCA CCR (S. cinna) F14 TGGAGTCCTCCGATGGCCACAACGATACTATGCTGGACC CCR (S. cinna) R15 CCAGATGCGCTGTTCCGGGTCCAGCATAGTATCGTTGTG CCR (S. cinna) F15 CGGAACAGCGCATCTGGGGTTTTGAAACCAACTTCGGC CCR (S. cinna) R16 GCAATTTCAGCCAGGCCGCCGAAGTTGGTTTCAAAACC CCR (S. cinna) F16 GGCCTGGCTGAAATTGCTCTGGTGAAATCTAACCAGCT CCR (S. cinna) R17 TGACCCGGTTTAGGCATCAGCTGGTTAGATTTCACCAGA CCR (S. cinna) F17 GATGCCTAAACCGGGTCATCTGTCTTGGGAAGAGGC CCR (S. cinna) R18 CAGACCTGGGCTGGCGGCCTCTTCCCAAGACAGA CCR (S. cinna) F18 CGCCAGCCCAGGTCTGGTTAATTCTACTGCATACCGTCAA CCR (S. cinna) R19 CGCCATTACGGGAAACCAGTTGACGGTATGCAGTAGAATTAAC CCR (S. cinna) F19 CTGGTTTCCCGTAATGGCGCAGGCATGAAACAGGGCG CCR (S. cinna) R20 ACCCCAAATCAGTACGTTGTCGCCCTGTTTCATGCCTG CCR (S. cinna) F20 ACAACGTACTGATTTGGGGTGCGTCCGGTGGCCTGG CCR (S. cinna) R21 CGCGAACTGAGTAGCATAAGAGCCCAGGCCACCGGACGC CCR (S. cinna) F21 GCTCTTATGCTACTCAGTTCGCGCTGGCGGGTGGTGCCA CCR (S. cinna) R22 GAAACGACGCAAATTGGGTTGGCACCACCCGCCAG CCR (S. cinna) F22 ACCCAATTTGCGTCGTTTCCTCCCCGCAGAAAGCG CCR (S. cinna) R23 CCATTGCACGGCAGATTTCCGCTTTCTGCGGGGAG CCR (S. cinna) F23 GAAATCTGCCGTGCAATGGGTGCCGAAGCAATCATTGA CCR (S. cinna) R24 GCCCTCCGCGTTACGATCAATGATTGCTTCGGCAC CCR (S. cinna) F24 TCGTAACGCGGAGGGCTACAAGTTTTGGAAAGACGAGC CCR (S. cinna) R25 TTTTGGGTCCTGCGTCTGCTCGTCTTTCCAAAACTTGTA CCR (S. cinna) F25 AGACGCAGGACCCAAAAGAATGGAAACGCTTTGGCA CCR (S. cinna) R26 AGCTCACGGATGCGCTTGCCAAAGCGTTTCCATTC CCR (S. cinna) F26 AGCGCATCCGTGAGCTGACTGGTCGCCGTGGT CCR (S. cinna) R27 GTGTTCGAAAACGATGTCCAGACCACGGCGACCAGTC CCR (S. cinna) F27 CTGGACATCGTTTTCGAACACCCTGGTCGTGAAACGTTCG CCR (S. cinna) R28 AACATAAACGCTCGCGCCGAACGTTTCACGACCAGG CCR (S. cinna) F28 GCGCGAGCGTTTATGTTACTCGCAAGGGTGGTAC CCR (S. cinna) R29 CTCGCGCAGGTGGTAATAGTACCACCCTTGCGAGT

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CCR (S. cinna) F29 TATTACCACCTGCGCGAGCACTTCCGGTTACATGCAC CCR (S. cinna) R30 CCACAGATAGCGGTTATCGTATTCGTGCATGTAACCGGAAGTG CCR (S. cinna) F30 GAATACGATAACCGCTATCTGTGGATGTCCCTGAAGCGTATTATCGCCR (S. cinna) R31 TTCGCGAAGTGGGAGCCGATAATACGCTTCAGGGACAT CCR (S. cinna) F31 GCTCCCACTTCGCGAACTATCGTGAGGCGTGGG CCR (S. cinna) R32 GATCAGACGGTTGGCCTCCCACGCCTCACGATAG CCR (S. cinna) F32 AGGCCAACCGTCTGATCGCGAAGGGTAAAATTCATCCA CCR (S. cinna) R33 GCGGTAAGTTTTAGACAGGGTTGGATGAATTTTACCCTTCGC CCR (S. cinna) F33 ACCCTGTCTAAAACTTACCGCCTGGAAGACACTGGCCA CCR (S. cinna) R34 GACGTCGTACGCCGCCTGGCCAGTGTCTTCCAG CCR (S. cinna) F34 GGCGGCGTACGACGTCCACCGCAACCTGCACC CCR (S. cinna) R35 AGTACGCCCACTTTACCTTGGTGCAGGTTGCGGTG CCR (S. cinna) F35 AAGGTAAAGTGGGCGTACTGGCACTGGCGCCGGAA CCR (S. cinna) R36 GGACGCCCAGACCCTCTTCCGGCGCCAGTGCC CCR (S. cinna) F36 GAGGGTCTGGGCGTCCGTGACCCTGAAAAGCGTG CCR (S. cinna) R37 GGTTAATAGCATCGATGTGTTGTGCACGCTTTTCAGGGTCAC CCR (S. cinna) F37 CACAACACATCGATGCTATTAACCGTTTCCGCAACGTCTAAagag CCR (S. cinna) R38 cctgttcgtaggtcaaacgactctTTAGACGTTGCGGAAAC CCR (S. cinna) R39 tcgtttgacctacgaacagg

138

Table A1.14. Oligonucleotides used for the synthesis of the dtsR1 gene. Name Sequence dtsR1 R1 TAGTTGTCGGTATCGCTACCag dtsR1 F1 ctGGTAGCGATACCGACAACTATAAGGAGGACCAAATGACCATT dtsR1 R2 GTCAATCAACGGGCTGGAAATGGTCATTTGGTCCTCCTTA dtsR1 F2 TCCAGCCCGTTGATTGACGTGGCGAACTTGCCGG dtsR1 R3 CCCGCGGTGGTGTTAATATCCGGCAAGTTCGCCAC dtsR1 F3 ATATTAACACCACCGCGGGCAAGATTGCGGACTTGAAAGC dtsR1 R4 TGCTTCTGCGCGACGGGCTTTCAAGTCCGCAATCTTG dtsR1 F4 CCGTCGCGCAGAAGCACATTTTCCGATGGGCGAAAA dtsR1 R5 CATGGACTTTTTCGACAGCCTTTTCGCCCATCGGAAAATG dtsR1 F5 GGCTGTCGAAAAAGTCCATGCGGCAGGCCGTCTGAC dtsR1 R6 CAGACGCTCACGGGCGGTCAGACGGCCTGCCG dtsR1 F6 CGCCCGTGAGCGTCTGGACTACCTGCTGGATGAAGG dtsR1 R7 GTCGGTCTCGATGAAGCTGCCTTCATCCAGCAGGTAGTC dtsR1 F7 CAGCTTCATCGAGACCGACCAGTTGGCGCGTCATCG dtsR1 R8 ACCGAAGGCCGTCGTGCGATGACGCGCCAACTG dtsR1 F8 CACGACGGCCTTCGGTTTGGGCGCTAAACGCCC dtsR1 R9 TCACAATACCGTCCGTAGCCGGGCGTTTAGCGCCCAA dtsR1 F9 GGCTACGGACGGTATTGTGACCGGCTGGGGCACCATT dtsR1 R10 GCAGACCTCACGGCCATCAATGGTGCCCCAGCCGG dtsR1 F10 GATGGCCGTGAGGTCTGCATCTTCTCCCAGGATGGCAC dtsR1 R11 AGAGCACCACCAAACACCGTGCCATCCTGGGAGAAGAT dtsR1 F11 GGTGTTTGGTGGTGCTCTGGGTGAGGTTTATGGTGAGAAA dtsR1 R12 CGCCAGCTCCATGATTTTAATCATTTTCTCACCATAAACCTCACCC dtsR1 F12 ATGATTAAAATCATGGAGCTGGCGATCGACACGGGCCGTCC dtsR1 R13 CCTCATACAGGCCGATCAGCGGACGGCCCGTGTCGAT dtsR1 F13 GCTGATCGGCCTGTATGAGGGCGCGGGTGCGCGTAT dtsR1 R14 TGACTGCACCGTCCTGAATACGCGCACCCGCGC dtsR1 F14 TCAGGACGGTGCAGTCAGCCTGGACTTTATCAGCCA dtsR1 R15 GCTTGAATGTTCTGGTAGAAGGTTTGGCTGATAAAGTCCAGGC dtsR1 F15 AACCTTCTACCAGAACATTCAAGCGAGCGGTGTTATCCCGC dtsR1 R16 GCACCCATAATCACGGAAATCTGCGGGATAACACCGCTC dtsR1 F16 AGATTTCCGTGATTATGGGTGCGTGCGCTGGTGGCAAC dtsR1 R17 GCGCCGGGCCGTAAGCGTTGCCACCAGCGCAC dtsR1 F17 GCTTACGGCCCGGCGCTGACCGATTTTGTGGTGATGG dtsR1 R18 ACATCTTGCTGGTCTTGTCAACCATCACCACAAAATCGGTCA dtsR1 F18 TTGACAAGACCAGCAAGATGTTCGTCACCGGCCCGGA dtsR1 R19 CGGTCACCGTCTTAATGACATCCGGGCCGGTGACGA dtsR1 F19 TGTCATTAAGACGGTGACCGGCGAGGAGATCACCCAGG dtsR1 R20 GCACCACCCAGCTCCTCCTGGGTGATCTCCTCGC dtsR1 F20 AGGAGCTGGGTGGTGCTACCACGCACATGGTCAC dtsR1 R21 GTGGGAGTTGCCTGCCGTGACCATGTGCGTGGTA dtsR1 F21 GGCAGGCAACTCCCACTACACCGCGGCAACCG dtsR1 R22 CAGTCCAGAGCTTCTTCGTCGGTTGCCGCGGTGTA dtsR1 F22 ACGAAGAAGCTCTGGACTGGGTGCAGGACCTGGTTAGC dtsR1 R23 GCTACGATTATTAGACGGCAGAAAGCTAACCAGGTCCTGCACC dtsR1 F23 TTTCTGCCGTCTAATAATCGTAGCTATGCACCGATGGAAGATTTCG dtsR1 R24 CACACCACCTTCCTCTTCATCGAAATCTTCCATCGGTGCATA dtsR1 F24 ATGAAGAGGAAGGTGGTGTGGAAGAGAATATTACCGCCGACG dtsR1 R25 GGATAATCTCGTCCAACTTCAGATCGTCGGCGGTAATATTCTCTTC dtsR1 F25 ATCTGAAGTTGGACGAGATTATCCCGGACAGCGCGACGGT dtsR1 R26 CATCACGAACATCGTACGGAACCGTCGCGCTGTCCG dtsR1 F26 TCCGTACGATGTTCGTGATGTTATTGAATGTCTGACCGATGACG dtsR1 R27 CGCCTGAATCTCCAGATATTCACCGTCATCGGTCAGACATTCAATAAdtsR1 F27 GTGAATATCTGGAGATTCAGGCGGACCGTGCCGAAAATGTTG dtsR1 R28 TGCGGCCAAAGGCAATAACAACATTTTCGGCACGGTC dtsR1 F28 TTATTGCCTTTGGCCGCATTGAGGGTCAGAGCGTCG dtsR1 R29 GGTTGATTGGCAACGAAGCCGACGCTCTGACCCTCAA dtsR1 F29 GCTTCGTTGCCAATCAACCGACCCAGTTCGCGGGT dtsR1 R30 GCTGTCGATGTCCAGGCAACCCGCGAACTGGGTC dtsR1 F30 TGCCTGGACATCGACAGCTCTGAGAAAGCGGCACG dtsR1 R31 CGTCACAGGTACGGACAAAGCGTGCCGCTTTCTCAGA

139

dtsR1 F31 CTTTGTCCGTACCTGTGACGCATTCAACATCCCGATTGTGATG dtsR1 R32 CCCGGAACATCCACCAGCATCACAATCGGGATGTTGAATG dtsR1 F32 CTGGTGGATGTTCCGGGCTTCCTGCCGGGTGCG dtsR1 R33 GAATACCACCATACTCCTGACCCGCACCCGGCAGGAAG dtsR1 F33 GGTCAGGAGTATGGTGGTATTCTGCGTCGTGGCGCGAA dtsR1 R34 CCGTAGGCGTACAGCAGTTTCGCGCCACGACGCA dtsR1 F34 ACTGCTGTACGCCTACGGCGAAGCGACCGTCCCG dtsR1 R35 GCATGGTGACGGTGATCTTCGGGACGGTCGCTTCG dtsR1 F35 AAGATCACCGTCACCATGCGCAAGGCGTACGGTGG dtsR1 R36 CCCATGACACAGTACGCGCCACCGTACGCCTTGC dtsR1 F36 CGCGTACTGTGTCATGGGTTCCAAAGGCCTGGGTAGC dtsR1 R37 GGCCATGCCAGATTAATGTCGCTACCCAGGCCTTTGGAA dtsR1 F37 GACATTAATCTGGCATGGCCGACCGCACAAATTGCGG dtsR1 R38 CCAGCCGCGCCCATCACCGCAATTTGTGCGGTC dtsR1 F38 TGATGGGCGCGGCTGGTGCAGTGGGCTTTATCTACC dtsR1 R39 GCCGCCATCAGCTCTTTACGGTAGATAAAGCCCACTGCA dtsR1 F39 GTAAAGAGCTGATGGCGGCAGATGCCAAGGGTCTGGA dtsR1 R40 CGCCAGCGCAACGGTGTCCAGACCCTTGGCATCT dtsR1 F40 CACCGTTGCGCTGGCGAAGAGCTTCGAACGCGAG dtsR1 R41 CGGATTCAGCATGTGATCTTCATACTCGCGTTCGAAGCTCTT dtsR1 F41 TATGAAGATCACATGCTGAATCCGTATCATGCGGCGGAACG dtsR1 R42 CCGCATCGATCAGACCGCGTTCCGCCGCATGATA dtsR1 F42 CGGTCTGATCGATGCGGTGATTTTGCCGAGCGAGA dtsR1 R43 GGCTAATCTGACCACGGGTCTCGCTCGGCAAAATCA dtsR1 F43 CCCGTGGTCAGATTAGCCGCAATCTGCGTCTGCTGA dtsR1 R44 GGACGGGTGACATTCTTATGTTTCAGCAGACGCAGATTGC dtsR1 F44 AACATAAGAATGTCACCCGTCCGGCTCGCAAGCATGGTA dtsR1 R45 cgctgTTACAGCGGCATATTACCATGCTTGCGAGCC dtsR1 F45 ATATGCCGCTGTAAcagcgacgctctctgcggact dtsR1 R46 agtccgcagagagcgt

140

Table A1.15. Oligonucleotides used for the synthesis of the jFar gene. Name Sequence Jfar R2 CTTCCATATGggattctactttgc Jfar F2 gcaaagtagaatccCATATGGAAGAAATGGGCAGCATCCTG Jfar R3 CGCTTTGTTATCCAGGAACTCCAGGATGCTGCCCATTT Jfar F3 GAGTTCCTGGATAACAAAGCGATCCTGGTAACCGGCGC Jfar R4 TTGCCAGGGAGCCGGTCGCGCCGGTTACCAGGAT Jfar F4 GACCGGCTCCCTGGCAAAAATCTTCGTTGAAAAAGTGCTG Jfar R5 ACGTTTGGCTGAGAACGCAGCACTTTTTCAACGAAGATTT Jfar F5 CGTTCTCAGCCAAACGTAAAAAAACTGTACCTGCTGCT Jfar R6 CATCGTCAGTGGCGCGCAGCAGCAGGTACAGTTTTTTT Jfar F6 GCGCGCCACTGACGATGAAACTGCGGCTCTGCGT Jfar R7 GCCAAATACCTCATTTTGCAGACGCAGAGCCGCAGTTT Jfar F7 CTGCAAAATGAGGTATTTGGCAAAGAACTGTTCAAAGTGCTGA Jfar R8 TCGCACCCAGATTCTGTTTCAGCACTTTGAACAGTTCTTT Jfar F8 AACAGAATCTGGGTGCGAACTTCTACTCCTTCGTTTCTGA Jfar R9 CGGTACGACAGTGACTTTCTCAGAAACGAAGGAGTAGAAGT Jfar F9 GAAAGTCACTGTCGTACCGGGTGACATTACCGGTGAGG Jfar R10 ACGTCTTTCAGACACAGGTCCTCACCGGTAATGTCACC Jfar F10 ACCTGTGTCTGAAAGACGTCAACCTGAAGGAAGAGATGTG Jfar R11 CGACGTCAATTTCGCGCCACATCTCTTCCTTCAGGTTG Jfar F11 GCGCGAAATTGACGTCGTCGTTAATCTGGCTGCAAC Jfar R12 TCGTAACGTTCGATGAAATTAATGGTTGCAGCCAGATTAACGA Jfar F12 CATTAATTTCATCGAACGTTACGATGTATCCCTGCTGATCAACA Jfar R13 GCACATATTTTGCACCGTAGGTGTTGATCAGCAGGGATACA Jfar F13 CCTACGGTGCAAAATATGTGCTGGACTTCGCTAAGAAGTGCA Jfar R14 ACATGTACAAAGATTTTCAGTTTGTTGCACTTCTTAGCGAAGTCCA Jfar F14 ACAAACTGAAAATCTTTGTACATGTGTCTACCGCTTATGTTAGCG Jfar R15 GAATCAGACCATTCTTTTCGCCGCTAACATAAGCGGTAGAC Jfar F15 GCGAAAAGAATGGTCTGATTCTGGAAAAACCGTACTATATGGG Jfar R16 GGCCGTTCAGGGACTCACCCATATAGTACGGTTTTTCCA Jfar F16 TGAGTCCCTGAACGGCCGTCTGGGTCTGGACATCA Jfar R17 CGACCAGTTTCTTTTCTACGTTGATGTCCAGACCCAGAC Jfar F17 ACGTAGAAAAGAAACTGGTCGAGGCGAAGATCAACGAAC Jfar R18 GGCACCAGCAGCTTGCAGTTCGTTGATCTTCGCCT Jfar F18 TGCAAGCTGCTGGTGCCACTGAAAAATCCATCAAATCTACGA Jfar R19 ACGCTCGATACCCATATCTTTCATCGTAGATTTGATGGATTTTTCAGTJfar F19 TGAAAGATATGGGTATCGAGCGTGCTCGTCATTGGGGCTGG Jfar R20 TGAAAACGTACACGTTCGGCCAGCCCCAATGACGAGC Jfar F20 CCGAACGTGTACGTTTTCACTAAAGCTCTGGGTGAAATGC Jfar R21 TCACCCTTATATTGCATCAGCAGCATTTCACCCAGAGCTTTAG Jfar F21 TGCTGATGCAATATAAGGGTGACATCCCACTGACCATCATTC Jfar R22 GGTAGACGTGATAATAGTCGGACGAATGATGGTCAGTGGGATG Jfar F22 GTCCGACTATTATCACGTCTACCTTTAAAGAACCGTTCCCAGG Jfar R23 GCACGCCTTCCACCCAGCCTGGGAACGGTTCTTTAAA Jfar F23 CTGGGTGGAAGGCGTGCGTACGATTGATAACGTGCCG Jfar R24 CGACCCTTGCCGTAGTAAACCGGCACGTTATCAATCGTAC Jfar F24 GTTTACTACGGCAAGGGTCGTCTGCGTTGCATGCTGTG Jfar R25 TAATGGTGCTCGGGCCGCACAGCATGCAACGCAGA Jfar F25 CGGCCCGAGCACCATTATCGACCTGATCCCGGCG Jfar R26 GTGGCGTTCACTACCATATCCGCCGGGATCAGGTCGA Jfar F26 GATATGGTAGTGAACGCCACCATCGTTGCTATGGTTGCG Jfar R27 TAACGCTGGTTCGCGTGCGCAACCATAGCAACGATG Jfar F27 CACGCGAACCAGCGTTACGTAGAGCCGGTTACTTACC Jfar R28 GGCAGAGCTGCCGACGTGGTAAGTAACCGGCTCTACG Jfar F28 ACGTCGGCAGCTCTGCCGCTAACCCAATGAAGCTGA Jfar R29 CATCTCCGGCAGTGCGCTCAGCTTCATTGGGTTAGC Jfar F29 GCGCACTGCCGGAGATGGCGCACCGTTACTTCAC Jfar R30 CGGGTTAATCCACGGGTTTTTGGTGAAGTAACGGTGCGC Jfar F30 CAAAAACCCGTGGATTAACCCGGACCGTAATCCGGTTCACG Jfar R31 GACCATCGCGCGACCCACGTGAACCGGATTACGGTC Jfar F31 TGGGTCGCGCGATGGTCTTTTCTAGCTTTTCTACTTTTCACC Jfar R32 GGAAGTTCAGAGTCAGGTACAGGTGAAAAGTAGAAAAGCTAGAAAA

141

Jfar F32 TGTACCTGACTCTGAACTTCCTGCTGCCGCTGAAAGTG Jfar R33 GGTGTTCGCGATTTCCAGCACTTTCAGCGGCAGCA Jfar F33 CTGGAAATCGCGAACACCATTTTCTGCCAGTGGTTCAA Jfar R34 CTTCAGATCCATGTATTTGCCTTTGAACCACTGGCAGAAAAT Jfar F34 AGGCAAATACATGGATCTGAAGCGTAAAACCCGTCTGCTG Jfar R35 GATGTCTACCAGGCGCAGCAGCAGACGGGTTTTACG Jfar F35 CTGCGCCTGGTAGACATCTATAAACCTTATCTGTTCTTCCAGG Jfar R36 GTGTTCATATCGTCGAAAATGCCCTGGAAGAACAGATAAGGTTTATA Jfar F36 GCATTTTCGACGATATGAACACGGAAAAACTGCGCATCGC Jfar R37 CACGATGGATTCTTTGGCTGCGATGCGCAGTTTTTCC Jfar F37 AGCCAAAGAATCCATCGTGGAAGCTGATATGTTCTATTTCGACC Jfar R38 CCAGTTGATCGCACGCGGGTCGAAATAGAACATATCAGCTTC Jfar F38 CGCGTGCGATCAACTGGGAAGATTATTTTCTGAAAACCCACT Jfar R39 CTCCACAACACCCGGAAAGTGGGTTTTCAGAAAATAATCTTC Jfar F39 TTCCGGGTGTTGTGGAGCATGTCCTGAACTAATCTAGAgc Jfar R40 tcgcctcacaggtttaaaacgcTCTAGATTAGTTCAGGACATG Jfar R41 gttttaaacctgtgaggcga

142

Table A1.16. Oligonucleotides used for the synthesis of the hbd gene. Name Sequence hbd R2 ATATCTCCTTGAATTCgcg hbd F2 cgcGAATTCAAGGAGATATATAATGAAAAAGGTTTGCGT hbd R3 TAGTACCCGCACCAATAACGCAAACCTTTTTCATTAT hbd F3 TATTGGTGCGGGTACTATGGGTTCTGGTATCGC hbd R4 CTGCGAAAGCCTGGGCGATACCAGAACCCA hbd F4 CCAGGCTTTCGCAGCAAAGGGCTTCGAAGT hbd R5 TGTCGCGCAGAACGACTTCGAAGCCCTTTG hbd F5 CGTTCTGCGCGACATTAAGGATGAATTTGTTGATCG hbd R6 TGAAGTCCAGGCCGCGATCAACAAATTCATCCTTAA hbd F6 CGGCCTGGACTTCATTAACAAAAACCTGTCTAAACT hbd R7 TCGATTTTACCTTTCTTTACCAGTTTAGACAGGTTTTTGTTAAhbd F7 GGTAAAGAAAGGTAAAATCGAGGAGGCTACGAAAGTAG hbd R8 AAATACGGGTCAGGATTTCTACTTTCGTAGCCTCC hbd F8 AAATCCTGACCCGTATTTCCGGCACCGTTGAC hbd R9 GGCAGCCATGTTCAGGTCAACGGTGCCGG hbd F9 CTGAACATGGCTGCCGATTGTGACCTGGTTATCG hbd R10 CGTTCTACTGCTGCTTCGATAACCAGGTCACAATC hbd F10 AAGCAGCAGTAGAACGCATGGACATCAAGAAACAG hbd R11 TCCAGATCAGCGAAAATCTGTTTCTTGATGTCCATG hbd F11 ATTTTCGCTGATCTGGACAACATCTGCAAGCCT hbd R12 CGCCAGGATCGTTTCAGGCTTGCAGATGTTG hbd F12 GAAACGATCCTGGCGTCTAACACTTCTTCCCTG hbd R13 CGACTTCAGTGATGCTCAGGGAAGAAGTGTTAGA hbd F13 AGCATCACTGAAGTCGCTTCCGCAACCAAACG hbd R14 CAATCACCTTATCCGGACGTTTGGTTGCGGAAG hbd F14 TCCGGATAAGGTGATTGGTATGCACTTCTTTAACCC hbd R15 AGTTTCATAACTGGTGCCGGGTTAAAGAAGTGCATAC hbd F15 GGCACCAGTTATGAAACTGGTCGAAGTGATCCG hbd R16 AGAAGTAGCGATGCCACGGATCACTTCGACC hbd F16 TGGCATCGCTACTTCTCAGGAAACCTTCGACG hbd R17 GGAAGTTTCCTTCACCGCGTCGAAGGTTTCCTG hbd F17 CGGTGAAGGAAACTTCCATCGCCATCGGTAAAG hbd R18 GCAACTTCTACCGGATCTTTACCGATGGCGAT hbd F18 ATCCGGTAGAAGTTGCTGAAGCACCGGGTT hbd R19 AGAATACGGTTAACTACGAAACCCGGTGCTTCA hbd F19 TCGTAGTTAACCGTATTCTGATCCCGATGATTAACGA hbd R20 AGGATGCCTACTGCTTCGTTAATCATCGGGATC hbd F20 AGCAGTAGGCATCCTGGCTGAGGGTATCGC hbd R21 TCGATGTCTTCAACAGATGCGATACCCTCAGCC hbd F21 ATCTGTTGAAGACATCGACAAGGCAATGAAGCTGG hbd R22 CCATAGGGTGATTTGCACCCAGCTTCATTGCCTTG hbd F22 GTGCAAATCACCCTATGGGCCCGCTGGAGCTG hbd R23 AGGCCGATAAAATCACCCAGCTCCAGCGGGC hbd F23 GGTGATTTTATCGGCCTGGACATCTGTCTGGC hbd R24 ACAGTACGTCCATGATGGCCAGACAGATGTCC hbd F24 CATCATGGACGTACTGTACTCTGAAACGGGCG hbd R25 GCGGACGGTACTTAGAATCGCCCGTTTCAGAGT hbd F25 ATTCTAAGTACCGTCCGCACACGCTGCTGAAAA hbd R26 CGGCACGAACATACTTTTTCAGCAGCGTGT hbd F26 AGTATGTTCGTGCCGGCTGGCTGGGTCGTA hbd R27 AAAAACCTTTGCCAGATTTACGACCCAGCCAGC hbd F27 AATCTGGCAAAGGTTTTTACGATTACTCCAAGTAACT hbd R28 TACCGACGTCCTCGAGTTACTTGGAGTAATCGT hbd F29 CGAGGACGTCGGTAa

143

Table A1.17. Oligonucleotides used for the synthesis of the nphT7 gene. Name Sequence nphT7 R1 AAACGAACGTCGGTCATggtg nphT7 F1 caccATGACCGACGTTCGTTTTCGTATCATTGGCACGGGT nphT7 R2 GCTCCGGCACGTACGCACCCGTGCCAATGATACGA nphT7 F2 GCGTACGTGCCGGAGCGTATTGTGTCCAACGACGAGGT nphT7 R3 ACCAGCCGGCGCACCCACCTCGTCGTTGGACACAATAC nphT7 F3 GGGTGCGCCGGCTGGTGTTGATGATGACTGGATTACCCGT nphT7 R4 CGTTGACGAATGCCGGTCTTACGGGTAATCCAGTCATCATCAACnphT7 F4 AAGACCGGCATTCGTCAACGTCGTTGGGCGGCGGAC nphT7 R5 TCGGAGGTCGCTTGGTCGTCCGCCGCCCAACGA nphT7 F5 GACCAAGCGACCTCCGACCTGGCAACCGCGGCG nphT7 R6 TCAACGCCGCACGACCCGCCGCGGTTGCCAGG nphT7 F6 GGTCGTGCGGCGTTGAAAGCAGCGGGTATTACGCC nphT7 R7 GCAATAACCGTCAGTTGCTCCGGCGTAATACCCGCTGCTT nphT7 F7 GGAGCAACTGACGGTTATTGCGGTCGCAACGTCCACCC nphT7 R8 GGCTGCGGACGGTCCGGGGTGGACGTTGCGACC nphT7 F8 CGGACCGTCCGCAGCCGCCGACGGCGGCCTAC nphT7 R9 CGCCCAGATGATGTTGCACGTAGGCCGCCGTCGGC nphT7 F9 GTGCAACATCATCTGGGCGCAACCGGCACCGCGGC nphT7 R10 TGCACACAGCGTTAACATCAAATGCCGCGGTGCCGGTTG nphT7 F10 ATTTGATGTTAACGCTGTGTGCAGCGGCACGGTTTTTGCT nphT7 R11 CCGCCACGCTGGACAGAGCAAAAACCGTGCCGC nphT7 F11 CTGTCCAGCGTGGCGGGCACGCTGGTGTATCGTGG nphT7 R12 CAATGACCAGTGCGTAACCGCCACGATACACCAGCGTGC nphT7 F12 CGGTTACGCACTGGTCATTGGTGCCGATCTGTATTCCCGTA nphT7 R13 GGTCCGCCGGATTCAGAATACGGGAATACAGATCGGCAC nphT7 F13 TTCTGAATCCGGCGGACCGCAAGACCGTTGTTCTGTTTGG nphT7 R14 CGCACCCGCGCCGTCACCAAACAGAACAACGGTCTTGC nphT7 F14 TGACGGCGCGGGTGCGATGGTGCTGGGTCCGAC nphT7 R15 ACCCGTACCCGTGCTGGTCGGACCCAGCACCAT nphT7 F15 CAGCACGGGTACGGGTCCGATCGTCCGTCGCG nphT7 R16 CAAACGTGTGCAGGGCAACGCGACGGACGATCGG nphT7 F16 TTGCCCTGCACACGTTTGGTGGTCTGACCGACCTGATT nphT7 R17 CACCCGCCGGCACACGAATCAGGTCGGTCAGACCAC nphT7 F17 CGTGTGCCGGCGGGTGGCAGCCGCCAACCGCT nphT7 R18 TCCAAGCCATCCGTGTCCAGCGGTTGGCGGCTGC nphT7 F18 GGACACGGATGGCTTGGACGCGGGTCTGCAATACTTCG nphT7 R19 CCTCGCGACCGTCCATAGCGAAGTATTGCAGACCCGCG nphT7 F19 CTATGGACGGTCGCGAGGTGCGTCGTTTTGTTACCGAAC nphT7 R20 CCTTTAATCAGTTGCGGCAAGTGTTCGGTAACAAAACGACGCA nphT7 F20 ACTTGCCGCAACTGATTAAAGGTTTCTTGCACGAGGCGGG nphT7 R21 GCTAATATCTGCCGCATCGACACCCGCCTCGTGCAAGAAA nphT7 F21 TGTCGATGCGGCAGATATTAGCCATTTTGTGCCGCACCAAGC nphT7 R22 CGTCCAGCATGACACCGTTCGCTTGGTGCGGCACAAAATG nphT7 F22 GAACGGTGTCATGCTGGACGAGGTCTTTGGTGAACTGCACC nphT7 R23 ATGGTCGCACGCGGCAGGTGCAGTTCACCAAAGACCT nphT7 F23 TGCCGCGTGCGACCATGCACCGTACCGTCGAAACC nphT7 R24 CGCACCCGTATTGCCGTAGGTTTCGACGGTACGGTGC nphT7 F24 TACGGCAATACGGGTGCGGCCAGCATTCCGATTACGATG nphT7 R25 TGCACGGACTGCTGCATCCATCGTAATCGGAATGCTGGC nphT7 F25 GATGCAGCAGTCCGTGCAGGTAGCTTCCGTCCGGG nphT7 R26 GCCAGCAGGACCAGTTCACCCGGACGGAAGCTACC nphT7 F26 TGAACTGGTCCTGCTGGCGGGTTTTGGTGGTGGCATG nphT7 R27 GCGCGAAGCTCGCTGCCATGCCACCACCAAAACCC nphT7 F27 GCAGCGAGCTTCGCGCTGATCGAGTGGTAAgtcagcc nphT7 R28 acccgctctagccgtcaggctgacTTACCACTCGATCA nphT7 F28 tgacggctagagcgggt

144

Table A1.18. Oligonucleotides used for the synthesis of the phaJ gene. Name Sequence phaJ R1 GCGCTCATACAAGAGGGGATTC phaJ F1 GAATCCCCTCTTGTATGAGCGCGCAGAGCCTGGAAGTGGGphaJ R2 GGACAGGCGAGCTTTTTGACCCACTTCCAGGCTCTGC phaJ F2 TCAAAAAGCTCGCCTGTCCAAACGTTTTGGTGCAGCAGA phaJ R3 TGCGAAGGCCGCAACTTCTGCTGCACCAAAACGTTT phaJ F3 AGTTGCGGCCTTCGCAGCACTGTCTGAAGACTTCAATCC phaJ R4 CCGGGTCCAGATGCAGCGGATTGAAGTCTTCAGACAGTGCphaJ F4 GCTGCATCTGGACCCGGCATTCGCGGCAACCACC phaJ R5 CGATCGGGCGTTCAAATGCGGTGGTTGCCGCGAATG phaJ F5 GCATTTGAACGCCCGATCGTTCATGGTATGCTGTTGGCA phaJ R6 GCAGACCGCTAAACAGGCTTGCCAACAGCATACCATGAA phaJ F6 AGCCTGTTTAGCGGTCTGCTGGGTCAGCAGCTGCC phaJ R7 AGGTAAATGCTACCTTTGCCCGGCAGCTGCTGACCCA phaJ F7 GGGCAAAGGTAGCATTTACCTGGGTCAGAGCCTGAGCT phaJ R8 GACGAACACCGGCAGTTTAAAGCTCAGGCTCTGACCC phaJ F8 TTAAACTGCCGGTGTTCGTCGGTGACGAGGTCACGGC phaJ R9 GCCGTAACCTCGACCTCCGCCGTGACCTCGTCACC phaJ F9 GGAGGTCGAGGTTACGGCCCTGCGTGAGGACAAGC phaJ R10 GGTCAGGGTCGCGATCGGCTTGTCCTCACGCAGG phaJ F10 CGATCGCGACCCTGACCACCCGTATTTTCACCCAGGG phaJ R11 CACGGCCAACGCGCCACCCTGGGTGAAAATACGGGT phaJ F11 TGGCGCGTTGGCCGTGACGGGTGAGGCCGTGG phaJ R12 TCAAGTTTCTTACGGCAGTTTGACCACGGCCTCACCCGT phaJ R13 TCAAACTGCCGTAAGAAACTTGA

145

Table A1.19. Oligonucleotides used for the synthesis of the ppPhaJ4 gene.

Name Sequence PpPhaJ4 R1 CGGAACGTGCGGCATt PpPhaJ4 F1 aATGCCGCACGTTCCGGTTACCGAGCTGAGCCA PpPhaJ4 R2 CCAGTTCTTTACCCACGTACTGGCTCAGCTCGGTAAC PpPhaJ4 F2 GTACGTGGGTAAAGAACTGGGTCATAGCGAATGGCTGAAG PpPhaJ4 R3 AGATTAATACGCTGTTGGTCAATCTTCAGCCATTCGCTATGACPpPhaJ4 F3 ATTGACCAACAGCGTATTAATCTGTTCGCGGAGGCGACC PpPhaJ4 R4 TGAATGAACTGGAAATCGCCGGTCGCCTCCGCGAAC PpPhaJ4 F4 GGCGATTTCCAGTTCATTCATGTTGACCCGGAGAAGG PpPhaJ4 R5 AACGGCGTCTTTGCGGCCTTCTCCGGGTCAACA PpPhaJ4 F5 CCGCAAAGACGCCGTTCGGCGGCACCATTGCG PpPhaJ4 R6 GACAGGGTCAGAAAACCATGCGCAATGGTGCCGCCG PpPhaJ4 F6 CATGGTTTTCTGACCCTGTCTCTGATCCCGAAACTGATCG PpPhaJ4 R7 GCAGGACCAGAATGTCCTCGATCAGTTTCGGGATCAGA PpPhaJ4 F7 AGGACATTCTGGTCCTGCCGCAAGGCCTGAAAATGG PpPhaJ4 R8 TCCAGACCGTAATTCACGACCATTTTCAGGCCTTGCG PpPhaJ4 F8 TCGTGAATTACGGTCTGGACTCTGTGCGTTTCATTCAGC PpPhaJ4 R9 GGCTATCGACCTTGACCGGCTGAATGAAACGCACAGAG PpPhaJ4 F9 CGGTCAAGGTCGATAGCCGCGTTCGTCTGAAAGTTAAACT PpPhaJ4 R10 TTTTCAACCACTTCGCCCAGTTTAACTTTCAGACGAACGC PpPhaJ4 F10 GGGCGAAGTGGTTGAAAAGAAGCCGGGTCAGTGG PpPhaJ4 R11 CGCGATTGCTTTCAGCAGCCACTGACCCGGCTTC PpPhaJ4 F11 CTGCTGAAAGCAATCGCGACCTTGGAAATCGAGGGC PpPhaJ4 R12 TACGCCGGTTTCTCCTCGCCCTCGATTTCCAAGGT PpPhaJ4 F12 GAGGAGAAACCGGCGTATATCGCAGAGTCCTTGAGC PpPhaJ4 R13 tccgGACGAAGCACAGGCTCAAGGACTCTGCGATA PpPhaJ4 F13 CTGTGCTTCGTCcggataaacgtgggcggtagta PpPhaJ4 R14 tactaccgcccacgttta

146

Table A1.20. Oligonucleotides used for the synthesis of the phaZ1 gene. Name Sequence sPhaZ1R1 GTGCAATTGATACAGCAT sPhaZ1F1 ATGCTGTATCAATTGCACGAGTTTCAACGTTCTATC sPhaZ1R2 TCAACGGGTGCAGGATAGAACGTTGAAACTC sPhaZ1F2 CTGCACCCGTTGACCGCCTGGGCA sPhaZ1R3 GCGGTCGCCTGTGCCCAGGCGG sPhaZ1F3 CAGGCGACCGCTAAGACGTTCACGAAC sPhaZ1R4 GGGCTCAGCGGGTTCGTGAACGTCTTA sPhaZ1F4 CCGCTGAGCCCGCTGTCCCTGGTG sPhaZ1R5 GGCGCACCCGGCACCAGGGACAGC sPhaZ1F5 CCGGGTGCGCCGCGCCTGGCCG sPhaZ1R6 AGCTCATAGCCCGCGGCCAGGCGC sPhaZ1F6 CGGGCTATGAGCTGCTGTATCGTTTGGG sPhaZ1R7 GGCTTCTCATACTCTTTACCCAAACGATACAGC sPhaZ1F7 TAAAGAGTATGAGAAGCCGGCGTTTGATATTAAGAG sPhaZ1R8 GTTGCTGCGAACGCTCTTAATATCAAACGCC sPhaZ1F8 CGTTCGCAGCAACGGTCGTGACATCCC sPhaZ1R9 GGTTTGTTCCACGATCGGGATGTCACGACC sPhaZ1F9 GATCGTGGAACAAACCGTTCTGGAAAAACCGT sPhaZ1R10 CGAACCAGCTTACAAAACGGTTTTTCCAGAAC sPhaZ1F10 TTTGTAAGCTGGTTCGTTTCAAACGCTACGC sPhaZ1R11 TTTCCGGGTCATCTGCGTAGCGTTTGAAA sPhaZ1F11 AGATGACCCGGAAACGATCAAGCTGCTG sPhaZ1R12 ACCGGCTCATCTTTCAGCAGCTTGATCG sPhaZ1F12 AAAGATGAGCCGGTTGTGCTGGTGGCG sPhaZ1R13 CCGCTCAACGGAGCCGCCACCAGCACA sPhaZ1F13 GCTCCGTTGAGCGGTCACCACGCGACC sPhaZ1R14 GTATCACGCAGCAGGGTCGCGTGGTGA sPhaZ1F14 CTGCTGCGTGATACCGTTCGTACGCTGC sPhaZ1R15 CCTTGTGGTCTTGCAGCAGCGTACGAACG sPhaZ1F15 TGCAAGACCACAAGGTTTATGTTACCGATTGGA sPhaZ1R16 CCATACGAGCGTCAATCCAATCGGTAACATAAA sPhaZ1F16 TTGACGCTCGTATGGTTCCGGTTGAGGAA sPhaZ1R17 TCAGATGAAACGCACCTTCCTCAACCGGAA sPhaZ1F17 GGTGCGTTTCATCTGAGCGACTACATCTACTATAT sPhaZ1R18 TGACGAATAAACTCTTGAATATAGTAGATGTAGTCGCsPhaZ1F18 TCAAGAGTTTATTCGTCATATTGGCGCGGAG sPhaZ1R19 GATGACATGCAGGTTCTCCGCGCCAATA sPhaZ1F19 AACCTGCATGTCATCTCTGTTTGCCAGCC sPhaZ1R20 ACCGGGACGGTCGGCTGGCAAACAGA sPhaZ1F20 GACCGTCCCGGTTCTGGCTGCGATC sPhaZ1R21 GCTTGCCATCAAAGAGATCGCAGCCAGA sPhaZ1F21 TCTTTGATGGCAAGCGCGGGTGAGAAAAC sPhaZ1R22 ATGGTACGCGGCGTTTTCTCACCCGC sPhaZ1F22 GCCGCGTACCATGACGATGATGGGCG sPhaZ1R23 CGTCGATCGGGCCGCCCATCATCGTC sPhaZ1F23 GCCCGATCGACGCCCGCAAAAGCCC sPhaZ1R24 TTCACCGCCGTCGGGCTTTTGCGGG sPhaZ1F24 GACGGCGGTGAATAGCCTGGCCACG sPhaZ1R25 CCACTCGAAAGACTTATTCGTGGCCAGGCTA sPhaZ1F25 AATAAGTCTTTCGAGTGGTTCGAAAATAATGTGATCTsPhaZ1R26 CCGGAACGGTGTAGATCACATTATTTTCGAA sPhaZ1F26 ACACCGTTCCGGCTAATTATCCGGGCC sPhaZ1R27 CACGACGACCGTGGCCCGGATAATTAG sPhaZ1F27 ACGGTCGTCGTGTGTATCCGGGTTTCC sPhaZ1R28 CCAGCGTGTTGCAGGAAACCCGGATACA sPhaZ1F28 TGCAACACGCTGGCTTCGTGGCGATGA sPhaZ1R29 GCGGTCCGGGTTCATCGCCACGAAG sPhaZ1F29 ACCCGGACCGCCATTTGAGCAGCCA sPhaZ1R30 GCTCAGGTAGAAATCATAGTGGCTGCTCAAATG sPhaZ1F30 CTATGATTTCTACCTGAGCCTGGTTGAGGGCG sPhaZ1R31 CGTCGTCGGCATCGCCCTCAACCAG

147

sPhaZ1F31 ATGCCGACGACGCAGAGGCGCATGT sPhaZ1R32 CGTCGTAAAAGCGAACATGCGCCTCTG sPhaZ1F32 TCGCTTTTACGACGAATATAACGCAGTCCTG sPhaZ1R33 TCCGCTGCCATATCCAGGACTGCGTTATATT sPhaZ1F33 GATATGGCAGCGGAATATTACCTGGACACCA sPhaZ1R34 GAAAACTTCGCGGATGGTGTCCAGGTAATAT sPhaZ1F34 TCCGCGAAGTTTTCCAGGAGTTCCGTCT sPhaZ1R35 CGTACCGTTCGCCAGACGGAACTCCTG sPhaZ1F35 GGCGAACGGTACGTGGGCGATCGATG sPhaZ1R36 GGACCGGGTTGCCATCGATCGCCCA sPhaZ1F36 GCAACCCGGTCCGTCCGCAGGACAT sPhaZ1R37 GTGCGGTGCTTTTAATGTCCTGCGGAC sPhaZ1F37 TAAAAGCACCGCACTGATGACGGTTGAGG sPhaZ1R38 TCGTCCAGCTCACCCTCAACCGTCATCA sPhaZ1F38 GTGAGCTGGACGATATTTCTGGTGCGGG sPhaZ1R39 CCGCGGTCTGGCCCGCACCAGAAATA sPhaZ1F39 CCAGACCGCGGCTGCGCACGATCT sPhaZ1R40 TGCCCGCACACAGATCGTGCGCAG sPhaZ1F40 GTGTGCGGGCATTCCGAAAATTCGTAAGC sPhaZ1R41 AGCGTTCAGGTGTTGCTTACGAATTTTCGGAA sPhaZ1F41 AACACCTGAACGCTGCGCATTGCGGT sPhaZ1R42 GAGAAAATGCCGTAATGACCGCAATGCGC sPhaZ1F42 CATTACGGCATTTTCTCCGGTCGTCGCTG sPhaZ1R43 ATAGATTTCCTCACGCCAGCGACGACCG sPhaZ1F43 GCGTGAGGAAATCTATCCGCAACTGCGT sPhaZ1R44 CTTGCGGATGAAGTCACGCAGTTGCGG sPhaZ1F44 GACTTCATCCGCAAGTATCATCAAGCGTCCG sPhaZ1R45 atatTTAGCGCGTCGCGGACGCTTGATGATA sPhaZ1F45 CGACGCGCTAAatatggaaacatgcgtgc sPhaZ1R46 gcacgcatgtttcc

148

Table A1.21. Oligonucleotides used for the synthesis of the egTer gene. Name Sequence egTer R2 TGGAATGATCagtgacagggg egTer F2 cccctgtcactGATCATTCCATATGTCTTGTCCGGCTTCTCC egTer R3 GGAAACAACAGCTGCGCTAGGAGAAGCCGGACAAGACATA egTer F3 TAGCGCAGCTGTTGTTTCCGCAGGTGCACTGTGTCT egTer R4 GCAGAACAGTAGCAACGCACAGACACAGTGCACCTGC egTer F4 GTGCGTTGCTACTGTTCTGCTGGCCACCGGCAGCAA egTer R5 TGGACAGGGCGGTCGGATTGCTGCCGGTGGCCA egTer F5 TCCGACCGCCCTGTCCACCGCGAGCACGCGCAG egTer R6 GCACCAGGCTAGTCGGGCTGCGCGTGCTCGCGG egTer F6 CCCGACTAGCCTGGTGCGTGGCGTTGACCGTGG egTer R7 TGGTCGGACGCATCAGACCACGGTCAACGCCAC egTer F7 TCTGATGCGTCCGACCACCGCTGCGGCTCTGAC egTer R8 CGGAACTTCACGCATCGTGGTCAGAGCCGCAGCGG egTer F8 CACGATGCGTGAAGTTCCGCAGATGGCGGAAGGCTT egTer R9 AGGTAGCCTCGCCAGAAAAGCCTTCCGCCATCTG egTer F9 TTCTGGCGAGGCTACCTCCGCATGGGCGGCAGC egTer R10 AGCCCACTGCGGACCAGCTGCCGCCCATGCGG egTer F10 TGGTCCGCAGTGGGCTGCTCCTCTGGTGGCAGC egTer R11 CCAGTGCGCTAGACGCAGCTGCCACCAGAGGAGC egTer F11 TGCGTCTAGCGCACTGGCTCTGTGGTGGTGGGCA egTer R12 GTACGGAACGACGGGCTGCCCACCACCACAGAG egTer F12 GCCCGTCGTTCCGTACGCCGCCCACTGGCTGC egTer R13 TGGCAGTTCGGCCAGGGCAGCCAGTGGGCGGC egTer F13 CCTGGCCGAACTGCCAACCGCTGTGACTCACCT egTer R14 TAGCCATCGGTGGGGCCAGGTGAGTCACAGCGGT egTer F14 GGCCCCACCGATGGCTATGTTTACCACTACCGCGAAAG egTer R15 CACGGATTTTCGGCTGGATAACTTTCGCGGTAGTGGTAAACA egTer F15 TTATCCAGCCGAAAATCCGTGGTTTTATCTGCACTACCACTCACC egTer R16 GCGTTTTTCGCAGCCAATTGGGTGAGTGGTAGTGCAGATAAAAC egTer F16 CAATTGGCTGCGAAAAACGCGTCCAGGAAGAAATTGCTTACGCT egTer R17 TTGGCGGGTGCGCACGAGCGTAAGCAATTTCTTCCTGGAC egTer F17 CGTGCGCACCCGCCAACCAGCCCTGGCCCTAA egTer R18 CCGATGACCAGTACACGCTTAGGGCCAGGGCTGG egTer F18 GCGTGTACTGGTCATCGGTTGTAGCACGGGTTACGGT egTer R19 AGCAGTGATACGGGTAGACAGACCGTAACCCGTGCTACAA egTer F19 CTGTCTACCCGTATCACTGCTGCGTTCGGCTACCAGGC egTer R20 AACGCCCAGGGTCGCCGCCTGGTAGCCGAACGC egTer F20 GGCGACCCTGGGCGTTTTCCTGGCGGGTCCAC egTer R21 GGGCGACCTTTGGTCGGTGGACCCGCCAGGAA egTer F21 CGACCAAAGGTCGCCCGGCAGCTGCGGGTTGG egTer R22 CTCGAAGGCAACAGTGTTGTACCAACCCGCAGCTGCC egTer F22 TACAACACTGTTGCCTTCGAGAAAGCAGCGCTGGAGG egTer R23 CGGGCATACAGGCCCGCCTCCAGCGCTGCTTT egTer F23 CGGGCCTGTATGCCCGTTCTCTGAACGGCGACGC egTer R24 GCGCTTTCGTAGTGGAATCAAAAGCGTCGCCGTTCAGAGAA egTer F24 TTTTGATTCCACTACGAAAGCGCGCACTGTTGAAGCTATCAAACG egTer R25 TACGGTGCCCAGGTCACGTTTGATAGCTTCAACAGTGC egTer F25 TGACCTGGGCACCGTAGACCTGGTAGTGTACTCTATCGC egTer R26 GGTACGCTTCGGGGCAGCGATAGAGTACACTACCAGGTC egTer F26 TGCCCCGAAGCGTACCGATCCGGCGACCGGCG egTer R27 AGACAAGCCTTGTGCAGAACGCCGGTCGCCGGATC egTer F27 TTCTGCACAAGGCTTGTCTGAAACCAATCGGCGCGA egTer R28 GACGGTACGGTTGGTGTAAGTCGCGCCGATTGGTTTC egTer F28 CTTACACCAACCGTACCGTCAACACCGACAAAGCGGAG egTer R29 TCGATGCTAACATCGGTCACCTCCGCTTTGTCGGTGTT egTer F29 GTGACCGATGTTAGCATCGAACCTGCCTCCCCGGAA egTer R30 ACCGTGTCCGCGATCTCTTCCGGGGAGGCAGGT egTer F30 GAGATCGCGGACACGGTTAAAGTGATGGGTGGTGAAGAC egTer R31 CCTGAATCCACAGCTCCCAGTCTTCACCACCCATCACTTTA egTer F31 TGGGAGCTGTGGATTCAGGCGCTGAGCGAAGCCGG egTer R32 ACCCTCCGCCAGAACACCGGCTTCGCTCAGCG

149

egTer F32 TGTTCTGGCGGAGGGTGCGAAAACCGTGGCGTA egTer R33 TCTCAGGGCCAATGTAGGAGTACGCCACGGTTTTCGC egTer F33 CTCCTACATTGGCCCTGAGATGACCTGGCCGGTATATTGG egTer R34 GGCTTCGCCAATAGTACCAGACCAATATACCGGCCAGGTCA egTer F34 TCTGGTACTATTGGCGAAGCCAAAAAGGATGTTGAAAAGGCGG egTer R35 ACTGCTGGGTGATACGTTTAGCCGCCTTTTCAACATCCTTTTT egTer F35 CTAAACGTATCACCCAGCAGTATGGTTGCCCAGCATACC egTer R36 GCGCTTTAGCGACTACCGGGTATGCTGGGCAACCAT egTer F36 CGGTAGTCGCTAAAGCGCTGGTCACCCAGGCCAG egTer R37 AACTACCGGAATTGCGGAGCTGGCCTGGGTGACCA egTer F37 CTCCGCAATTCCGGTAGTTCCACTGTACATTTGCCTGCT egTer R38 CCTTTTTCTTTCATCACACGGTACAGCAGGCAAATGTACAGTGG egTer F38 GTACCGTGTGATGAAAGAAAAAGGTACTCATGAAGGTTGCATTGAACAegTer R39 GGTCAGCAGACGAACCATCTGTTCAATGCAACCTTCATGAGTA egTer F39 GATGGTTCGTCTGCTGACCACTAAACTGTACCCTGAGAACGG egTer R40 CGTCCACGATCGGAGCACCGTTCTCAGGGTACAGTTTAGT egTer F40 TGCTCCGATCGTGGACGAAGCGGGCCGTGTTCG egTer R41 GCCATTTCCCAGTCATCAACACGAACACGGCCCGCTT egTer F41 TGTTGATGACTGGGAAATGGCTGAAGACGTGCAGCAAGC egTer R42 TGGGACCACAGGTCTTTAACAGCTTGCTGCACGTCTTCA egTer F42 TGTTAAAGACCTGTGGTCCCAGGTGTCTACGGCTAACCTGA egTer R43 GCGAAGTCGCTGATGTCTTTCAGGTTAGCCGTAGACACC egTer F43 AAGACATCAGCGACTTCGCTGGCTACCAAACTGAGTTCC egTer R44 TACCAAAACCAAACAGACGCAGGAACTCAGTTTGGTAGCCA egTer F44 TGCGTCTGTTTGGTTTTGGTATCGACGGTGTAGACTACGAC egTer R45 TCAACGTCAACCGGCTGGTCGTAGTCTACACCGTCGA egTer F45 CAGCCGGTTGACGTTGAAGCGGACCTGCCGAGC egTer R46 AATTCTTATTGCTGCGCTGCGCTCGGCAGGTCCGCT egTer F46 GCAGCGCAGCAATAAGAATTCCTCGAGTCATGACAggcc egTer R47 taccgcgaggggttgcggccTGTCATGACTCGAGG egTer R48 gcaacccctcgcggta

150

Table A1.22. Oligonucleotides used for the synthesis of the tdTer gene. Name Sequence TdTER R2 TCgtggcttgctggac TdTER F2 gtccagcaagccacGAGATATACATATGATCGTCAAGCC TdTER R3 AGATATTATTGCGCACCATTGGCTTGACGATCATATGTATATCTdTER F3 AATGGTGCGCAATAATATCTGTCTGAACGCTCACCC TdTER R4 CCCTTTTTACAACCCTGCGGGTGAGCGTTCAGAC TdTER F4 GCAGGGTTGTAAAAAGGGTGTAGAAGACCAGATTGAATACA TdTER R5 CGGTGATGCGTTTCTTAGTGTATTCAATCTGGTCTTCTACA TdTER F5 CTAAGAAACGCATCACCGCAGAAGTTAAAGCAGGTGC TdTER R6 ACGTTTTTCGGTGCTTTGGCACCTGCTTTAACTTCTG TdTER F6 CAAAGCACCGAAAAACGTCCTGGTGCTGGGCTG TdTER R7 CCGTAGCCGTTGCTGCAGCCCAGCACCAGG TdTER F7 CAGCAACGGCTACGGTCTGGCAAGCCGCAT TdTER R8 CCGAATGCAGCCGTAATGCGGCTTGCCAGA TdTER F8 TACGGCTGCATTCGGTTACGGCGCTGCTAC TdTER R9 TTTTCGAAGCTAACACCAATAGTAGCAGCGCCGTAA TdTER F9 TATTGGTGTTAGCTTCGAAAAGGCGGGTTCTGAAACC TdTER R10 CCTGGAGTGCCGTATTTGGTTTCAGAACCCGCC TdTER F10 AAATACGGCACTCCAGGCTGGTACAACAACCTGGC TdTER R11 CGCTGCTTCGTCGAATGCCAGGTTGTTGTACCAG TdTER F11 ATTCGACGAAGCAGCGAAGCGTGAGGGTCTGTA TdTER R12 CCGTCGATGGTAACAGAGTACAGACCCTCACGCTT TdTER F12 CTCTGTTACCATCGACGGTGACGCGTTCTCTGAC TdTER R13 GATAACCTGAGCTTTGATCTCGTCAGAGAACGCGTCA TdTER F13 GAGATCAAAGCTCAGGTTATCGAGGAAGCTAAAAAGAAAGGTATdTER R14 CACAATCAGGTCGAATTTGATACCTTTCTTTTTAGCTTCCTC TdTER F14 TCAAATTCGACCTGATTGTGTACTCCCTGGCCTCTC TdTER R15 GGTCGGTACGAACCGGAGAGGCCAGGGAGTA TdTER F15 CGGTTCGTACCGACCCGGATACCGGCATCAT TdTER R16 TTCAGTACGCTTTTGTGCATGATGCCGGTATCCG TdTER F16 GCACAAAAGCGTACTGAAGCCGTTTGGCAAAACC TdTER R17 CAACGGTTTTACCAGTGAAGGTTTTGCCAAACGGC TdTER F17 TTCACTGGTAAAACCGTTGATCCTTTCACCGGCG TdTER R18 CGGAGATTTCCTTCAGCTCGCCGGTGAAAGGAT TdTER F18 AGCTGAAGGAAATCTCCGCCGAGCCAGCTAACG TdTER R19 GCAGCAGCCTCCTCATCGTTAGCTGGCTCGG TdTER F19 ATGAGGAGGCTGCTGCGACCGTTAAAGTGATGGGT TdTER R20 GTTCCCAGTCTTCGCCACCCATCACTTTAACGGTC TdTER F20 GGCGAAGACTGGGAACGTTGGATCAAACAACTGTCC TdTER R21 CCAGCAGACCTTCCTTGGACAGTTGTTTGATCCAAC TdTER F21 AAGGAAGGTCTGCTGGAGGAGGGCTGTATTACTCT TdTER R22 GCCGATGTAAGAATATGCCAGAGTAATACAGCCCTCCT TdTER F22 GGCATATTCTTACATCGGCCCGGAGGCGACTCAG TdTER R23 GCCCTTACGATACAGTGCCTGAGTCGCCTCCGG TdTER F23 GCACTGTATCGTAAGGGCACCATCGGTAAAGCGAA TdTER R24 TGGCCTCCAGATGTTCTTTCGCTTTACCGATGGT TdTER F24 AGAACATCTGGAGGCCACCGCTCACCGTCTGA TdTER R25 GCTCGGGTTTTCCTTGTTCAGACGGTGAGCGG TdTER F25 ACAAGGAAAACCCGAGCATCCGTGCTTTCGTGT TdTER R26 AGGCCCTTGTTAACGGACACGAAAGCACGGAT TdTER F26 CCGTTAACAAGGGCCTGGTTACGCGCGCTTC TdTER R27 TGACCGGAATTACTGCGGAAGCGCGCGTAACC TdTER F27 CGCAGTAATTCCGGTCATTCCGCTGTACCTGGC TdTER R28 TTCATGACTTTAAACAGGGAAGCCAGGTACAGCGGAA TdTER F28 TTCCCTGTTTAAAGTCATGAAAGAAAAAGGCAACCACG TdTER R29 AGTAATTTGTTCGATACAACCTTCGTGGTTGCCTTTTTCT TdTER F29 AAGGTTGTATCGAACAAATTACTCGCCTGTATGCGGAG TdTER R30 CCTTACGGTACAGGCGCTCCGCATACAGGCG TdTER F30 CGCCTGTACCGTAAGGATGGCACTATCCCGGT TdTER R31 TGCGGTTCTCTTCATCAACCGGGATAGTGCCAT TdTER F31 TGATGAAGAGAACCGCATCCGCATTGACGATTGG TdTER R32 TGTACATCCTCTTCCAGTTCCCAATCGTCAATGCGGA

151

TdTER F32 GAACTGGAAGAGGATGTACAGAAAGCGGTTTCCGC TdTER R33 CGTCACTTTTTCCATCAGCGCGGAAACCGCTTTC TdTER F33 GCTGATGGAAAAAGTGACGGGCGAAAACGCGGAA TdTER R34 CCAGATCCGTCAGGGATTCCGCGTTTTCGCC TdTER F34 TCCCTGACGGATCTGGCAGGTTACCGTCACGA TdTER R35 ACCATTAGACGCCAGAAAGTCGTGACGGTAACCTG TdTER F35 CTTTCTGGCGTCTAATGGTTTCGACGTTGAGGGTATTA TdTER R36 CAACTTCTGCCTCGTAGTTAATACCCTCAACGTCGAA TdTER F36 ACTACGAGGCAGAAGTTGAACGTTTCGATCGTATTTAATCT TdTER R37 atgccctggCGTTCTAGATTAAATACGATCGAAACGTT TdTER F37 AGAACGccagggcat

152

Figure A1.1. Oligonucleotide maps for the assembly of synthetic genes (A) accA3, (B) accBC, (C) accD4, (D) accD5, (E) accD6, (F) accE, (G) tbACS4, (H) aldh593, (I) alkK, (J) atfA, (K) bdhA, (L) bdhB, (M) ccr (from Streptomyces cinnamonensis), (N) dtsR1 (O) jFar, (P) hbd, (Q) nphT7, (R) phaJ, (S) ppPhaJ4, (T) PhaZ1, (U) egTer, (V) tdTer.

A. AccA3: Gene name from Mycobacterium tuberculosis H37Rv (Accession NP217802)

5'– ATGGCAAGCC ACGCAGGTAG CCGTATCGCC CGCATTAGCA AAGTCCTGGT GGCCAACCGT GGTGAGATCG CAGTTCGTGT 3'– TACCGTTCGG TGCGTCCATC GGCATAGCGG GCGTAATCGT TTCAGGACCA CCGGTTGGCA CCACTCTAGC GTCAAGCACA

CATCCGTGCC GCACGTGATG CAGGTTTGCC GTCTGTGGCG GTCTACGCAG AGCCGGATGC CGAATCCCCG CACGTCCGTC GTAGGCACGG CGTGCACTAC GTCCAAACGG CAGACACCGC CAGATGCGTC TCGGCCTACG GCTTAGGGGC GTGCAGGCAG TGGCCGACGA GGCTTTTGCA CTGGGTGGTC AGACGAGCGC GGAAAGCTAC CTGGACTTTG CGAAAATTTT GGACGCCGCG ACCGGCTGCT CCGAAAACGT GACCCACCAG TCTGCTCGCG CCTTTCGATG GACCTGAAAC GCTTTTAAAA CCTGCGGCGC GCAAAGAGCG GTGCCAACGC GATTCATCCG GGTTATGGCT TTCTGGCCGA AAACGCGGAC TTTGCACAAG CGGTTATTGA CGTTTCTCGC CACGGTTGCG CTAAGTAGGC CCAATACCGA AAGACCGGCT TTTGCGCCTG AAACGTGTTC GCCAATAACT CGCCGGTCTG ATTTGGATCG GCCCGAGCCC GCAAAGCATC CGCGACCTGG GTGACAAAGT CACGGCACGT CACATCGCGG GCGGCCAGAC TAAACCTAGC CGGGCTCGGG CGTTTCGTAG GCGCTGGACC CACTGTTTCA GTGCCGTGCA GTGTAGCGCC CGCGTGCCCA AGCGCCGCTG GTGCCGGGTA CGCCGGACCC GGTGAAAGGT GCAGACGAAG TCGTTGCGTT CGCAGAAGAG GCGCACGGGT TCGCGGCGAC CACGGCCCAT GCGGCCTGGG CCACTTTCCA CGTCTGCTTC AGCAACGCAA GCGTCTTCTC TACGGTCTGC CGATCGCTAT CAAGGCAGCC CACGGCGGCG GCGGTAAAGG TATGAAAGTT GCGCGTACCA TCGACGAAAT ATGCCAGACG GCTAGCGATA GTTCCGTCGG GTGCCGCCGC CGCCATTTCC ATACTTTCAA CGCGCATGGT AGCTGCTTTA CCCGGAGCTG TACGAAAGCG CAGTGCGTGA GGCGACGGCG GCCTTCGGTC GTGGCGAATG CTACGTTGAA CGTTATTTGG GGGCCTCGAC ATGCTTTCGC GTCACGCACT CCGCTGCCGC CGGAAGCCAG CACCGCTTAC GATGCAACTT GCAATAAACC ATAAGCCGCG CCACGTGGAG GCCCAGGTGA TTGCCGACCA GCACGGTAAC GTTGTGGTGG CCGGTACGCG CGACTGCTCC TATTCGGCGC GGTGCACCTC CGGGTCCACT AACGGCTGGT CGTGCCATTG CAACACCACC GGCCATGCGC GCTGACGAGG TTGCAGCGTC GCTATCAGAA ACTGGTCGAA GAGGCACCGG CCCCGTTCCT GACCGACTTT CAACGTAAGG AGATCCACGA AACGTCGCAG CGATAGTCTT TGACCAGCTT CTCCGTGGCC GGGGCAAGGA CTGGCTGAAA GTTGCATTCC TCTAGGTGCT CAGCGCGAAA CGCATTTGCA AGGAGGCGCA TTACCACGGT GCGGGCACCG TCGAGTATCT GGTGGGTCAA GATGGCCTGA GTCGCGCTTT GCGTAAACGT TCCTCCGCGT AATGGTGCCA CGCCCGTGGC AGCTCATAGA CCACCCAGTT CTACCGGACT TTTCTTTTCT GGAAGTGAAC ACGCGCCTGC AAGTTGAACA CCCGGTCACG GAAGAAACGG CGGGTATTGA TCTGGTGCTG AAAGAAAAGA CCTTCACTTG TGCGCGGACG TTCAACTTGT GGGCCAGTGC CTTCTTTGCC GCCCATAACT AGACCACGAC CAACAATTCC GCATTGCGAA CGGCGAAAAA CTGGACATCA CCGAGGACCC GACCCCGCGT GGTCACGCGA TCGAGTTCCG GTTGTTAAGG CGTAACGCTT GCCGCTTTTT GACCTGTAGT GGCTCCTGGG CTGGGGCGCA CCAGTGCGCT AGCTCAAGGC CATTAATGGT GAGGATGCGG GTCGTAACTT TCTGCCGGCT CCGGGTCCGG TGACGAAGTT TCATCCGCCG AGCGGTCCGG GTAATTACCA CTCCTACGCC CAGCATTGAA AGACGGCCGA GGCCCAGGCC ACTGCTTCAA AGTAGGCGGC TCGCCAGGCC GCGTTCGTGT GGATAGCGGT GTTGAGACGG GCAGCGTCAT TGGTGGCCAG TTTGATAGCA TGTTGGCGAA ACTGATTGTT CGCAAGCACA CCTATCGCCA CAACTCTGCC CGTCGCAGTA ACCACCGGTC AAACTATCGT ACAACCGCTT TGACTAACAA CACGGTGCAG ATCGTGCGGA GGCGCTGGCG CGTGCGCGTC GTGCGCTGAA CGAGTTCGGT GTGGAAGGTT TGGCTACCGT GTGCCACGTC TAGCACGCCT CCGCGACCGC GCACGCGCAG CACGCGACTT GCTCAAGCCA CACCTTCCAA ACCGATGGCA GATTCCGTTC CATCGCGCAG TTGTGAGCGA CCCGGCTTTT ATTGGTGATG CTAATGGCTT CTCCGTTCAC ACGCGCTGGA CTAAGGCAAG GTAGCGCGTC AACACTCGCT GGGCCGAAAA TAACCACTAC GATTACCGAA GAGGCAAGTG TGCGCGACCT TCGAGACCGA GTGGAACAAT ACCATCGAGC CGTTCACCGA TGGTGAGCCG CTGGACGAGG ATGCGCGTCC GCGTCAAAAA AGCTCTGGCT CACCTTGTTA TGGTAGCTCG GCAAGTGGCT ACCACTCGGC GACCTGCTCC TACGCGCAGG CGCAGTTTTT GTCGTGGTCG AAATCGACGG TCGTCGCGTC GAGGTGAGCC TGCCGGCGGA TCTGGCGCTG AGCAACGGTG GTGGCTGTGA CAGCACCAGC TTTAGCTGCC AGCAGCGCAG CTCCACTCGG ACGGCCGCCT AGACCGCGAC TCGTTGCCAC CACCGACACT CCCGGTCGGT GTTATTCGCC GTAAGCCGAA GCCGCGTAAG CGCGGTGCTC ACACCGGCGC GGCGGCCAGC GGCGACGCCG GGGCCAGCCA CAATAAGCGG CATTCGGCTT CGGCGCATTC GCGCCACGAG TGTGGCCGCG CCGCCGGTCG CCGCTGCGGC

153

TGACGGCACC GATGCAGGGC ACCGTTGTGA AATTTGCAGT TGAGGAAGGC CAAGAGGTCG TGGCCGGCGA CCTGGTGGTC ACTGCCGTGG CTACGTCCCG TGGCAACACT TTAAACGTCA ACTCCTTCCG GTTCTCCAGC ACCGGCCGCT GGACCACCAG GTCCTGGAGG CAATGAAGAT GGAGAACCCG GTTACCGCGC ACAAAGACGG CACCATCACG GGTCTGGCGG TGGAGGCGGG CAGGACCTCC GTTACTTCTA CCTCTTGGGC CAATGGCGCG TGTTTCTGCC GTGGTAGTGC CCAGACCGCC ACCTCCGCCC TGCGGCGATT ACCCAAGGCA CGGTCCTGGC GGAGATTAAA TAAtagaagc tctaacgtag acgtga -3' ACGCCGCTAA TGGGTTCCGT GCCAGGACCG CCTCTAATTT ATTatcttcg agattgcatc tgcact -5'

154

B. AccBC: Gene name from Corynebacterium glutamicum ATCC 13032 (Accession NP599932)

5'– GGGCTGCCGA AGCCACGGCT AATAGAAGGA AGGATAAGGC ATGAGCGTTG AAACCCGCAA GATTACGAAA GTTCTGGTGG 3'– CCCGACGGCT TCGGTGCCGA TTATCTTCCT TCCTATTCCG TACTCGCAAC TTTGGGCGTT CTAATGCTTT CAAGACCACC

CAAACCGTGG TGAAATCGCC ATTCGTGTTT TCCGTGCGGC ACGTGACGAA GGCATCGGCA GCGTCGCAGT TTACGCCGAG GTTTGGCACC ACTTTAGCGG TAAGCACAAA AGGCACGCCG TGCACTGCTT CCGTAGCCGT CGCAGCGTCA AATGCGGCTC

CCGGATGCGG ATGCGCCGTT CGTGAGCTAT GCCGATGAGG CGTTCGCGTT GGGTGGTCAG ACGAGCGCAG AGAGCTATCT GGCCTACGCC TACGCGGCAA GCACTCGATA CGGCTACTCC GCAAGCGCAA CCCACCAGTC TGCTCGCGTC TCTCGATAGA

GGTTATCGAC AAAATTATCG ATGCGGCGCG TAAGAGCGGC GCGGATGCGA TCCATCCGGG CTACGGTTTT TTGGCCGAAA CCAATAGCTG TTTTAATAGC TACGCCGCGC ATTCTCGCCG CGCCTACGCT AGGTAGGCCC GATGCCAAAA AACCGGCTTT

ATGCAGACTT TGCGGAAGCA GTCATTAACG AAGGTCTGAT TTGGATCGGC CCGAGCCCGG AATCCATCCG CAGCCTGGGT TACGTCTGAA ACGCCTTCGT CAGTAATTGC TTCCAGACTA AACCTAGCCG GGCTCGGGCC TTAGGTAGGC GTCGGACCCA

GACAAAGTCA CCGCGCGTCA CATCGCAGAT ACCGCAAAAG CCCCGATGGC TCCGGGCACC AAGGAGCCGG TTAAGGATGC CTGTTTCAGT GGCGCGCAGT GTAGCGTCTA TGGCGTTTTC GGGGCTACCG AGGCCCGTGG TTCCTCGGCC AATTCCTACG

GGCGGAGGTC GTTGCGTTTG CGGAGGAGTT CGGCCTGCCG ATCGCAATTA AAGCAGCGTT TGGCGGCGGT GGTCGTGGTA CCGCCTCCAG CAACGCAAAC GCCTCCTCAA GCCGGACGGC TAGCGTTAAT TTCGTCGCAA ACCGCCGCCA CCAGCACCAT

TGAAGGTCGC GTACAAGATG GAAGAAGTCG CAGATTTGTT CGAAAGCGCT ACCCGTGAAG CGACCGCGGC GTTCGGTCGT ACTTCCAGCG CATGTTCTAC CTTCTTCAGC GTCTAAACAA GCTTTCGCGA TGGGCACTTC GCTGGCGCCG CAAGCCAGCA

GGTGAATGTT TCGTGGAGCG CTATCTGGAT AAGGCTCGCC ACGTTGAAGC TCAGGTCATT GCGGACAAAC ATGGCAATGT CCACTTACAA AGCACCTCGC GATAGACCTA TTCCGAGCGG TGCAACTTCG AGTCCAGTAA CGCCTGTTTG TACCGTTACA

TGTCGTGGCG GGCACGCGCG ACTGTAGCCT GCAACGCCGT TTCCAGAAAC TGGTCGAGGA AGCACCGGCA CCGTTTCTGA ACAGCACCGC CCGTGCGCGC TGACATCGGA CGTTGCGGCA AAGGTCTTTG ACCAGCTCCT TCGTGGCCGT GGCAAAGACT

CGGACGACCA ACGCGAGCGC CTGCATAGCT CTGCAAAAGC AATTTGCAAG GAGGCCGGTT ACTATGGCGC AGGCACCGTT GCCTGCTGGT TGCGCTCGCG GACGTATCGA GACGTTTTCG TTAAACGTTC CTCCGGCCAA TGATACCGCG TCCGTGGCAA

GAGTATCTGG TCGGTAGCGA CGGTCTGATC TCCTTTCTGG AGGTCAACAC CCGCTTGCAA GTGGAGCACC CGGTTACCGA CTCATAGACC AGCCATCGCT GCCAGACTAG AGGAAAGACC TCCAGTTGTG GGCGAACGTT CACCTCGTGG GCCAATGGCT

GGAGACCACC GGCATTGACC TGGTGCGTGA GATGTTTCGT ATTGCCGAAG GTCATGAGCT GAGCATCAAG GAGGACCCGG CCTCTGGTGG CCGTAACTGG ACCACGCACT CTACAAAGCA TAACGGCTTC CAGTACTCGA CTCGTAGTTC CTCCTGGGCC

CACCGCGTGG TCACGCATTC GAGTTCCGTA TCAATGGCGA AGATGCGGGC AGCAACTTCA TGCCGGCACC GGGCAAGATC GTGGCGCACC AGTGCGTAAG CTCAAGGCAT AGTTACCGCT TCTACGCCCG TCGTTGAAGT ACGGCCGTGG CCCGTTCTAG

ACCAGCTACC GCGAGCCGCA GGGTCCGGGC GTGCGTATGG ACAGCGGCGT GGTCGAGGGC AGCGAGATTA GCGGTCAGTT TGGTCGATGG CGCTCGGCGT CCCAGGCCCG CACGCATACC TGTCGCCGCA CCAGCTCCCG TCGCTCTAAT CGCCAGTCAA

CGACTCTATG CTGGCAAAAC TGATTGTTTG GGGCGATACC CGTGAGCAGG CACTGCAACG TTCTCGTCGT GCGCTGGCGG GCTGAGATAC GACCGTTTTG ACTAACAAAC CCCGCTATGG GCACTCGTCC GTGACGTTGC AAGAGCAGCA CGCGACCGCC

AATATGTCGT GGAAGGTATG CCGACCGTTA TTCCGTTCCA CCAACATATT GTCGAAAACC CGGCGTTCGT CGGTAATGAT TTATACAGCA CCTTCCATAC GGCTGGCAAT AAGGCAAGGT GGTTGTATAA CAGCTTTTGG GCCGCAAGCA GCCATTACTA

GAGGGTTTCG AAATCTACAC GAAGTGGATT GAGGAGGTGT GGGATAACCC GATCGCCCCG TACGTTGATG CGTCCGAGTT CTCCCAAAGC TTTAGATGTG CTTCACCTAA CTCCTCCACA CCCTATTGGG CTAGCGGGGC ATGCAACTAC GCAGGCTCAA

GGATGAAGAC GAGGATAAAA CCCCGGCCCA AAAAGTTGTC GTCGAAATTA ACGGTCGCCG TGTTGAAGTG GCCCTGCCGG CCTACTTCTG CTCCTATTTT GGGGCCGGGT TTTTCAACAG CAGCTTTAAT TGCCAGCGGC ACAACTTCAC CGGGACGGCC

GCGACTTGGC ATTGGGTGGT ACGGCAGGTC CGAAAAAAAA AGCGAAGAAG CGCCGCGCAG GTGGCGCTAA AGCAGGCGTG CGCTGAACCG TAACCCACCA TGCCGTCCAG GCTTTTTTTT TCGCTTCTTC GCGGCGCGTC CACCGCGATT TCGTCCGCAC

AGCGGCGATG CGGTCGCAGC TCCGATGCAA GGCACGGTTA TTAAGGTCAA TGTTGAAGAA GGTGCAGAAG TCAACGAAGG TCGCCGCTAC GCCAGCGTCG AGGCTACGTT CCGTGCCAAT AATTCCAGTT ACAACTTCTT CCACGTCTTC AGTTGCTTCC

TGACACCGTT GTGGTTCTGG AGGCAATGAA AATGGAGAAT CCGGTGAAGG CCCACAAAAG CGGCACCGTG ACGGGTCTGA ACTGTGGCAA CACCAAGACC TCCGTTACTT TTACCTCTTA GGCCACTTCC GGGTGTTTTC GCCGTGGCAC TGCCCAGACT

CGGTTGCGGC GGGTGAGGGT GTTAATAAAG GTGTCGTTCT GCTGGAGATT AAGTAAgccg agcctcagcc ccatggg -3' GCCAACGCCG CCCACTCCCA CAATTATTTC CACAGCAAGA CGACCTCTAA TTCATTcggc tcggagtcgg ggtaccc -5'

155

C. AccD4: Gene name from Mycobacterium tuberculosis H37Rv (Accession NP218316)

5'– ctggaaaggc gggaATGACG GTTACGGAGC CGGTTCTGCA CACGACGGCG GAAAAGCTGG CTGAGCTGCG TGAGCGCCTG 3'– gacctttccg ccctTACTGC CAATGCCTCG GCCAAGACGT GTGCTGCCGC CTTTTCGACC GACTCGACGC ACTCGCGGAC

GAACTGGCTA AAGAGCCGGG TGGCGAGAAA GCTGCGGCGA AGCGCGATAA GAAAGGTATC CCGTCCGCGC GTGCACGTAT CTTGACCGAT TTCTCGGCCC ACCGCTCTTT CGACGCCGCT TCGCGCTATT CTTTCCATAG GGCAGGCGCG CACGTGCATA CTATGAGCTG GTCGATCCGG GTAGCTTTAT GGAGATTGGT GCGCTGTGCC GCACGCCGGG TGACCCGAAC GCCTTGTACG GATACTCGAC CAGCTAGGCC CATCGAAATA CCTCTAACCA CGCGACACGG CGTGCGGCCC ACTGGGCTTG CGGAACATGC GTGACGGCGT GGTGACCGGC CACGGTCTGA TTAATGGTCG TCCGGTGGGC GTTTTTAGCC ATGACCAGAC CGTGTTCGGT CACTGCCGCA CCACTGGCCG GTGCCAGACT AATTACCAGC AGGCCACCCG CAAAAATCGG TACTGGTCTG GCACAAGCCA GGCACGGTGG GTGAAATGTT TGGTCGTAAA GTTGCACGTC TGATGGAGTG GTGTGCGATG GTCGGCTGTC CGATTGTCGG CCGTGCCACC CACTTTACAA ACCAGCATTT CAACGTGCAG ACTACCTCAC CACACGCTAC CAGCCGACAG GCTAACAGCC CATCAACGAC TCTGGTGGTG CCCGTATTCA GGATGCAGTG ACCAGCTTGG CATGGTACGC CGAGCTGGGC CGTCGTCACG GTAGTTGCTG AGACCACCAC GGGCATAAGT CCTACGTCAC TGGTCGAACC GTACCATGCG GCTCGACCCG GCAGCAGTGC AACTGCTGAG CGGCCTGGTC CCGCAAATCT CCATCATTCT GGGTAAGTGT GCAGGTGGCG CGGTTTACAG CCCGATCCAA TTGACGACTC GCCGGACCAG GGCGTTTAGA GGTAGTAAGA CCCATTCACA CGTCCACCGC GCCAAATGTC GGGCTAGGTT ACCGATCTGG TCGTTGCCGT CCGCGATCAG GGCTATATGT TCGTCACCGG CCCGGACGTT ATCAAGGATG TTACGGGCGA TGGCTAGACC AGCAACGGCA GGCGCTAGTC CCGATATACA AGCAGTGGCC GGGCCTGCAA TAGTTCCTAC AATGCCCGCT GGACGTTAGC CTGGATGAGC TGGGTGGTGC CGACCACCAG GCCAGCTACG GTAACATTCA TCAGGTGGTC GAAAGCGAAG CCTGCAATCG GACCTACTCG ACCCACCACG GCTGGTGGTC CGGTCGATGC CATTGTAAGT AGTCCACCAG CTTTCGCTTC CGGCAGCCTA CCAGTATGTG CGCGACTTCC TGAGCTTCCT GCCGTCTAAT TGCTTCGATA AACCGCCGGT GGTCAATCCG GCCGTCGGAT GGTCATACAC GCGCTGAAGG ACTCGAAGGA CGGCAGATTA ACGAAGCTAT TTGGCGGCCA CCAGTTAGGC GGCCTGGAGC CGGAAATTAC GGGTCACGAT TTGGAGCTGG ACAGCATCGT GCCGGATAGC GATAACATGG CGTATGACAT CCGGACCTCG GCCTTTAATG CCCAGTGCTA AACCTCGACC TGTCGTAGCA CGGCCTATCG CTATTGTACC GCATACTGTA GCACGAGGTT CTGCTGCGTA TCTTTGACGA TGGTGATTTT CTGGATGTCG CGGCACAGGC AGGCCAAGCA ATCATTACGG CGTGCTCCAA GACGACGCAT AGAAACTGCT ACCACTAAAA GACCTACAGC GCCGTGTCCG TCCGGTTCGT TAGTAATGCC GTTACGCCCG TGTCGATGGT CGTACGGTTG GCGTTGTTGC GAATCAACCG ATGCACATGA GCGGCGCGAT TGACAATGAG CAATGCGGGC ACAGCTACCA GCATGCCAAC CGCAACAACG CTTAGTTGGC TACGTGTACT CGCCGCGCTA ACTGTTACTC GCGAGCGACA AGGCTGCACG TTTCATTCGC TTCAGCGATG CGTTCGATAT TCCGTTGGTG TTCGTCGTCG ATACGCCGGG CGCTCGCTGT TCCGACGTGC AAAGTAAGCG AAGTCGCTAC GCAAGCTATA AGGCAACCAC AAGCAGCAGC TATGCGGCCC TTTCCTGCCG GGCGTCGAGC AAGAAAAGAA TGGTATCATC AAGCGCGGCG GTCGCTTCCT GTATGCGGTC GTGGAAGCTG AAAGGACGGC CCGCAGCTCG TTCTTTTCTT ACCATAGTAG TTCGCGCCGC CAGCGAAGGA CATACGCCAG CACCTTCGAC ATGTGCCGAA AGTTACGATT ACGATTCGCA AGAGCTATGG CGGCGCGTAC GCTGTGATGG GCAGCAAGCA ACTGACCGCC TACACGGCTT TCAATGCTAA TGCTAAGCGT TCTCGATACC GCCGCGCATG CGACACTACC CGTCGTTCGT TGACTGGCGG GATCTGAATT TCGCGTGGCC GACCGCACGC ATCGCAGTGA TTGGTGCAGA TGGCGCTGCC CAATTGCTGA TGAAACGCTT CTAGACTTAA AGCGCACCGG CTGGCGTGCG TAGCGTCACT AACCACGTCT ACCGCGACGG GTTAACGACT ACTTTGCGAA CCCGGACCCG AATGCCCCGG AGGCGCAGGC GATCCGTAAG TCCTTCGTTG AGAATTACAA TCTGAATATG GCTATTCCGT GGGCCTGGGC TTACGGGGCC TCCGCGTCCG CTAGGCATTC AGGAAGCAAC TCTTAATGTT AGACTTATAC CGATAAGGCA GGATCGCAGC GGAACGCGGT TTTATCGATG CCGTCATTGA CCCGCATGAA ACGCGCTTGC TGCTGCGCAA ATCTATGCAC CCTAGCGTCG CCTTGCGCCA AAATAGCTAC GGCAGTAACT GGGCGTACTT TGCGCGAACG ACGACGCGTT TAGATACGTG CTGCTGCGCG ACAAACAACT GTGGTGGCGT GTCGGTCGTA AACATGGTCT GATCCCGGTC TAAccccatt acgcgg -3' GACGACGCGC TGTTTGTTGA CACCACCGCA CAGCCAGCAT TTGTACCAGA CTAGGGCCAG ATTggggtaa tgcgcc -5'

156

D. AccD5 : Gene name from Mycobacterium tuberculosis H37Rv (Accession #NP217797) 5’- tcccacagtc gatcATGACC AGCGTGACCG ATCGTAGCGC TCACTCTGCT GAACGTTCTA CCGAACACAC CATTGACATC 3’- agggtgtcag ctagTACTGG TCGCACTGGC TAGCATCGCG AGTGAGACGA CTTGCAAGAT GGCTTGTGTG GTAACTGTAG

CACACCACCG CGGGTAAGCT GGCGGAGCTG CATAAACGTC GTGAAGAGAG CCTGCATCCG GTCGGTGAAG ATGCCGTTGA GTGTGGTGGC GCCCATTCGA CCGCCTCGAC GTATTTGCAG CACTTCTCTC GGACGTAGGC CAGCCACTTC TACGGCAACT GAAGGTCCAC GCGAAAGGTA AGCTGACGGC TCGTGAGCGT ATCTACGCGC TGTTGGACGA AGACAGCTTC GTTGAACTGG CTTCCAGGTG CGCTTTCCAT TCGACTGCCG AGCACTCGCA TAGATGCGCG ACAACCTGCT TCTGTCGAAG CAACTTGACC ACGCGCTGGC GAAACATCGT AGCACGAACT TCAATCTGGG TGAGAAACGT CCGCTGGGCG ACGGTGTGGT CACGGGCTAC TGCGCGACCG CTTTGTAGCA TCGTGCTTGA AGTTAGACCC ACTCTTTGCA GGCGACCCGC TGCCACACCA GTGCCCGATG GGCACCATCG ACGGTCGTGA CGTGTGCATC TTTTCCCAAG ACGCCACCGT GTTTGGTGGC AGCCTGGGTG AAGTTTATGG CCGTGGTAGC TGCCAGCACT GCACACGTAG AAAAGGGTTC TGCGGTGGCA CAAACCACCG TCGGACCCAC TTCAAATACC TGAAAAGATC GTCAAGGTCC AAGAGTTGGC AATTAAAACG GGTCGTCCGC TGATCGGTAT CAACGATGGC GCTGGTGCTC ACTTTTCTAG CAGTTCCAGG TTCTCAACCG TTAATTTTGC CCAGCAGGCG ACTAGCCATA GTTGCTACCG CGACCACGAG GTATTCAAGA AGGTGTGGTT AGCCTGGGCC TGTACAGCCG TATCTTTCGT AATAATATTC TGGCGAGCGG TGTTATTCCG CATAAGTTCT TCCACACCAA TCGGACCCGG ACATGTCGGC ATAGAAAGCA TTATTATAAG ACCGCTCGCC ACAATAAGGC CAAATCTCTC TGATTATGGG CGCGGCTGCC GGTGGTCACG TCTATAGCCC GGCACTGACC GACTTCGTTA TCATGGTTGA GTTTAGAGAG ACTAATACCC GCGCCGACGG CCACCAGTGC AGATATCGGG CCGTGACTGG CTGAAGCAAT AGTACCAACT CCAGACCTCC CAGATGTTTA TTACCGGCCC GGATGTTATC AAGACGGTGA CGGGTGAGGA GGTCACGATG GAAGAGCTGG GGTCTGGAGG GTCTACAAAT AATGGCCGGG CCTACAATAG TTCTGCCACT GCCCACTCCT CCAGTGCTAC CTTCTCGACC GCGGTGCGCA TACGCACATG GCGAAGTCCG GCACGGCACA TTACGCAGCA AGCGGTGAGC AAGATGCCTT CGATTACGTC CGCCACGCGT ATGCGTGTAC CGCTTCAGGC CGTGCCGTGT AATGCGTCGT TCGCCACTCG TTCTACGGAA GCTAATGCAG CGTGAACTGC TGAGCTACTT GCCGCCGAAT AATTCCACGG ATGCCCCGCG CTATCAGGCA GCGGCTCCGA CGGGTCCGAT GCACTTGACG ACTCGATGAA CGGCGGCTTA TTAAGGTGCC TACGGGGCGC GATAGTCCGT CGCCGAGGCT GCCCAGGCTA TGAAGAGAAT CTGACGGACG AGGATCTGGA ACTGGATACG CTGATCCCGG ATAGCCCGAA TCAGCCGTAC GACATGCACG ACTTCTCTTA GACTGCCTGC TCCTAGACCT TGACCTATGC GACTAGGGCC TATCGGGCTT AGTCGGCATG CTGTACGTGC AGGTTATTAC CCGTTTGCTG GACGATGAGT TCCTGGAGAT TCAAGCCGGT TACGCACAGA ACATTGTGGT GGGCTTTGGC TCCAATAATG GGCAAACGAC CTGCTACTCA AGGACCTCTA AGTTCGGCCA ATGCGTGTCT TGTAACACCA CCCGAAACCG CGCATCGATG GCCGCCCGGT GGGCATTGTC GCCAACCAAC CGACCCATTT TGCAGGCTGT CTGGACATTA ACGCCAGCGA GCGTAGCTAC CGGCGGGCCA CCCGTAACAG CGGTTGGTTG GCTGGGTAAA ACGTCCGACA GACCTGTAAT TGCGGTCGCT AAAAGCCGCA CGTTTCGTTC GTACGTGTGA CTGCTTCAAT ATCCCGATCG TTATGCTGGT GGATGTTCCG GGTTTCCTGC TTTTCGGCGT GCAAAGCAAG CATGCACACT GACGAAGTTA TAGGGCTAGC AATACGACCA CCTACAAGGC CCAAAGGACG CGGGTACGGA TCAAGAGTAT AATGGCATTA TCCGCCGCGG TGCGAAATTG CTGTATGCCT ATGGTGAGGC AACGGTTCCG GCCCATGCCT AGTTCTCATA TTACCGTAAT AGGCGGCGCC ACGCTTTAAC GACATACGGA TACCACTCCG TTGCCAAGGC AAAATTACGG TCATCACCCG TAAAGCGTAT GGTGGCGCGT ACTGCGTTAT GGGCAGCAAG GACATGGGTT GCGACGTTAA TTTTAATGCC AGTAGTGGGC ATTTCGCATA CCACCGCGCA TGACGCAATA CCCGTCGTTC CTGTACCCAA CGCTGCAATT CCTGGCCTGG CCGACGGCGC AAATCGCGGT GATGGGTGCC AGCGGCGCGG TGGGCTTCGT CTATCGTCAA CAACTGGCCG GGACCGGACC GGCTGCCGCG TTTAGCGCCA CTACCCACGG TCGCCGCGCC ACCCGAAGCA GATAGCAGTT GTTGACCGGC AAGCAGCCGC TAACGGTGAG GATATTGATA AGTTGCGTCT GCGCCTGCAA CAGGAGTATG AAGATACCTT GGTCAATCCG TTCGTCGGCG ATTGCCACTC CTATAACTAT TCAACGCAGA CGCGGACGTT GTCCTCATAC TTCTATGGAA CCAGTTAGGC TACGTTGCGG CGGAACGCGG CTATGTTGAC GCCGTGATCC CGCCGAGCCA TACCCGTGGT TACATTGGCA CCGCGTTGCG ATGCAACGCC GCCTTGCGCC GATACAACTG CGGCACTAGG GCGGCTCGGT ATGGGCACCA ATGTAACCGT GGCGCAACGC CCTGTTGGAA CGTAAGATTG CGCAACTGCC GCCGAAAAAG CATGGTAACG TCCCGCTGTA Aagggtaacg tctacgaggt GGACAACCTT GCATTCTAAC GCGTTGACGG CGGCTTTTTC GTACCATTGC AGGGCGACAT Ttcccattgc agatgctcca a -3’ t -5’

157

E. AccD6: Gene name from Mycobacterium tuberculosis H37Rv (Accession NP216763)

5'– gATGACGATT ATGGCACCGG AAGCAGTCGG CGAAAGCCTG GACCCGCGCG ACCCGCTGCT GCGTCTGAGC AATTTTTTTG 3'– cTACTGCTAA TACCGTGGCC TTCGTCAGCC GCTTTCGGAC CTGGGCGCGC TGGGCGACGA CGCAGACTCG TTAAAAAAAC

ATGATGGTAG CGTGGAGTTG CTGCACGAAC GTGACCGTAG CGGCGTCCTG GCGGCAGCAG GCACCGTCAA CGGTGTGCGC TACTACCATC GCACCTCAAC GACGTGCTTG CACTGGCATC GCCGCAGGAC CGCCGTCGTC CGTGGCAGTT GCCACACGCG ACCATCGCGT TTTGCACGGA TGGCACCGTT ATGGGTGGTG CAATGGGCGT TGAAGGCTGT ACCCACATTG TTAATGCCTA TGGTAGCGCA AAACGTGCCT ACCGTGGCAA TACCCACCAC GTTACCCGCA ACTTCCGACA TGGGTGTAAC AATTACGGAT CGATACGGCG ATTGAAGACC AAAGCCCGAT TGTTGGCATC TGGCACTCTG GCGGTGCACG TCTGGCGGAA GGTGTTCGCG GCTATGCCGC TAACTTCTGG TTTCGGGCTA ACAACCGTAG ACCGTGAGAC CGCCACGTGC AGACCGCCTT CCACAAGCGC CTTTGCACGC TGTGGGCCAG GTGTTCGAGG CGATGATCCG CGCTTCTGGC TATATTCCGC AGATTTCCGT TGTGGTGGGT GAAACGTGCG ACACCCGGTC CACAAGCTCC GCTACTAGGC GCGAAGACCG ATATAAGGCG TCTAAAGGCA ACACCACCCA TTCGCGGCGG GTGGTGCGGC CTACGGTCCG GCCTTGACCG ACGTTGTGGT TATGGCCCCG GAAAGCCGCG TTTTTGTCAC AAGCGCCGCC CACCACGCCG GATGCCAGGC CGGAACTGGC TGCAACACCA ATACCGGGGC CTTTCGGCGC AAAAACAGTG CGGCCCGGAC GTGGTTCGTA GCGTCACGGG TGAGGACGTT GATATGGCGA GCCTGGGCGG TCCGGAAACC CATCATAAAA GCCGGGCCTG CACCAAGCAT CGCAGTGCCC ACTCCTGCAA CTATACCGCT CGGACCCGCC AGGCCTTTGG GTAGTATTTT AGAGCGGCGT TTGTCACATC GTGGCGGATG ACGAACTGGA CGCATACGAC CGTGGCCGTC GCCTGGTGGG TCTGTTCTGC TCTCGCCGCA AACAGTGTAG CACCGCCTAC TGCTTGACCT GCGTATGCTG GCACCGGCAG CGGACCACCC AGACAAGACG CAGCAGGGTC ACTTTGACCG CAGCAAGGCG GAGGCGGGCG ACACCGACAT CCACGCGCTG CTGCCGGAAA GCTCCCGTCG GTCGTCCCAG TGAAACTGGC GTCGTTCCGC CTCCGCCCGC TGTGGCTGTA GGTGCGCGAC GACGGCCTTT CGAGGGCAGC TGCGTACGAC GTTCGCCCGA TCGTTACGGC AATCCTGGAT GCAGATACGC CGTTCGACGA ATTTCAAGCC AACTGGGCAC ACGCATGCTG CAAGCGGGCT AGCAATGCCG TTAGGACCTA CGTCTATGCG GCAAGCTGCT TAAAGTTCGG TTGACCCGTG CGAGCATGGT TGTCGGTTTG GGTCGTCTGA GCGGTCGTAC CGTTGGTGTC CTGGCGAATA ATCCGTTGCG TTTGGGCGGC GCTCGTACCA ACAGCCAAAC CCAGCAGACT CGCCAGCATG GCAACCACAG GACCGCTTAT TAGGCAACGC AAACCCGCCG TGTCTGAATA GCGAGAGCGC CGAGAAAGCT GCTCGCTTTG TCCGCCTGTG CGACGCTTTT GGTATCCCGC TGGTCGTTGT ACAGACTTAT CGCTCTCGCG GCTCTTTCGA CGAGCGAAAC AGGCGGACAC GCTGCGAAAA CCATAGGGCG ACCAGCAACA GGTCGATGTT CCGGGCTACC TGCCGGGTGT GGACCAGGAA TGGGGTGGTG TGGTGCGCCG TGGTGCGAAA TTGTTGCACG CCAGCTACAA GGCCCGATGG ACGGCCCACA CCTGGTCCTT ACCCCACCAC ACCACGCGGC ACCACGCTTT AACAACGTGC CATTCGGCGA GTGCACCGTT CCGCGCGTGA CCCTGGTTAC CCGTAAGACC TACGGTGGCG CATATATCGC AATGAATAGC GTAAGCCGCT CACGTGGCAA GGCGCGCACT GGGACCAATG GGCATTCTGG ATGCCACCGC GTATATAGCG TTACTTATCG CGTTCTCTGA ACGCGACGAA GGTCTTTGCC TGGCCGGATG CGGAGGTGGC CGTCATGGGT GCCAAAGCGG CGGTCGGCAT GCAAGAGACT TGCGCTGCTT CCAGAAACGG ACCGGCCTAC GCCTCCACCG GCAGTACCCA CGGTTTCGCC GCCAGCCGTA TCTGCACAAG AAGAAGCTGG CAGCCGCACC GGAGCATGAA CGTGAGGCTC TGCACGATCA ACTGGCGGCG GAACATGAAC AGACGTGTTC TTCTTCGACC GTCGGCGTGG CCTCGTACTT GCACTCCGAG ACGTGCTAGT TGACCGCCGC CTTGTACTTG GCATCGCGGG CGGTGTTGAT TCTGCTCTGG ATATTGGTGT GGTTGATGAA AAAATTGATC CGGCTCATAC CCGTAGCAAG CGTAGCGCCC GCCACAACTA AGACGAGACC TATAACCACA CCAACTACTT TTTTAACTAG GCCGAGTATG GGCATCGTTC CTGACCGAAG CCCTGGCTCA AGCGCCGGCT CGTCGTGGTC GTCATAAAAA CATCCCGCTG TAAccgaggg ggtggtgtag GACTGGCTTC GGGACCGAGT TCGCGGCCGA GCAGCACCAG CAGTATTTTT GTAGGGCGAC ATTggctccc ccaccacatc ccg -3' ggc -5'

158

F. AccE: Gene name from Mycobacterium tuberculosis H37Rv (Accession NP217798)

5'– aaggaagtcg cgggATGGGT ACGTGTCCGT GCGAGAGCAG CGAGCGTAAC GAGCCGGTGA GCCGCGTTAG CGGCACCAAC 3'– ttccttcagc gcccTACCCA TGCACAGGCA CGCTCTCGTC GCTCGCATTG CTCGGCCACT CGGCGCAATC GCCGTGGTTG

GAAGTCAGCG ACGGCAACGA GACGAACAAT CCGGCTGAAG TCTCCGACGG TAACGAAACC AACAACCCGG CGGAAGTTTC CTTCAGTCGC TGCCGTTGCT CTGCTTGTTA GGCCGACTTC AGAGGCTGCC ATTGCTTTGG TTGTTGGGCC GCCTTCAAAG TGACGGTAAT GAAACCAATA ACCCGGCCCC GGTCTCCCGT GTGTCTGGCA CGAATGAAGT TAGCGATGGT AACGAGACCA ACTGCCATTA CTTTGGTTAT TGGGCCGGGG CCAGAGGGCA CACAGACCGT GCTTACTTCA ATCGCTACCA TTGCTCTGGT ACAATCCGGC ACCGGTCAGC CGTGTGAGCG GTACGAATGA GGTGTCCGAC GGCAATGAGA CCAATAATCC GGCTCCGGTT TGTTAGGCCG TGGCCAGTCG GCACACTCGC CATGCTTACT CCACAGGCTG CCGTTACTCT GGTTATTAGG CCGAGGCCAA ACGGAAAAGC CGCTGCATCC GCACGAACCG CACATTGAGA TCCTGCGTGG CCAACCGACC GATCAGGAGT TGGCCGCACT TGCCTTTTCG GCGACGTAGG CGTGCTTGGC GTGTAACTCT AGGACGCACC GGTTGGCTGG CTAGTCCTCA ACCGGCGTGA GATCGCTGTT TTGGGTAGCA TCAGCGGTTC TACCCCGCCG GCGCAGCCGG AGCCGACCCG TTGGGGCCTG CCGGTCGATC CTAGCGACAA AACCCATCGT AGTCGCCAAG ATGGGGCGGC CGCGTCGGCC TCGGCTGGGC AACCCCGGAC GGCCAGCTAG AACTGCGCTA CCCGGTGTTC TCTTGGCAGC GTATTACCTT GCAAGAAATG ACCCACATGC GCCGCTAAcc cctcgc -3' TTGACGCGAT GGGCCACAAG AGAACCGTCG CATAATGGAA CGTTCTTTAC TGGGTGTACG CGGCGATTgg ggagcg -5'

159

G. ACS4 : Gene name from Trypanosoma brucei brucei strain 927/4 (Accession #XP803726) 5'– tgaacgcATG GGTGGCTGCG TAATCTCTGT GATGGACTAT ATGAATAACC GTTCCGAAGT AGAAAACGAA CATGTTAAAA 3'– acttgcgTAC CCACCGACGC ATTAGAGACA CTACCTGATA TACTTATTGG CAAGGCTTCA TCTTTTGCTT GTACAATTTT

AGTTCCGTGC GCTGGGCAAA GTGGCTGTTC CGGTTCCTGG TTCTGAAACC TCTGATTGCT CTCCAATCTA CCGCCTGGTT TCAAGGCACG CGACCCGTTT CACCGACAAG GCCAAGGACC AAGACTTTGG AGACTAACGA GAGGTTAGAT GGCGGACCAA ACTGACGACG GTAAGGACAT CGAGGAAGTT CGCCGTGAAT GGTACGAAGG CGAATGTCTG CCGCAGCGTT TTGCGGCACT TGACTGCTGC CATTCCTGTA GCTCCTTCAA GCGGCACTTA CCATGCTTCC GCTTACAGAC GGCGTCGCAA AACGCCGTGA GTGCAAACGT CAGCCAAAAC AACGTGCACT GGCGTATCGT CCAGTGGACC GCGTTGAGAA AGCGGTGATC AAGGATCTGC CACGTTTGCA GTCGGTTTTG TTGCACGTGA CCGCATAGCA GGTCACCTGG CGCAACTCTT TCGCCACTAG TTCCTAGACG ATACCGGTAC GGAAAAGATG ATGAATGTTA CGCACTTCAA GGAAACCAAA TATCTGGACT ACGGTACGTT CTGGGACTAT TATGGCCATG CCTTTTCTAC TACTTACAAT GCGTGAAGTT CCTTTGGTTT ATAGACCTGA TGCCATGCAA GACCCTGATA ATCGAATCCT TCGGTCGTGG TCTGGTAGAG CTGGGTATTT CTCCGTCTTC CCGTGTCGCC ATTTACGAAG AAACTCGTTG TAGCTTAGGA AGCCAGCACC AGACCATCTC GACCCATAAA GAGGCAGAAG GGCACAGCGG TAAATGCTTC TTTGAGCAAC GGAATGGCTG GCTACCATCT ACGGTATCTG GAGCCAGAAT ATGGTTGCGA CCACTGTTTA TGCAAACCTG GGCGAGGACG CCTTACCGAC CGATGGTAGA TGCCATAGAC CTCGGTCTTA TACCAACGCT GGTGACAAAT ACGTTTGGAC CCGCTCCTGC CGCTGGCTTA TGCTCTGCGT GAAACGGGCT GCAAAGGTAT TATCTGCAAC GCTAAAAACG TGTCCGTGGT TATCAAATTC GCGACCGAAT ACGAGACGCA CTTTGCCCGA CGTTTCCATA ATAGACGTTG CGATTTTTGC ACAGGCACCA ATAGTTTAAG ATGTCTGAAG GCATTACCCC GAGCGCTCCG ATCATTTATA ACGGTAGCCT GCCGGCTTCT GTTGACCAGG AAGCCTGTCA TACAGACTTC CGTAATGGGG CTCGCGAGGC TAGTAAATAT TGCCATCGGA CGGCCGAAGA CAACTGGTCC TTCGGACAGT CCTGGTTTCT TGGGAAGAAG TGGTGAAGCT GGGCCGTGAA GCACGCGACC GTCTGCCGCT GAACAATTCT GGTCGTGCAG GGACCAAAGA ACCCTTCTTC ACCACTTCGA CCCGGCACTT CGTGCGCTGG CAGACGGCGA CTTGTTAAGA CCAGCACGTC ACGATCTGGC ACTGATTATG TATACGAGCG GCACCACTGG CGATCCGAAG GGTGTTATCC ACACTCATGG CTCTCTGATG TGCTAGACCG TGACTAATAC ATATGCTCGC CGTGGTGACC GCTAGGCTTC CCACAATAGG TGTGAGTACC GAGAGACTAC TCCGGCGTGC ATGCTCTGGA CCATCGCCTG AATGCGGTGA TGGGTCCTCT GCGCGACGGC GAGACTTACC TGAGCTACCT AGGCCGCACG TACGAGACCT GGTAGCGGAC TTACGCCACT ACCCAGGAGA CGCGCTGCCG CTCTGAATGG ACTCGATGGA GCCACTGGCT CACATCCTGG AACTGGGTGT CCTGTCCGTT TTCATTGCGC GTGGTGCACT GATCTGCTTT GGCTCTCCGT CGGTGACCGA GTGTAGGACC TTGACCCACA GGACAGGCAA AAGTAACGCG CACCACGTGA CTAGACGAAA CCGAGAGGCA TCACCCTGAC CGACCTGACT GCTCGCCCTC GCGGTGACCT GGCTGAATAC AACCCGTCTC TGCTGATTGG TGTTCCGCGT AGTGGGACTG GCTGGACTGA CGAGCGGGAG CGCCACTGGA CCGACTTATG TTGGGCAGAG ACGACTAACC ACAAGGCGCA ATTTATGATA CCCTGAAAAA GGCAATCCAG GCGAAACTGC CTGCGCCTGG CACCTTTAAA CGTCGTGCTT TCGACCACGC TAAATACTAT GGGACTTTTT CCGTTAGGTC CGCTTTGACG GACGCGGACC GTGGAAATTT GCAGCACGAA AGCTGGTGCG TTTTCAGTCT CGTCTGCGTG CGTTCAAAGA TGGTAAAGAC TCTCCGTACT GGGACGCGAA AGTCTTTGCG GCGACCCGTG AAAAGTCAGA GCAGACGCAC GCAAGTTTCT ACCATTTCTG AGAGGCATGA CCCTGCGCTT TCAGAAACGC CGCTGGGCAC CGGTACTGGG CAAAAACATG TATATGGTGC TGTCCGGCGG TGGCCCGCTG TCTACTGCCA CCCAGGATTT CCTGAACGTT GCCATGACCC GTTTTTGTAC ATATACCACG ACAGGCCGCC ACCGGGCGAC AGATGACGGT GGGTCCTAAA GGACTTGCAA GCCGTGGTTC GTATTATCCA AGGTTGGGGT CTGACCGAAA CCGTTTGCGT AGGCGGCGTT CAGCTGACCG GTGATATTGA CGGCACCAAG CATAATAGGT TCCAACCCCA GACTGGCTTT GGCAAACGCA TCCGCCGCAA GTCGACTGGC CACTATAACT AACTGGCGCT GTTGGTCCGC CTCTGCTGAG CGAAGAGGTA AAACTGCTGG ATGTGGAAGG CTACAAGCAC ACTGACGAAC TTGACCGCGA CAACCAGGCG GAGACGACTC GCTTCTCCAT TTTGACGACC TACACCTTCC GATGTTCGTG TGACTGCTTG CGGACCCGCG TGGCGAGATC CTGCTGCGTG GCCCGTTCCT GTTCAAGGGT TACTACAAAC AGGAAGAACT GACTAAAGAA GCCTGGGCGC ACCGCTCTAG GACGACGCAC CGGGCAAGGA CAAGTTCCCA ATGATGTTTG TCCTTCTTGA CTGATTTCTT GCTATCGACG AAGACGGTTG GTTTCACACT GGTGACGTAG GTTCTATTGG TCCGAACGGC ACCCTGCGTA TTATCGGCCG CGATAGCTGC TTCTGCCAAC CAAAGTGTGA CCACTGCATC CAAGATAACC AGGCTTGCCG TGGGACGCAT AATAGCCGGC TGTAAAAGCA CTGGCTAAAA ATGTCCTGGG CGAATACGTG GCTATGGAGA CTCTGGAATC TATGTACGCT CATAACTCTC ACATTTTCGT GACCGATTTT TACAGGACCC GCTTATGCAC CGATACCTCT GAGACCTTAG ATACATGCGA GTATTGAGAG TGAGCATGCC AAACGGCGTG TGCGTCCTGG TCCATCCGGA TCGCCCTTAC ATCTGTGCCC TGGTGCTGAC CGATGAAGCA ACTCGTACGG TTTGCCGCAC ACGCAGGACC AGGTAGGCCT AGCGGGAATG TAGACACGGG ACCACGACTG GCTACTTCGT

160

AAAGTGGTGG CATTTACTCG TGAGCATGGT CTGAAAGGTA AGTACCCGGA AGTACTGCAG GACCCGGAGT TCCAAAAAAA TTTCACCACC GTAAATGAGC ACTCGTACCA GACTTTCCAT TCATGGGCCT TCATGACGTC CTGGGCCTCA AGGTTTTTTT AGCTACTGCG TCCTTCCAGG AAACTGCGCG CGCTTCTGAT CGTCAGAAAT TCGAAATCGT GCGTCACGTT CGTCTGCTGT TCGATGACGC AGGAAGGTCC TTTGACGCGC GCGAAGACTA GCAGTCTTTA AGCTTTAGCA CGCAGTGCAA GCAGACGACA CCGACGAGTG GACGCCGGAA AACGGTGTTC TGACCGCTGC GGGCAAACTG AAACGCCGTG TTATCGACGA GAAGTACACC GGCTGCTCAC CTGCGGCCTT TTGCCACAAG ACTGGCGACG CCCGTTTGAC TTTGCGGCAC AATAGCTGCT CTTCATGTGG GACACCATTG TGTCTCTGTT CGTAGAAGAA TGCTAAggcg gcatgctgtt tcactcatat t -3' CTGTGGTAAC ACAGAGACAA GCATCTTCTT ACGATTccgc cgtacgacaa agtgagtata a -5'

161

H. Aldh593 : Gene name from Clostridium beijerinckii NRRL B593 (Accession #AAD31841) 5'– cgacaataga gcgaGAGATA TACATATGAA CAAAGATACC CTGATCCCGA CTACGAAAGA CCTGAAACTG AAAACCAACG 3'– gctgttatct cgctCTCTAT ATGTATACTT GTTTCTATGG GACTAGGGCT GATGCTTTCT GGACTTTGAC TTTTGGTTGC

TGGAAAACAT CAACCTGAAA AATTACAAAG ACAATTCCTC TTGTTTCGGC GTTTTTGAGA ACGTTGAGAA CGCAATCAAC ACCTTTTGTA GTTGGACTTT TTAATGTTTC TGTTAAGGAG AACAAAGCCG CAAAAACTCT TGCAACTCTT GCGTTAGTTG AGCGCCGTTC ATGCGCAGAA AATTCTGTCC CTGCATTATA CCAAAGAACA GCGTGAGAAA ATCATTACCG AAATCCGTAA TCGCGGCAAG TACGCGTCTT TTAAGACAGG GACGTAATAT GGTTTCTTGT CGCACTCTTT TAGTAATGGC TTTAGGCATT AGCTGCTCTG GAAAACAAAG AAGTACTGGC GACCATGATC CTGGAAGAAA CCCACATGGG TCGCTACGAG GATAAGATTC TCGACGAGAC CTTTTGTTTC TTCATGACCG CTGGTACTAG GACCTTCTTT GGGTGTACCC AGCGATGCTC CTATTCTAAG TGAAACACGA ACTGGTTGCC AAATATACCC CTGGTACTGA AGACCTGACT ACCACGGCTT GGAGCGGCGA CAACGGTCTG ACTTTGTGCT TGACCAACGG TTTATATGGG GACCATGACT TCTGGACTGA TGGTGCCGAA CCTCGCCGCT GTTGCCAGAC ACCGTAGTAG AGATGTCTCC GTACGGCGTT ATTGGTGCGA TCACTCCGAG CACCAACCCA ACGGAGACTG TTATCTGCAA TGGCATCATC TCTACAGAGG CATGCCGCAA TAACCACGCT AGTGAGGCTC GTGGTTGGGT TGCCTCTGAC AATAGACGTT TTCCATCGGT ATGATTGCAG CGGGCAATGC TGTTGTGTTT AACGGTCACC CTGGCGCGAA GAAATGTGTT GCTTTCGCAA AAGGTAGCCA TACTAACGTC GCCCGTTACG ACAACACAAA TTGCCAGTGG GACCGCGCTT CTTTACACAA CGAAAGCGTT TTGAAATGAT CAATAAAGCT ATCATCAGCT GCGGTGGCCC AGAGAACCTG GTAACGACTA TCAAAAACCC GACGATGGAA AACTTTACTA GTTATTTCGA TAGTAGTCGA CGCCACCGGG TCTCTTGGAC CATTGCTGAT AGTTTTTGGG CTGCTACCTT AGCCTGGACG CCATCATCAA ACACCCGCTG ATTAAACTGC TGTGCGGCAC TGGCGGCCCA GGTATGGTGA AAACCCTGCT TCGGACCTGC GGTAGTAGTT TGTGGGCGAC TAATTTGACG ACACGCCGTG ACCGCCGGGT CCATACCACT TTTGGGACGA GAACAGCGGT AAAAAGGCAA TTGGCGCGGG TGCTGGCAAC CCGCCGGTTA TTGTTGATGA TACCGCCGAT ATTGAAAAAG CTTGTCGCCA TTTTTCCGTT AACCGCGCCC ACGACCGTTG GGCGGCCAAT AACAACTACT ATGGCGGCTA TAACTTTTTC CTGGTAAGAG CATTATTGAA GGCTGTTCTT TTGATAATAA CCTGCCGTGC ATTGCTGAAA AAGAAGTATT CGTCTTCGAA GACCATTCTC GTAATAACTT CCGACAAGAA AACTATTATT GGACGGCACG TAACGACTTT TTCTTCATAA GCAGAAGCTT AACGTCGCTG ATGATCTGAT CAGCAACATG CTGAAAAACA ACGCTGTTAT CATCAACGAA GACCAGGTAT CTAAACTGAT TTGCAGCGAC TACTAGACTA GTCGTTGTAC GACTTTTTGT TGCGACAATA GTAGTTGCTT CTGGTCCATA GATTTGACTA TGATCTGGTA CTGCAGAAAA ACAACGAAAC TCAAGAATAC TTCATCAACA AGAAATGGGT GGGCAAAGAC GCTAAACTGT ACTAGACCAT GACGTCTTTT TGTTGCTTTG AGTTCTTATG AAGTAGTTGT TCTTTACCCA CCCGTTTCTG CGATTTGACA TCTCCGACGA AATCGACGTA GAATCCCCGT CCAACATCAA ATGTATCGTG TGCGAAGTTA ATGCAAACCA CCCATTCGTT AGAGGCTGCT TTAGCTGCAT CTTAGGGGCA GGTTGTAGTT TACATAGCAC ACGCTTCAAT TACGTTTGGT GGGTAAGCAA ATGACCGAGC TGATGATGCC GATTCTGCCA ATCGTCCGTG TTAAAGACAT CGACGAAGCG GTTAAATACA CCAAAATTGC TACTGGCTCG ACTACTACGG CTAAGACGGT TAGCAGGCAC AATTTCTGTA GCTGCTTCGC CAATTTATGT GGTTTTAACG CGAACAGAAC CGCAAACACT CTGCATACAT CTATTCCAAG AACATTGACA ACCTGAACCG TTTTGAGCGC GAAATTGACA GCTTGTCTTG GCGTTTGTGA GACGTATGTA GATAAGGTTC TTGTAACTGT TGGACTTGGC AAAACTCGCG CTTTAACTGT CTACCATCTT CGTCAAAAAC GCCAAATCCT TCGCAGGTGT CGGTTATGAG GCTGAGGGCT TTACCACTTT CACTATCGCT GATGGTAGAA GCAGTTTTTG CGGTTTAGGA AGCGTCCACA GCCAATACTC CGACTCCCGA AATGGTGAAA GTGATAGCGA GGCAGCACCG GCGAGGGCAT CACCTCTGCG CGCAACTTCA CCCGTCAACG TCGTTGCGTA CTGGCCGGCT GATCTAGAGA CCGTCGTGGC CGCTCCCGTA GTGGAGACGC GCGTTGAAGT GGGCAGTTGC AGCAACGCAT GACCGGCCGA CTAGATCTCT Tattacgca -3' Ataatgcgt -5'

162

I. AlkK: Gene name from Pseudomona oleoverans (Accession #Q00594) 5'– tagcgttacg ccacATGCTG GGTCAGATGA TGCGTAATCA ACTGGTTATC GGTAGCCTGG TGGAGCACGC CGCTCGTTAC 3'– atcgcaatgc ggtgTACGAC CCAGTCTACT ACGCATTAGT TGACCAATAG CCATCGGACC ACCTCGTGCG GCGAGCAATG

CACGGTGCCC GTGAGGTGGT GTCTGTTGAA ACGAGCGGTG AGGTCACCCG TTCTTGCTGG AAAGAAGTTG AACTGCGTGC GTGCCACGGG CACTCCACCA CAGACAACTT TGCTCGCCAC TCCAGTGGGC AAGAACGACC TTTCTTCAAC TTGACGCACG GCGTAAACTG GCGAGCGCAT TGGGTAAAAT GGGTTTGACC CCGAGCGACC GCTGTGCGAC CATTGCCTGG AATAATATCC CGCATTTGAC CGCTCGCGTA ACCCATTTTA CCCAAACTGG GGCTCGCTGG CGACACGCTG GTAACGGACC TTATTATAGG GCCACCTGGA GGTCTATTAC GCGGTTAGCG GCGCAGGCAT GGTTTGCCAC ACGATCAACC CGCGTCTGTT TATCGAACAG CGGTGGACCT CCAGATAATG CGCCAATCGC CGCGTCCGTA CCAAACGGTG TGCTAGTTGG GCGCAGACAA ATAGCTTGTC ATTACGTATG TCATTAATCA CGCGGAAGAT AAGGTGGTTT TGCTGGACGA CACCTTCCTG CCGATTATCG CAGAGATTCA TAATGCATAC AGTAATTAGT GCGCCTTCTA TTCCACCAAA ACGACCTGCT GTGGAAGGAC GGCTAATAGC GTCTCTAAGT TGGTAGCCTG CCGAAAGTGA AGGCCTTTGT TCTGATGGCA CACAATAACT CCAATGCGAG CGCCCAAATG CCGGGTCTGA ACCATCGGAC GGCTTTCACT TCCGGAAACA AGACTACCGT GTGTTATTGA GGTTACGCTC GCGGGTTTAC GGCCCAGACT TCGCCTACGA GGATCTGATT GGTCAAGGTG ATGATAACTA TATCTGGCCG GATGTCGATG AGAACGAGGC AAGCAGCCTG AGCGGATGCT CCTAGACTAA CCAGTTCCAC TACTATTGAT ATAGACCGGC CTACAGCTAC TCTTGCTCCG TTCGTCGGAC TGCTACACCA GCGGCACCAC GGGTAACCCG AAAGGTGTTT TGTACTCCCA TCGTAGCACC GTGTTGCACT CCATGACCAC ACGATGTGGT CGCCGTGGTG CCCATTGGGC TTTCCACAAA ACATGAGGGT AGCATCGTGG CACAACGTGA GGTACTGGTG GGCGATGCCG GACACCCTGA ACCTGAGCGC ACGTGACACG ATCCTGCCGG TCGTCCCGAT GTTTCATGTT AATGCCTGGG CCGCTACGGC CTGTGGGACT TGGACTCGCG TGCACTGTGC TAGGACGGCC AGCAGGGCTA CAAAGTACAA TTACGGACCC GCACCCCGTA TTCCGCCGCG ATGGTTGGTG CCAAGCTGGT GCTGCCGGGT CCGGCGCTGG ACGGTGCAAG CCTGAGCAAA CGTGGGGCAT AAGGCGGCGC TACCAACCAC GGTTCGACCA CGACGGCCCA GGCCGCGACC TGCCACGTTC GGACTCGTTT CTGATTGCAA GCGAAGGCGT CAGCATCGCT CTGGGCGTTC CGGTGGTGTG GCAGGGCCTG CTGGCGGCGC AAGCTGGTAA GACTAACGTT CGCTTCCGCA GTCGTAGCGA GACCCGCAAG GCCACCACAC CGTCCCGGAC GACCGCCGCG TTCGACCATT TGGTAGCAAG TCCCAGAGCC TGACCCGCGT TGTTGTGGGT GGCTCCGCAT GTCCGGCGTC CATGATTCGT GAGTTCAATG ACCATCGTTC AGGGTCTCGG ACTGGGCGCA ACAACACCCA CCGAGGCGTA CAGGCCGCAG GTACTAAGCA CTCAAGTTAC ACATCTATGG TGTTGAGGTC ATCCATGCGT GGGGTATGAC CGAGCTGAGC CCGTTCGGCA CCGCGAATAC CCCGCTGGCG TGTAGATACC ACAACTCCAG TAGGTACGCA CCCCATACTG GCTCGACTCG GGCAAGCCGT GGCGCTTATG GGGCGACCGC CACCACGTTG ATCTGAGCCC GGACGAAAAA CTGAGCCTGC GTAAAAGCCA GGGCCGTCCG CCGTACGGTG TTGAGCTGAA GTGGTGCAAC TAGACTCGGG CCTGCTTTTT GACTCGGACG CATTTTCGGT CCCGGCAGGC GGCATGCCAC AACTCGACTT AATCGTGAAT GATGAGGGCA TCCGTCTGCC GGAGGACGGT CGCAGCAAAG GTAACTTGAT GGCGCGTGGT CACTGGGTGA TTAGCACTTA CTACTCCCGT AGGCAGACGG CCTCCTGCCA GCGTCGTTTC CATTGAACTA CCGCGCACCA GTGACCCACT TTAAGGACTA CTTCCACAGC GATCCGGGCA GCACCCTGTC CGACGGCTGG TTTAGCACCG GCGATGTCGC CACCATCGAT AATTCCTGAT GAAGGTGTCG CTAGGCCCGT CGTGGGACAG GCTGCCGACC AAATCGTGGC CGCTACAGCG GTGGTAGCTA AGCGACGGTT TTATGACCAT TTGCGATCGT GCGAAGGACA TCATCAAGAG CGGTGGTGAG TGGATCAGCA CCGTCGAGCT TCGCTGCCAA AATACTGGTA AACGCTAGCA CGCTTCCTGT AGTAGTTCTC GCCACCACTC ACCTAGTCGT GGCAGCTCGA GGAGAGCATC GCCATTGCGC ACCCGCACAT CGTCGATGCT GCGGTGATTG CGGCACGTCA CGAGAAATGG GACGAACGTC CCTCTCGTAG CGGTAACGCG TGGGCGTGTA GCAGCTACGA CGCCACTAAC GCCGTGCAGT GCTCTTTACC CTGCTTGCAG CGCTGCTGAT CGCGGTGAAA AGCCCGAACT CTGAGCTGAC CAGCGGCGAA GTCTGTAACT ACTTTGCGGA CAAAGTCGCC GCGACGACTA GCGCCACTTT TCGGGCTTGA GACTCGACTG GTCGCCGCTT CAGACATTGA TGAAACGCCT GTTTCAGCGG CGTTGGCAAA TCCCGGACGC TGCAATTTTC GTGGAGGAAT TGCCGCGTAA CGGTACGGGT AAGATTTTGA AGAATCGTCT GCAACCGTTT AGGGCCTGCG ACGTTAAAAG CACCTCCTTA ACGGCGCATT GCCATGCCCA TTCTAAAACT TCTTAGCAGA GCGTGAAAAA TACGGTGATA TTCTGCTGCG TTCTAGCAGC AGCGTCTGCG AATAAtatta acgtggtgtc g -3' CGCACTTTTT ATGCCACTAT AAGACGACGC AAGATCGTCG TCGCAGACGC TTATTataat tgcaccacag c -5'

163

J. AtfA : Gene name from Acinetobacter sp. ADP1 (Accession #YP045555) 5'– cttcccagca attcATGCGC CCACTGCACC CGATTGACTT TATTTTCCTG TCTCTGGAAA AACGTCAGCA GCCAATGCAC 3'– gaagggtcgt taagTACGCG GGTGACGTGG GCTAACTGAA ATAAAAGGAC AGAGACCTTT TTGCAGTCGT CGGTTACGTG

GTTGGTGGCC TGTTTCTGTT CCAGATCCCG GATAACGCTC CAGACACCTT CATCCAGGAT CTGGTAAACG ACATCCGTAT CAACCACCGG ACAAAGACAA GGTCTAGGGC CTATTGCGAG GTCTGTGGAA GTAGGTCCTA GACCATTTGC TGTAGGCATA CAGCAAATCT ATCCCGGTCC CGCCATTTAA CAACAAACTG AACGGTCTGT TCTGGGACGA AGATGAGGAG TTCGATCTGG GTCGTTTAGA TAGGGCCAGG GCGGTAAATT GTTGTTTGAC TTGCCAGACA AGACCCTGCT TCTACTCCTC AAGCTAGACC ATCACCACTT CCGTCACATT GCGCTGCCGC ATCCGGGTCG TATTCGTGAG CTGCTGATCT ACATTTCTCA GGAACACTCT TAGTGGTGAA GGCAGTGTAA CGCGACGGCG TAGGCCCAGC ATAAGCACTC GACGACTAGA TGTAAAGAGT CCTTGTGAGA ACTCTGCTGG ACCGTGCGAA ACCACTGTGG ACCTGCAACA TTATTGAGGG CATCGAAGGC AACCGTTTCG CGATGTATTT TGAGACGACC TGGCACGCTT TGGTGACACC TGGACGTTGT AATAACTCCC GTAGCTTCCG TTGGCAAAGC GCTACATAAA CAAAATCCAC CACGCGATGG TCGATGGTGT TGCTGGTATG CGTCTGATTG AAAAGTCCCT GAGCCACGAC GTAACCGAGA GTTTTAGGTG GTGCGCTACC AGCTACCACA ACGACCATAC GCAGACTAAC TTTTCAGGGA CTCGGTGCTG CATTGGCTCT AATCCATCGT GCCGCCGTGG TGCGTGGAAG GCAAACGTGC TAAACGTCTG CGTGAACCAA AGACCGGCAA GATCAAGAAA TTAGGTAGCA CGGCGGCACC ACGCACCTTC CGTTTGCACG ATTTGCAGAC GCACTTGGTT TCTGGCCGTT CTAGTTCTTT ATCATGTCCG GCATCAAATC TCAGCTGCAG GCAACCCCGA CCGTTATTCA GGAACTGTCT CAGACGGTAT TCAAAGACAT TAGTACAGGC CGTAGTTTAG AGTCGACGTC CGTTGGGGCT GGCAATAAGT CCTTGACAGA GTCTGCCATA AGTTTCTGTA CGGTCGTAAC CCGGACCACG TAAGCAGCTT CCAGGCGCCA TGCTCCATCC TGAACCAGCG TGTCTCCTCT TCTCGCCGCT GCCAGCATTG GGCCTGGTGC ATTCGTCGAA GGTCCGCGGT ACGAGGTAGG ACTTGGTCGC ACAGAGGAGA AGAGCGGCGA TCGCCGCTCA GAGCTTCGAC CTGGATCGCT TCCGTAACAT CGCTAAATCC CTGAATGTCA CGATCAACGA CGTTGTACTG AGCGGCGAGT CTCGAAGCTG GACCTAGCGA AGGCATTGTA GCGATTTAGG GACTTACAGT GCTAGTTGCT GCAACATGAC GCAGTTTGCA GCGGTGCGCT GCGTGCTTAC CTGATGAGCC ATAACTCTCT GCCGTCTAAA CCGCTGATCG CTATGGTGCC CGTCAAACGT CGCCACGCGA CGCACGAATG GACTACTCGG TATTGAGAGA CGGCAGATTT GGCGACTAGC GATACCACGG GGCGAGCATT CGTAACGACG ACAGCGACGT CTCTAACCGT ATCACCATGA TCCTGGCTAA CCTGGCTACC CACAAGGACG CCGCTCGTAA GCATTGCTGC TGTCGCTGCA GAGATTGGCA TAGTGGTACT AGGACCGATT GGACCGATGG GTGTTCCTGC ATCCACTGCA GCGTCTGGAA ATCATCCGTC GTTCCGTACA GAATAGCAAA CAGCGTTTCA AGCGCATGAC GTCCGATCAG TAGGTGACGT CGCAGACCTT TAGTAGGCAG CAAGGCATGT CTTATCGTTT GTCGCAAAGT TCGCGTACTG CAGGCTAGTC ATTCTGAACT ATTCCGCCGT TGTTTACGGT CCAGCGGGCC TGAACATCAT CTCCGGTATG ATGCCTAAAC GCCAGGCATT TAAGACTTGA TAAGGCGGCA ACAAATGCCA GGTCGCCCGG ACTTGTAGTA GAGGCCATAC TACGGATTTG CGGTCCGTAA TAATCTGGTC ATTTCCAATG TTCCGGGTCC TCGTGAACCT CTGTATTGGA ACGGCGCTAA ACTGGACGCG CTGTACCCGG ATTAGACCAG TAAAGGTTAC AAGGCCCAGG AGCACTTGGA GACATAACCT TGCCGCGATT TGACCTGCGC GACATGGGCC CGAGCATCGT TCTGGACGGT CAAGCACTGA ACATTACTAT GACTAGCTAC CTGGACAAGC TGGAAGTTGG TCTGATCGCC GCTCGTAGCA AGACCTGCCA GTTCGTGACT TGTAATGATA CTGATCGATG GACCTGTTCG ACCTTCAACC AGACTAGCGG TGCCGCAATG CTCTGCCGCG TATGCAGAAC CTGCTGACCC ACCTGGAAGA GGAGATTCAA CTGTTTGAGG GCGTTATCGC ACGGCGTTAC GAGACGGCGC ATACGTCTTG GACGACTGGG TGGACCTTCT CCTCTAAGTT GACAAACTCC CGCAATAGCG CAAACAGGAA GACATTAAAA CCGCGAACTA Aaagggtcg -3' GTTTGTCCTT CTGTAATTTT GGCGCTTGAT Tttcccagc -5'

164

K. BdhA : Gene name from Clostridium acetobutylicum ATCC 824 (Accession # NP349892) 5'– GCTTGGTACC AAGAAGGAGA TATCATATGC TGTCCTTCGA TTATTCTATC CCGACCAAAG TATTCTTCGG TAAAGGCAAA 5'– CGAACCATGG TTCTTCCTCT ATAGTATACG ACAGGAAGCT AATAAGATAG GGCTGGTTTC ATAAGAAGCC ATTTCCGTTT

ATCGACGTAA TTGGCGAAGA GATCAAGAAA TATGGTTCCC GCGTTCTGAT CGTGTATGGT GGCGGCAGCA TTAAACGTAA TAGCTGCATT AACCGCTTCT CTAGTTCTTT ATACCAAGGG CGCAAGACTA GCACATACCA CCGCCGTCGT AATTTGCATT CGGCATTTAT GATCGCGCTA CGGCCATCCT GAAAGAAAAC AACATCGCGT TCTACGAGCT GTCTGGCGTC GAACCTAACC GCCGTAAATA CTAGCGCGAT GCCGGTAGGA CTTTCTTTTG TTGTAGCGCA AGATGCTCGA CAGACCGCAG CTTGGATTGG CTCGTATCAC TACCGTGAAA AAGGGTATTG AAATCTGTCG TGAGAACAAT GTCGATCTGG TTCTGGCTAT CGGTGGCGGT GAGCATAGTG ATGGCACTTT TTCCCATAAC TTTAGACAGC ACTCTTGTTA CAGCTAGACC AAGACCGATA GCCACCGCCA TCTGCAATCG ACTGCTCCAA GGTCATCGCT GCGGGCGTAT ATTATGACGG CGATACCTGG GACATGGTGA AAGACCCGTC AGACGTTAGC TGACGAGGTT CCAGTAGCGA CGCCCGCATA TAATACTGCC GCTATGGACC CTGTACCACT TTCTGGGCAG TAAGATCACC AAAGTTCTGC CGATTGCTTC CATCCTGACT CTGAGCGCTA CGGGCTCTGA AATGGATCAG ATCGCGGTTA ATTCTAGTGG TTTCAAGACG GCTAACGAAG GTAGGACTGA GACTCGCGAT GCCCGAGACT TTACCTAGTC TAGCGCCAAT TTAGCAATAT GGAAACCAAC GAAAAACTGG GCGTCGGTCA TGACGACATG CGTCCGAAAT TTTCTGTACT GGACCCTACC AATCGTTATA CCTTTGGTTG CTTTTTGACC CGCAGCCAGT ACTGCTGTAC GCAGGCTTTA AAAGACATGA CCTGGGATGG TATACGTTCA CCGTACCGAA AAATCAGACC GCAGCGGGCA CCGCAGATAT TATGAGCCAC ACCTTCGAAT CCTACTTTAG ATATGCAAGT GGCATGGCTT TTTAGTCTGG CGTCGCCCGT GGCGTCTATA ATACTCGGTG TGGAAGCTTA GGATGAAATC CGGCGTAGAA GGTGCCTACG TACAGGATGG TATCGCCGAA GCCATCCTGC GTACCTGTAT CAAATACGGT AAAATCGCTA GCCGCATCTT CCACGGATGC ATGTCCTACC ATAGCGGCTT CGGTAGGACG CATGGACATA GTTTATGCCA TTTTAGCGAT TGGAGAAAAC TGATGATTAT GAAGCCCGTG CGAACCTGAT GTGGGCCTCT TCTCTGGCTA TCAATGGTCT GCTGAGCCTG ACCTCTTTTG ACTACTAATA CTTCGGGCAC GCTTGGACTA CACCCGGAGA AGAGACCGAT AGTTACCAGA CGACTCGGAC GGTAAAGATC GCAAATGGTC TTGCCACCCG ATGGAACATG AACTGAGCGC TTATTACGAC ATCACCCACG GCGTCGGTCT CCATTTCTAG CGTTTACCAG AACGGTGGGC TACCTTGTAC TTGACTCGCG AATAATGCTG TAGTGGGTGC CGCAGCCAGA GGCGATCCTG ACCCCGAATT GGATGGAGTA CATTCTGAAT GACGACACCC TGCACAAGTT CGTGTCTTAC GGTATTAACG CCGCTAGGAC TGGGGCTTAA CCTACCTCAT GTAAGACTTA CTGCTGTGGG ACGTGTTCAA GCACAGAATG CCATAATTGC TCTGGGGTAT CGACAAAAAC AAAGACAACT ACGAAATTGC ACGTGAAGCC ATCAAAAACA CTCGCGAGTA CTTCAACAGC AGACCCCATA GCTGTTTTTG TTTCTGTTGA TGCTTTAACG TGCACTTCGG TAGTTTTTGT GAGCGCTCAT GAAGTTGTCG CTGGGTATCC CTTCTAAACT GCGTGAAGTG GGCATCGGTA AAGATAAACT GGAACTGATG GCTAAACAGG CGGTTCGCAA GACCCATAGG GAAGATTTGA CGCACTTCAC CCGTAGCCAT TTCTATTTGA CCTTGACTAC CGATTTGTCC GCCAAGCGTT CTCTGGTGGC ACCATCGGCT CTCTGCGCCC AATCAACGCC GAGGACGTTC TGGAAATTTT CAAGAAAAGC TATTAAAAGC GAGACCACCG TGGTAGCCGA GAGACGCGGG TTAGTTGCGG CTCCTGCAAG ACCTTTAAAA GTTCTTTTCG ATAATTTTCG TTATCcgcaa cccgctggtg cctagctc -3' AATAGgcgtt gggcgaccac ggatcgag -5'

165

L. BdhB : Gene name from Clostridium acetobutylicum ATCC 824 (Accession #NP349891) 5'– attgccgtag ggcgGAGCTC GGCGCGCCGA ATTCGGATCC AAGGAGATAT ACCATGGTGG ACTTCGAGTA TTCTATCCCA 3'– taacggcatc ccgcCTCGAG CCGCGCGGCT TAAGCCTAGG TTCCTCTATA TGGTACCACC TGAAGCTCAT AAGATAGGGT

ACCCGTATCT TTTTCGGTAA AGATAAAATC AATGTGCTGG GTCGTGAGCT GAAGAAATAC GGCTCTAAAG TTCTGATCGT TGGGCATAGA AAAAGCCATT TCTATTTTAG TTACACGACC CAGCACTCGA CTTCTTTATG CCGAGATTTC AAGACTAGCA TTATGGTGGC GGTAGCATCA AACGTAACGG TATCTACGAC AAGGCGGTGT CTATTCTGGA AAAGAACTCC ATTAAATTCT AATACCACCG CCATCGTAGT TTGCATTGCC ATAGATGCTG TTCCGCCACA GATAAGACCT TTTCTTGAGG TAATTTAAGA ACGAACTGGC TGGCGTCGAA CCAAACCCGC GTGTGACGAC CGTTGAAAAA GGCGTAAAAA TCTGCCGTGA GAACGGCGTA TGCTTGACCG ACCGCAGCTT GGTTTGGGCG CACACTGCTG GCAACTTTTT CCGCATTTTT AGACGGCACT CTTGCCGCAT GAAGTGGTGC TGGCCATCGG TGGTGGCTCT GCAATCGACT GCGCGAAAGT GATTGCGGCA GCGTGCGAAT ATGACGGCAA CTTCACCACG ACCGGTAGCC ACCACCGAGA CGTTAGCTGA CGCGCTTTCA CTAACGCCGT CGCACGCTTA TACTGCCGTT CCCGTGGGAC ATTGTGCTGG ACGGCAGCAA AATTAAGCGC GTACTGCCGA TTGCTTCCAT TCTGACTATT GCTGCCACGG GGGCACCCTG TAACACGACC TGCCGTCGTT TTAATTCGCG CATGACGGCT AACGAAGGTA AGACTGATAA CGACGGTGCC GTAGCGAAAT GGATACCTGG GCAGTAATTA ACAACATGGA CACTAACGAA AAGCTGATCG CGGCCCACCC GGACATGGCA CATCGCTTTA CCTATGGACC CGTCATTAAT TGTTGTACCT GTGATTGCTT TTCGACTAGC GCCGGGTGGG CCTGTACCGT CCAAAATTCT CTATTCTGGA CCCGACCTAC ACCTACACTG TGCCGACTAA TCAGACTGCT GCTGGCACTG CGGATATTAT GGTTTTAAGA GATAAGACCT GGGCTGGATG TGGATGTGAC ACGGCTGATT AGTCTGACGA CGACCGTGAC GCCTATAATA GTCCCACATC TTCGAAGTCT ATTTTTCTAA TACCAAAACT GCGTATCTGC AAGACCGCAT GGCGGAAGCT CTGCTGCGTA CAGGGTGTAG AAGCTTCAGA TAAAAAGATT ATGGTTTTGA CGCATAGACG TTCTGGCGTA CCGCCTTCGA GACGACGCAT CCTGCATTAA ATACGGCGGT ATTGCGCTGG AAAAACCGGA CGACTACGAG GCTCGCGCTA ACCTGATGTG GGCATCTTCT GGACGTAATT TATGCCGCCA TAACGCGACC TTTTTGGCCT GCTGATGCTC CGAGCGCGAT TGGACTACAC CCGTAGAAGA CTGGCTATCA ACGGTCTGCT GACCTACGGT AAGGACACGA ACTGGAGCGT CCACCTGATG GAGCATGAAC TGTCTGCATA GACCGATAGT TGCCAGACGA CTGGATGCCA TTCCTGTGCT TGACCTCGCA GGTGGACTAC CTCGTACTTG ACAGACGTAT TTACGACATC ACTCACGGTG TAGGTCTGGC AATTCTGACG CCAAACTGGA TGGAATATAT CCTGAACAAC GATACTGTTT AATGCTGTAG TGAGTGCCAC ATCCAGACCG TTAAGACTGC GGTTTGACCT ACCTTATATA GGACTTGTTG CTATGACAAA ACAAGTTCGT TGAGTATGGT GTTAACGTGT GGGGTATCGA CAAAGAGAAA AACCACTACG ACATCGCACA CCAGGCGATC TGTTCAAGCA ACTCATACCA CAATTGCACA CCCCATAGCT GTTTCTCTTT TTGGTGATGC TGTAGCGTGT GGTCCGCTAG CAGAAGACCC GTGATTACTT CGTGAACGTT CTGGGTCTGC CGTCCCGCCT GCGTGACGTT GGTATCGAAG AAGAAAAACT GTCTTCTGGG CACTAATGAA GCACTTGCAA GACCCAGACG GCAGGGCGGA CGCACTGCAA CCATAGCTTC TTCTTTTTGA GGATATTATG GCAAAAGAAA GCGTCAAGCT GACCGGCGGC ACCATCGGCA ACCTGCGCCC GGTTAACGCT TCCGAGGTAC CCTATAATAC CGTTTTCTTT CGCAGTTCGA CTGGCCGCCG TGGTAGCCGT TGGACGCGGG CCAATTGCGA AGGCTCCATG TGCAAATTTT CAAGAAATCC GTTTAAGGTA CCGAATTCAA GCTTaaatcc agtaaga -3' ACGTTTAAAA GTTCTTTAGG CAAATTCCAT GGCTTAAGTT CGAAtttagg tcattct -5'

166

M. Ccr : Gene name from Streptomyces cinnamonensis (Accession #AAD53915) 5'– gATGAAAGAA ATCCTGGACG CTATTCAGGC ACAAACGGCA ACCGCAAGCG GTACGGCGGC TGTTACCTCT GCTGATTTTG 3'– cTACTTTCTT TAGGACCTGC GATAAGTCCG TGTTTGCCGT TGGCGTTCGC CATGCCGCCG ACAATGGAGA CGACTAAAAC

CGGCCCTGCC GCTGCCGGAC TCTTACCGTG CGATCACGGT TCACAAGGAT GAAACCGAGA TGTTCGCCGG TCTGGAATCC GCCGGGACGG CGACGGCCTG AGAATGGCAC GCTAGTGCCA AGTGTTCCTA CTTTGGCTCT ACAAGCGGCC AGACCTTAGG CGTGATAAAG ATCCTCGTAA ATCTCTGCAC CTGGATGACG TGCCAATTCC GGAACTGGGT CCGGGCGAAG CGCTGGTTGC GCACTATTTC TAGGAGCATT TAGAGACGTG GACCTACTGC ACGGTTAAGG CCTTGACCCA GGCCCGCTTC GCGACCAACG TGTGATGGCT TCCTCTGTGA ATTATAACAG CGTCTGGACT TCCATCTTCG AACCGGTATC TACCTTCAGC TTCCTGGAAC ACACTACCGA AGGAGACACT TAATATTGTC GCAGACCTGA AGGTAGAAGC TTGGCCATAG ATGGAAGTCG AAGGACCTTG GTTACGGTCG TCTGTCCGAT CTGAGCAAAC GTCATGACCT GCCGTACCAC ATCATCGGTT CTGATCTGGC CGGTGTCGTT CAATGCCAGC AGACAGGCTA GACTCGTTTG CAGTACTGGA CGGCATGGTG TAGTAGCCAA GACTAGACCG GCCACAGCAA CTGCGTACCG GTCCTGGTGT CAACGCTTGG AACCCAGGTG ACGAAGTGGT GGCGCATTGC CTGAGCGTGG AACTGGAGTC GACGCATGGC CAGGACCACA GTTGCGAACC TTGGGTCCAC TGCTTCACCA CCGCGTAACG GACTCGCACC TTGACCTCAG CTCCGATGGC CACAACGATA CTATGCTGGA CCCGGAACAG CGCATCTGGG GTTTTGAAAC CAACTTCGGC GGCCTGGCTG GAGGCTACCG GTGTTGCTAT GATACGACCT GGGCCTTGTC GCGTAGACCC CAAAACTTTG GTTGAAGCCG CCGGACCGAC AAATTGCTCT GGTGAAATCT AACCAGCTGA TGCCTAAACC GGGTCATCTG TCTTGGGAAG AGGCCGCCAG CCCAGGTCTG TTTAACGAGA CCACTTTAGA TTGGTCGACT ACGGATTTGG CCCAGTAGAC AGAACCCTTC TCCGGCGGTC GGGTCCAGAC GTTAATTCTA CTGCATACCG TCAACTGGTT TCCCGTAATG GCGCAGGCAT GAAACAGGGC GACAACGTAC TGATTTGGGG CAATTAAGAT GACGTATGGC AGTTGACCAA AGGGCATTAC CGCGTCCGTA CTTTGTCCCG CTGTTGCATG ACTAAACCCC TGCGTCCGGT GGCCTGGGCT CTTATGCTAC TCAGTTCGCG CTGGCGGGTG GTGCCAACCC AATTTGCGTC GTTTCCTCCC ACGCAGGCCA CCGGACCCGA GAATACGATG AGTCAAGCGC GACCGCCCAC CACGGTTGGG TTAAACGCAG CAAAGGAGGG CGCAGAAAGC GGAAATCTGC CGTGCAATGG GTGCCGAAGC AATCATTGAT CGTAACGCGG AGGGCTACAA GTTTTGGAAA GCGTCTTTCG CCTTTAGACG GCACGTTACC CACGGCTTCG TTAGTAACTA GCATTGCGCC TCCCGATGTT CAAAACCTTT GACGAGCAGA CGCAGGACCC AAAAGAATGG AAACGCTTTG GCAAGCGCAT CCGTGAGCTG ACTGGTCGCC GTGGTCTGGA CTGCTCGTCT GCGTCCTGGG TTTTCTTACC TTTGCGAAAC CGTTCGCGTA GGCACTCGAC TGACCAGCGG CACCAGACCT CATCGTTTTC GAACACCCTG GTCGTGAAAC GTTCGGCGCG AGCGTTTATG TTACTCGCAA GGGTGGTACT ATTACCACCT GTAGCAAAAG CTTGTGGGAC CAGCACTTTG CAAGCCGCGC TCGCAAATAC AATGAGCGTT CCCACCATGA TAATGGTGGA GCGCGAGCAC TTCCGGTTAC ATGCACGAAT ACGATAACCG CTATCTGTGG ATGTCCCTGA AGCGTATTAT CGGCTCCCAC CGCGCTCGTG AAGGCCAATG TACGTGCTTA TGCTATTGGC GATAGACACC TACAGGGACT TCGCATAATA GCCGAGGGTG TTCGCGAACT ATCGTGAGGC GTGGGAGGCC AACCGTCTGA TCGCGAAGGG TAAAATTCAT CCAACCCTGT CTAAAACTTA AAGCGCTTGA TAGCACTCCG CACCCTCCGG TTGGCAGACT AGCGCTTCCC ATTTTAAGTA GGTTGGGACA GATTTTGAAT CCGCCTGGAA GACACTGGCC AGGCGGCGTA CGACGTCCAC CGCAACCTGC ACCAAGGTAA AGTGGGCGTA CTGGCACTGG GGCGGACCTT CTGTGACCGG TCCGCCGCAT GCTGCAGGTG GCGTTGGACG TGGTTCCATT TCACCCGCAT GACCGTGACC CGCCGGAAGA GGGTCTGGGC GTCCGTGACC CTGAAAAGCG TGCACAACAC ATCGATGCTA TTAACCGTTT CCGCAACGTC GCGGCCTTCT CCCAGACCCG CAGGCACTGG GACTTTTCGC ACGTGTTGTG TAGCTACGAT AATTGGCAAA GGCGTTGCAG TAAagagtcg tttgacctac gaacagg -3' ATTtctcagc aaactggatg cttgtcc -5'

167

N. DtsR1: Gene name from Corynebacterium glutamicum ATCC 13032 (Accession NP599940)

5'– ctGGTAGCGA TACCGACAAC TATAAGGAGG ACCAAATGAC CATTTCCAGC CCGTTGATTG ACGTGGCGAA CTTGCCGGAT 3'– gaCCATCGCT ATGGCTGTTG ATATTCCTCC TGGTTTACTG GTAAAGGTCG GGCAACTAAC TGCACCGCTT GAACGGCCTA

ATTAACACCA CCGCGGGCAA GATTGCGGAC TTGAAAGCCC GTCGCGCAGA AGCACATTTT CCGATGGGCG AAAAGGCTGT TAATTGTGGT GGCGCCCGTT CTAACGCCTG AACTTTCGGG CAGCGCGTCT TCGTGTAAAA GGCTACCCGC TTTTCCGACA CGAAAAAGTC CATGCGGCAG GCCGTCTGAC CGCCCGTGAG CGTCTGGACT ACCTGCTGGA TGAAGGCAGC TTCATCGAGA GCTTTTTCAG GTACGCCGTC CGGCAGACTG GCGGGCACTC GCAGACCTGA TGGACGACCT ACTTCCGTCG AAGTAGCTCT CCGACCAGTT GGCGCGTCAT CGCACGACGG CCTTCGGTTT GGGCGCTAAA CGCCCGGCTA CGGACGGTAT TGTGACCGGC GGCTGGTCAA CCGCGCAGTA GCGTGCTGCC GGAAGCCAAA CCCGCGATTT GCGGGCCGAT GCCTGCCATA ACACTGGCCG TGGGGCACCA TTGATGGCCG TGAGGTCTGC ATCTTCTCCC AGGATGGCAC GGTGTTTGGT GGTGCTCTGG GTGAGGTTTA ACCCCGTGGT AACTACCGGC ACTCCAGACG TAGAAGAGGG TCCTACCGTG CCACAAACCA CCACGAGACC CACTCCAAAT TGGTGAGAAA ATGATTAAAA TCATGGAGCT GGCGATCGAC ACGGGCCGTC CGCTGATCGG CCTGTATGAG GGCGCGGGTG ACCACTCTTT TACTAATTTT AGTACCTCGA CCGCTAGCTG TGCCCGGCAG GCGACTAGCC GGACATACTC CCGCGCCCAC CGCGTATTCA GGACGGTGCA GTCAGCCTGG ACTTTATCAG CCAAACCTTC TACCAGAACA TTCAAGCGAG CGGTGTTATC GCGCATAAGT CCTGCCACGT CAGTCGGACC TGAAATAGTC GGTTTGGAAG ATGGTCTTGT AAGTTCGCTC GCCACAATAG CCGCAGATTT CCGTGATTAT GGGTGCGTGC GCTGGTGGCA ACGCTTACGG CCCGGCGCTG ACCGATTTTG TGGTGATGGT GGCGTCTAAA GGCACTAATA CCCACGCACG CGACCACCGT TGCGAATGCC GGGCCGCGAC TGGCTAAAAC ACCACTACCA TGACAAGACC AGCAAGATGT TCGTCACCGG CCCGGATGTC ATTAAGACGG TGACCGGCGA GGAGATCACC CAGGAGGAGC ACTGTTCTGG TCGTTCTACA AGCAGTGGCC GGGCCTACAG TAATTCTGCC ACTGGCCGCT CCTCTAGTGG GTCCTCCTCG TGGGTGGTGC TACCACGCAC ATGGTCACGG CAGGCAACTC CCACTACACC GCGGCAACCG ACGAAGAAGC TCTGGACTGG ACCCACCACG ATGGTGCGTG TACCAGTGCC GTCCGTTGAG GGTGATGTGG CGCCGTTGGC TGCTTCTTCG AGACCTGACC GTGCAGGACC TGGTTAGCTT TCTGCCGTCT AATAATCGTA GCTATGCACC GATGGAAGAT TTCGATGAAG AGGAAGGTGG CACGTCCTGG ACCAATCGAA AGACGGCAGA TTATTAGCAT CGATACGTGG CTACCTTCTA AAGCTACTTC TCCTTCCACC TGTGGAAGAG AATATTACCG CCGACGATCT GAAGTTGGAC GAGATTATCC CGGACAGCGC GACGGTTCCG TACGATGTTC ACACCTTCTC TTATAATGGC GGCTGCTAGA CTTCAACCTG CTCTAATAGG GCCTGTCGCG CTGCCAAGGC ATGCTACAAG GTGATGTTAT TGAATGTCTG ACCGATGACG GTGAATATCT GGAGATTCAG GCGGACCGTG CCGAAAATGT TGTTATTGCC CACTACAATA ACTTACAGAC TGGCTACTGC CACTTATAGA CCTCTAAGTC CGCCTGGCAC GGCTTTTACA ACAATAACGG TTTGGCCGCA TTGAGGGTCA GAGCGTCGGC TTCGTTGCCA ATCAACCGAC CCAGTTCGCG GGTTGCCTGG ACATCGACAG AAACCGGCGT AACTCCCAGT CTCGCAGCCG AAGCAACGGT TAGTTGGCTG GGTCAAGCGC CCAACGGACC TGTAGCTGTC CTCTGAGAAA GCGGCACGCT TTGTCCGTAC CTGTGACGCA TTCAACATCC CGATTGTGAT GCTGGTGGAT GTTCCGGGCT GAGACTCTTT CGCCGTGCGA AACAGGCATG GACACTGCGT AAGTTGTAGG GCTAACACTA CGACCACCTA CAAGGCCCGA TCCTGCCGGG TGCGGGTCAG GAGTATGGTG GTATTCTGCG TCGTGGCGCG AAACTGCTGT ACGCCTACGG CGAAGCGACC AGGACGGCCC ACGCCCAGTC CTCATACCAC CATAAGACGC AGCACCGCGC TTTGACGACA TGCGGATGCC GCTTCGCTGG GTCCCGAAGA TCACCGTCAC CATGCGCAAG GCGTACGGTG GCGCGTACTG TGTCATGGGT TCCAAAGGCC TGGGTAGCGA CAGGGCTTCT AGTGGCAGTG GTACGCGTTC CGCATGCCAC CGCGCATGAC ACAGTACCCA AGGTTTCCGG ACCCATCGCT CATTAATCTG GCATGGCCGA CCGCACAAAT TGCGGTGATG GGCGCGGCTG GTGCAGTGGG CTTTATCTAC CGTAAAGAGC GTAATTAGAC CGTACCGGCT GGCGTGTTTA ACGCCACTAC CCGCGCCGAC CACGTCACCC GAAATAGATG GCATTTCTCG TGATGGCGGC AGATGCCAAG GGTCTGGACA CCGTTGCGCT GGCGAAGAGC TTCGAACGCG AGTATGAAGA TCACATGCTG ACTACCGCCG TCTACGGTTC CCAGACCTGT GGCAACGCGA CCGCTTCTCG AAGCTTGCGC TCATACTTCT AGTGTACGAC AATCCGTATC ATGCGGCGGA ACGCGGTCTG ATCGATGCGG TGATTTTGCC GAGCGAGACC CGTGGTCAGA TTAGCCGCAA TTAGGCATAG TACGCCGCCT TGCGCCAGAC TAGCTACGCC ACTAAAACGG CTCGCTCTGG GCACCAGTCT AATCGGCGTT TCTGCGTCTG CTGAAACATA AGAATGTCAC CCGTCCGGCT CGCAAGCATG GTAATATGCC GCTGTAAcag cgacgctctc AGACGCAGAC GACTTTGTAT TCTTACAGTG GGCAGGCCGA GCGTTCGTAC CATTATACGG CGACATTgtc gctgcgagag tgcggact -3' acgcctga -5'

168

O. JFar : Gene name from Simmondsia chinensis (Accession #Q9XGY7) 5'– gcaaagtaga atccCATATG GAAGAAATGG GCAGCATCCT GGAGTTCCTG GATAACAAAG CGATCCTGGT AACCGGCGCG 3'– cgtttcatct taggGTATAC CTTCTTTACC CGTCGTAGGA CCTCAAGGAC CTATTGTTTC GCTAGGACCA TTGGCCGCGC

ACCGGCTCCC TGGCAAAAAT CTTCGTTGAA AAAGTGCTGC GTTCTCAGCC AAACGTAAAA AAACTGTACC TGCTGCTGCG TGGCCGAGGG ACCGTTTTTA GAAGCAACTT TTTCACGACG CAAGAGTCGG TTTGCATTTT TTTGACATGG ACGACGACGC CGCCACTGAC GATGAAACTG CGGCTCTGCG TCTGCAAAAT GAGGTATTTG GCAAAGAACT GTTCAAAGTG CTGAAACAGA GCGGTGACTG CTACTTTGAC GCCGAGACGC AGACGTTTTA CTCCATAAAC CGTTTCTTGA CAAGTTTCAC GACTTTGTCT ATCTGGGTGC GAACTTCTAC TCCTTCGTTT CTGAGAAAGT CACTGTCGTA CCGGGTGACA TTACCGGTGA GGACCTGTGT TAGACCCACG CTTGAAGATG AGGAAGCAAA GACTCTTTCA GTGACAGCAT GGCCCACTGT AATGGCCACT CCTGGACACA CTGAAAGACG TCAACCTGAA GGAAGAGATG TGGCGCGAAA TTGACGTCGT CGTTAATCTG GCTGCAACCA TTAATTTCAT GACTTTCTGC AGTTGGACTT CCTTCTCTAC ACCGCGCTTT AACTGCAGCA GCAATTAGAC CGACGTTGGT AATTAAAGTA CGAACGTTAC GATGTATCCC TGCTGATCAA CACCTACGGT GCAAAATATG TGCTGGACTT CGCTAAGAAG TGCAACAAAC GCTTGCAATG CTACATAGGG ACGACTAGTT GTGGATGCCA CGTTTTATAC ACGACCTGAA GCGATTCTTC ACGTTGTTTG TGAAAATCTT TGTACATGTG TCTACCGCTT ATGTTAGCGG CGAAAAGAAT GGTCTGATTC TGGAAAAACC GTACTATATG ACTTTTAGAA ACATGTACAC AGATGGCGAA TACAATCGCC GCTTTTCTTA CCAGACTAAG ACCTTTTTGG CATGATATAC GGTGAGTCCC TGAACGGCCG TCTGGGTCTG GACATCAACG TAGAAAAGAA ACTGGTCGAG GCGAAGATCA ACGAACTGCA CCACTCAGGG ACTTGCCGGC AGACCCAGAC CTGTAGTTGC ATCTTTTCTT TGACCAGCTC CGCTTCTAGT TGCTTGACGT AGCTGCTGGT GCCACTGAAA AATCCATCAA ATCTACGATG AAAGATATGG GTATCGAGCG TGCTCGTCAT TGGGGCTGGC TCGACGACCA CGGTGACTTT TTAGGTAGTT TAGATGCTAC TTTCTATACC CATAGCTCGC ACGAGCAGTA ACCCCGACCG CGAACGTGTA CGTTTTCACT AAAGCTCTGG GTGAAATGCT GCTGATGCAA TATAAGGGTG ACATCCCACT GACCATCATT GCTTGCACAT GCAAAAGTGA TTTCGAGACC CACTTTACGA CGACTACGTT ATATTCCCAC TGTAGGGTGA CTGGTAGTAA CGTCCGACTA TTATCACGTC TACCTTTAAA GAACCGTTCC CAGGCTGGGT GGAAGGCGTG CGTACGATTG ATAACGTGCC GCAGGCTGAT AATAGTGCAG ATGGAAATTT CTTGGCAAGG GTCCGACCCA CCTTCCGCAC GCATGCTAAC TATTGCACGG GGTTTACTAC GGCAAGGGTC GTCTGCGTTG CATGCTGTGC GGCCCGAGCA CCATTATCGA CCTGATCCCG GCGGATATGG CCAAATGATG CCGTTCCCAG CAGACGCAAC GTACGACACG CCGGGCTCGT GGTAATAGCT GGACTAGGGC CGCCTATACC TAGTGAACGC CACCATCGTT GCTATGGTTG CGCACGCGAA CCAGCGTTAC GTAGAGCCGG TTACTTACCA CGTCGGCAGC ATCACTTGCG GTGGTAGCAA CGATACCAAC GCGTGCGCTT GGTCGCAATG CATCTCGGCC AATGAATGGT GCAGCCGTCG TCTGCCGCTA ACCCAATGAA GCTGAGCGCA CTGCCGGAGA TGGCGCACCG TTACTTCACC AAAAACCCGT GGATTAACCC AGACGGCGAT TGGGTTACTT CGACTCGCGT GACGGCCTCT ACCGCGTGGC AATGAAGTGG TTTTTGGGCA CCTAATTGGG GGACCGTAAT CCGGTTCACG TGGGTCGCGC GATGGTCTTT TCTAGCTTTT CTACTTTTCA CCTGTACCTG ACTCTGAACT CCTGGCATTA GGCCAAGTGC ACCCAGCGCG CTACCAGAAA AGATCGAAAA GATGAAAAGT GGACATGGAC TGAGACTTGA TCCTGCTGCC GCTGAAAGTG CTGGAAATCG CGAACACCAT TTTCTGCCAG TGGTTCAAAG GCAAATACAT GGATCTGAAG AGGACGACGG CGACTTTCAC GACCTTTAGC GCTTGTGGTA AAAGACGGTC ACCAAGTTTC CGTTTATGTA CCTAGACTTC CGTAAAACCC GTCTGCTGCT GCGCCTGGTA GACATCTATA AACCTTATCT GTTCTTCCAG GGCATTTTCG ACGATATGAA GCATTTTGGG CAGACGACGA CGCGGACCAT CTGTAGATAT TTGGAATAGA CAAGAAGGTC CCGTAAAAGC TGCTATACTT CACGGAAAAA CTGCGCATCG CAGCCAAAGA ATCCATCGTG GAAGCTGATA TGTTCTATTT CGACCCGCGT GCGATCAACT GTGCCTTTTT GACGCGTAGC GTCGGTTTCT TAGGTAGCAC CTTCGACTAT ACAAGATAAA GCTGGGCGCA CGCTAGTTGA GGGAAGATTA TTTTCTGAAA ACCCACTTTC CGGGTGTTGT GGAGCATGTC CTGAACTAAT CTAGAgcgtt ttaaacctgt CCCTTCTAAT AAAAGACTTT TGGGTGAAAG GCCCACAACA CCTCGTACAG GACTTGATTA GATCTcgcaa aatttggaca gaggcga -3' ctccgct -5'

169

P. Hbd: Gene name from Clostridium acetobutylicum ATCC 824 (Accession #NP349314) 5’- cgcGAATTCA AGGAGATATA TAatgaaaaa ggtttgcgtt attggtgcgg gtactatggg ttctggtatc gcccaggctt 3’- gcgCTTAAGT TCCTCTATAT ATtacttttt ccaaacgcaa taaccacgcc catgataccc aagaccatag cgggtccgaa

tcgcagcaaa gggcttcgaa gtcgttctgc gcgacattaa ggatgaattt gttgatcgcg gcctggactt cattaacaaa agcgtcgttt cccgaagctt cagcaagacg cgctgtaatt cctacttaaa caactagcgc cggacctgaa gtaattgttt aacctgtcta aactggtaaa gaaaggtaaa atcgaggagg ctacgaaagt agaaatcctg acccgtattt ccggcaccgt ttggacagat ttgaccattt ctttccattt tagctcctcc gatgctttca tctttaggac tgggcataaa ggccgtggca tgacctgaac atggctgccg attgtgacct ggttatcgaa gcagcagtag aacgcatgga catcaagaaa cagattttcg actggacttg taccgacggc taacactgga ccaatagctt cgtcgtcatc ttgcgtacct gtagttcttt gtctaaaagc ctgatctgga caacatctgc aagcctgaaa cgatcctggc gtctaacact tcttccctga gcatcactga agtcgcttcc gactagacct gttgtagacg ttcggacttt gctaggaccg cagattgtga agaagggact cgtagtgact tcagcgaagg gcaaccaaac gtccggataa ggtgattggt atgcacttct ttaacccggc accagttatg aaactggtcg aagtgatccg cgttggtttg caggcctatt ccactaacca tacgtgaaga aattgggccg tggtcaatac tttgaccagc ttcactaggc tggcatcgct acttctcagg aaaccttcga cgcggtgaag gaaacttcca tcgccatcgg taaagatccg gtagaagttg accgtagcga tgaagagtcc tttggaagct gcgccacttc ctttgaaggt agcggtagcc atttctaggc catcttcaac ctgaagcacc gggtttcgta gttaaccgta ttctgatccc gatgattaac gaagcagtag gcatcctggc tgagggtatc gacttcgtgg cccaaagcat caattggcat aagactaggg ctactaattg cttcgtcatc cgtaggaccg actcccatag gcatctgttg aagacatcga caaggcaatg aagctgggtg caaatcaccc tatgggcccg ctggagctgg gtgattttat cgtagacaac ttctgtagct gttccgttac ttcgacccac gtttagtggg atacccgggc gacctcgacc cactaaaata cggcctggac atctgtctgg ccatcatgga cgtactgtac tctgaaacgg gcgattctaa gtaccgtccg cacacgctgc gccggacctg tagacagacc ggtagtacct gcatgacatg agactttgcc cgctaagatt catggcaggc gtgtgcgacg tgaaaaagta tgttcgtgcc ggctggctgg gtcgtaaatc tggcaaaggt ttttacgatt actccaagta aCTCGAGGAC actttttcat acaagcacgg ccgaccgacc cagcatttag accgtttcca aaaatgctaa tgaggttcat tGAGCTCCTG GTCGGTAa -3’ CAGCCATt -5’

170

Q. NphT7: Gene name from Streptomycs sp. Strain CL190 (Accession #BAJ10048)

5'– caccATGACC GACGTTCGTT TTCGTATCAT TGGCACGGGT GCGTACGTGC CGGAGCGTAT TGTGTCCAAC GACGAGGTGG 3'– gtggTACTGG CTGCAAGCAA AAGCATAGTA ACCGTGCCCA CGCATGCACG GCCTCGCATA ACACAGGTTG CTGCTCCACC

GTGCGCCGGC TGGTGTTGAT GATGACTGGA TTACCCGTAA GACCGGCATT CGTCAACGTC GTTGGGCGGC GGACGACCAA CACGCGGCCG ACCACAACTA CTACTGACCT AATGGGCATT CTGGCCGTAA GCAGTTGCAG CAACCCGCCG CCTGCTGGTT GCGACCTCCG ACCTGGCAAC CGCGGCGGGT CGTGCGGCGT TGAAAGCAGC GGGTATTACG CCGGAGCAAC TGACGGTTAT CGCTGGAGGC TGGACCGTTG GCGCCGCCCA GCACGCCGCA ACTTTCGTCG CCCATAATGC GGCCTCGTTG ACTGCCAATA TGCGGTCGCA ACGTCCACCC CGGACCGTCC GCAGCCGCCG ACGGCGGCCT ACGTGCAACA TCATCTGGGC GCAACCGGCA ACGCCAGCGT TGCAGGTGGG GCCTGGCAGG CGTCGGCGGC TGCCGCCGGA TGCACGTTGT AGTAGACCCG CGTTGGCCGT CCGCGGCATT TGATGTTAAC GCTGTGTGCA GCGGCACGGT TTTTGCTCTG TCCAGCGTGG CGGGCACGCT GGTGTATCGT GGCGCCGTAA ACTACAATTG CGACACACGT CGCCGTGCCA AAAACGAGAC AGGTCGCACC GCCCGTGCGA CCACATAGCA GGCGGTTACG CACTGGTCAT TGGTGCCGAT CTGTATTCCC GTATTCTGAA TCCGGCGGAC CGCAAGACCG TTGTTCTGTT CCGCCAATGC GTGACCAGTA ACCACGGCTA GACATAAGGG CATAAGACTT AGGCCGCCTG GCGTTCTGGC AACAAGACAA TGGTGACGGC GCGGGTGCGA TGGTGCTGGG TCCGACCAGC ACGGGTACGG GTCCGATCGT CCGTCGCGTT GCCCTGCACA ACCACTGCCG CGCCCACGCT ACCACGACCC AGGCTGGTCG TGCCCATGCC CAGGCTAGCA GGCAGCGCAA CGGGACGTGT CGTTTGGTGG TCTGACCGAC CTGATTCGTG TGCCGGCGGG TGGCAGCCGC CAACCGCTGG ACACGGATGG CTTGGACGCG GCAAACCACC AGACTGGCTG GACTAAGCAC ACGGCCGCCC ACCGTCGGCG GTTGGCGACC TGTGCCTACC GAACCTGCGC GGTCTGCAAT ACTTCGCTAT GGACGGTCGC GAGGTGCGTC GTTTTGTTAC CGAACACTTG CCGCAACTGA TTAAAGGTTT CCAGACGTTA TGAAGCGATA CCTGCCAGCG CTCCACGCAG CAAAACAATG GCTTGTGAAC GGCGTTGACT AATTTCCAAA CTTGCACGAG GCGGGTGTCG ATGCGGCAGA TATTAGCCAT TTTGTGCCGC ACCAAGCGAA CGGTGTCATG CTGGACGAGG GAACGTGCTC CGCCCACAGC TACGCCGTCT ATAATCGGTA AAACACGGCG TGGTTCGCTT GCCACAGTAC GACCTGCTCC TCTTTGGTGA ACTGCACCTG CCGCGTGCGA CCATGCACCG TACCGTCGAA ACCTACGGCA ATACGGGTGC GGCCAGCATT AGAAACCACT TGACGTGGAC GGCGCACGCT GGTACGTGGC ATGGCAGCTT TGGATGCCGT TATGCCCACG CCGGTCGTAA CCGATTACGA TGGATGCAGC AGTCCGTGCA GGTAGCTTCC GTCCGGGTGA ACTGGTCCTG CTGGCGGGTT TTGGTGGTGG GGCTAATGCT ACCTACGTCG TCAGGCACGT CCATCGAAGG CAGGCCCACT TGACCAGGAC GACCGCCCAA AACCACCACC CATGGCAGCG AGCTTCGCGC TGATCGAGTG GTAAgtcagc ctgacggcta gagcgggt -3' GTACCGTCGC TCGAAGCGCG ACTAGCTCAC CATTcagtcg gactgccgat ctcgccca -5'

171

R. PhaJ: Gene name from Aeromonas caviae (Accession #O32472) 5’- GAATCCCCTC TTGTatgagc gcgcagagcc tggaagtggg tcaaaaagct cgcctgtcca aacgttttgg tgcagcagaa 3’- CTTAGGGGAG AACAtactcg cgcgtctcgg accttcaccc agtttttcga gcggacaggt ttgcaaaacc acgtcgtctt

gttgcggcct tcgcagcact gtctgaagac ttcaatccgc tgcatctgga cccggcattc gcggcaacca ccgcatttga caacgccgga agcgtcgtga cagacttctg aagttaggcg acgtagacct gggccgtaag cgccgttggt ggcgtaaact acgcccgatc gttcatggta tgctgttggc aagcctgttt agcggtctgc tgggtcagca gctgccgggc aaaggtagca tgcgggctag caagtaccat acgacaaccg ttcggacaaa tcgccagacg acccagtcgt cgacggcccg tttccatcgt tttacctggg tcagagcctg agctttaaac tgccggtgtt cgtcggtgac gaggtcacgg cggaggtcga ggttacggcc aaatggaccc agtctcggac tcgaaatttg acggccacaa gcagccactg ctccagtgcc gcctccagct ccaatgccgg ctgcgtgagg acaagccgat cgcgaccctg accacccgta ttttcaccca gggtggcgcg ttggccgtga cgggtgaggc gacgcactcc tgttcggcta gcgctgggac tggtgggcat aaaagtgggt cccaccgcgc aaccggcact gcccactccg cgtggtcaaa ctgccgtaaG AAACTTGA -3’ gcaccagttt gacggcattC TTTGAACT -5’

172

S. PhaJ4 : Gene name from Pseudomonas putida KT2440 (Accession #NP746922) 5'– aATGCCGCAC GTTCCGGTTA CCGAGCTGAG CCAGTACGTG GGTAAAGAAC TGGGTCATAG CGAATGGCTG AAGATTGACC 3'– tTACGGCGTG CAAGGCCAAT GGCTCGACTC GGTCATGCAC CCATTTCTTG ACCCAGTATC GCTTACCGAC TTCTAACTGG

AACAGCGTAT TAATCTGTTC GCGGAGGCGA CCGGCGATTT CCAGTTCATT CATGTTGACC CGGAGAAGGC CGCAAAGACG TTGTCGCATA ATTAGACAAG CGCCTCCGCT GGCCGCTAAA GGTCAAGTAA GTACAACTGG GCCTCTTCCG GCGTTTCTGC CCGTTCGGCG GCACCATTGC GCATGGTTTT CTGACCCTGT CTCTGATCCC GAAACTGATC GAGGACATTC TGGTCCTGCC GGCAAGCCGC CGTGGTAACG CGTACCAAAA GACTGGGACA GAGACTAGGG CTTTGACTAG CTCCTGTAAG ACCAGGACGG GCAAGGCCTG AAAATGGTCG TGAATTACGG TCTGGACTCT GTGCGTTTCA TTCAGCCGGT CAAGGTCGAT AGCCGCGTTC CGTTCCGGAC TTTTACCAGC ACTTAATGCC AGACCTGAGA CACGCAAAGT AAGTCGGCCA GTTCCAGCTA TCGGCGCAAG GTCTGAAAGT TAAACTGGGC GAAGTGGTTG AAAAGAAGCC GGGTCAGTGG CTGCTGAAAG CAATCGCGAC CTTGGAAATC CAGACTTTCA ATTTGACCCG CTTCACCAAC TTTTCTTCGG CCCAGTCACC GACGACTTTC GTTAGCGCTG GAACCTTTAG GAGGGCGAGG AGAAACCGGC GTATATCGCA GAGTCCTTGA GCCTGTGCTT CGTCcggata aacgtgggcg gtagta -3' CTCCCGCTCC TCTTTGGCCG CATATAGCGT CTCAGGAACT CGGACACGAA GCAGgcctat ttgcacccgc catcat -5'

173

T. PhaZ1 : Gene name from Ralstonia eutropha H16 (Accession #YP725659) 5'– ATGCTGTATC AATTGCACGA GTTTCAACGT TCTATCCTGC ACCCGTTGAC CGCCTGGGCA CAGGCGACCG CTAAGACGTT 5'– TACGACATAG TTAACGTGCT CAAAGTTGCA AGATAGGACG TGGGCAACTG GCGGACCCGT GTCCGCTGGC GATTCTGCAA

CACGAACCCG CTGAGCCCGC TGTCCCTGGT GCCGGGTGCG CCGCGCCTGG CCGCGGGCTA TGAGCTGCTG TATCGTTTGG GTGCTTGGGC GACTCGGGCG ACAGGGACCA CGGCCCACGC GGCGCGGACC GGCGCCCGAT ACTCGACGAC ATAGCAAACC GTAAAGAGTA TGAGAAGCCG GCGTTTGATA TTAAGAGCGT TCGCAGCAAC GGTCGTGACA TCCCGATCGT GGAACAAACC CATTTCTCAT ACTCTTCGGC CGCAAACTAT AATTCTCGCA AGCGTCGTTG CCAGCACTGT AGGGCTAGCA CCTTGTTTGG GTTCTGGAAA AACCGTTTTG TAAGCTGGTT CGTTTCAAAC GCTACGCAGA TGACCCGGAA ACGATCAAGC TGCTGAAAGA CAAGACCTTT TTGGCAAAAC ATTCGACCAA GCAAAGTTTG CGATGCGTCT ACTGGGCCTT TGCTAGTTCG ACGACTTTCT TGAGCCGGTT GTGCTGGTGG CGGCTCCGTT GAGCGGTCAC CACGCGACCC TGCTGCGTGA TACCGTTCGT ACGCTGCTGC ACTCGGCCAA CACGACCACC GCCGAGGCAA CTCGCCAGTG GTGCGCTGGG ACGACGCACT ATGGCAAGCA TGCGACGACG AAGACCACAA GGTTTATGTT ACCGATTGGA TTGACGCTCG TATGGTTCCG GTTGAGGAAG GTGCGTTTCA TCTGAGCGAC TTCTGGTGTT CCAAATACAA TGGCTAACCT AACTGCGAGC ATACCAAGGC CAACTCCTTC CACGCAAAGT AGACTCGCTG TACATCTACT ATATTCAAGA GTTTATTCGT CATATTGGCG CGGAGAACCT GCATGTCATC TCTGTTTGCC AGCCGACCGT ATGTAGATGA TATAAGTTCT CAAATAAGCA GTATAACCGC GCCTCTTGGA CGTACAGTAG AGACAAACGG TCGGCTGGCA CCCGGTTCTG GCTGCGATCT CTTTGATGGC AAGCGCGGGT GAGAAAACGC CGCGTACCAT GACGATGATG GGCGGCCCGA GGGCCAAGAC CGACGCTAGA GAAACTACCG TTCGCGCCCA CTCTTTTGCG GCGCATGGTA CTGCTACTAC CCGCCGGGCT TCGACGCCCG CAAAAGCCCG ACGGCGGTGA ATAGCCTGGC CACGAATAAG TCTTTCGAGT GGTTCGAAAA TAATGTGATC AGCTGCGGGC GTTTTCGGGC TGCCGCCACT TATCGGACCG GTGCTTATTC AGAAAGCTCA CCAAGCTTTT ATTACACTAG TACACCGTTC CGGCTAATTA TCCGGGCCAC GGTCGTCGTG TGTATCCGGG TTTCCTGCAA CACGCTGGCT TCGTGGCGAT ATGTGGCAAG GCCGATTAAT AGGCCCGGTG CCAGCAGCAC ACATAGGCCC AAAGGACGTT GTGCGACCGA AGCACCGCTA GAACCCGGAC CGCCATTTGA GCAGCCACTA TGATTTCTAC CTGAGCCTGG TTGAGGGCGA TGCCGACGAC GCAGAGGCGC CTTGGGCCTG GCGGTAAACT CGTCGGTGAT ACTAAAGATG GACTCGGACC AACTCCCGCT ACGGCTGCTG CGTCTCCGCG ATGTTCGCTT TTACGACGAA TATAACGCAG TCCTGGATAT GGCAGCGGAA TATTACCTGG ACACCATCCG CGAAGTTTTC TACAAGCGAA AATGCTGCTT ATATTGCGTC AGGACCTATA CCGTCGCCTT ATAATGGACC TGTGGTAGGC GCTTCAAAAG CAGGAGTTCC GTCTGGCGAA CGGTACGTGG GCGATCGATG GCAACCCGGT CCGTCCGCAG GACATTAAAA GCACCGCACT GTCCTCAAGG CAGACCGCTT GCCATGCACC CGCTAGCTAC CGTTGGGCCA GGCAGGCGTC CTGTAATTTT CGTGGCGTGA GATGACGGTT GAGGGTGAGC TGGACGATAT TTCTGGTGCG GGCCAGACCG CGGCTGCGCA CGATCTGTGT GCGGGCATTC CTACTGCCAA CTCCCACTCG ACCTGCTATA AAGACCACGC CCGGTCTGGC GCCGACGCGT GCTAGACACA CGCCCGTAAG CGAAAATTCG TAAGCAACAC CTGAACGCTG CGCATTGCGG TCATTACGGC ATTTTCTCCG GTCGTCGCTG GCGTGAGGAA GCTTTTAAGC ATTCGTTGTG GACTTGCGAC GCGTAACGCC AGTAATGCCG TAAAAGAGGC CAGCAGCGAC CGCACTCCTT ATCTATCCGC AACTGCGTGA CTTCATCCGC AAGTATCATC AAGCGTCCGC GACGCGCTAA atatggaaac atgcgtgc -3' TAGATAGGCG TTGACGCACT GAAGTAGGCG TTCATAGTAG TTCGCAGGCG CTGCGCGATT tatacctttg tacgcacg -5'

174

U. Ter : Gene name from Euglen gracilis (Accession #Q5EU90) 5'– cccctgtcac tGATCATTCC ATATGTCTTG TCCGGCTTCT CCTAGCGCAG CTGTTGTTTC CGCAGGTGCA CTGTGTCTGT 3'– ggggacagtg aCTAGTAAGG TATACAGAAC AGGCCGAAGA GGATCGCGTC GACAACAAAG GCGTCCACGT GACACAGACA

GCGTTGCTAC TGTTCTGCTG GCCACCGGCA GCAATCCGAC CGCCCTGTCC ACCGCGAGCA CGCGCAGCCC GACTAGCCTG CGCAACGATG ACAAGACGAC CGGTGGCCGT CGTTAGGCTG GCGGGACAGG TGGCGCTCGT GCGCGTCGGG CTGATCGGAC GTGCGTGGCG TTGACCGTGG TCTGATGCGT CCGACCACCG CTGCGGCTCT GACCACGATG CGTGAAGTTC CGCAGATGGC CACGCACCGC AACTGGCACC AGACTACGCA GGCTGGTGGC GACGCCGAGA CTGGTGCTAC GCACTTCAAG GCGTCTACCG GGAAGGCTTT TCTGGCGAGG CTACCTCCGC ATGGGCGGCA GCTGGTCCGC AGTGGGCTGC TCCTCTGGTG GCAGCTGCGT CCTTCCGAAA AGACCGCTCC GATGGAGGCG TACCCGCCGT CGACCAGGCG TCACCCGACG AGGAGACCAC CGTCGACGCA CTAGCGCACT GGCTCTGTGG TGGTGGGCAG CCCGTCGTTC CGTACGCCGC CCACTGGCTG CCCTGGCCGA ACTGCCAACC GATCGCGTGA CCGAGACACC ACCACCCGTC GGGCAGCAAG GCATGCGGCG GGTGACCGAC GGGACCGGCT TGACGGTTGG GCTGTGACTC ACCTGGCCCC ACCGATGGCT ATGTTTACCA CTACCGCGAA AGTTATCCAG CCGAAAATCC GTGGTTTTAT CGACACTGAG TGGACCGGGG TGGCTACCGA TACAAATGGT GATGGCGCTT TCAATAGGTC GGCTTTTAGG CACCAAAATA CTGCACTACC ACTCACCCAA TTGGCTGCGA AAAACGCGTC CAGGAAGAAA TTGCTTACGC TCGTGCGCAC CCGCCAACCA GACGTGATGG TGAGTGGGTT AACCGACGCT TTTTGCGCAG GTCCTTCTTT AACGAATGCG AGCACGCGTG GGCGGTTGGT GCCCTGGCCC TAAGCGTGTA CTGGTCATCG GTTGTAGCAC GGGTTACGGT CTGTCTACCC GTATCACTGC TGCGTTCGGC CGGGACCGGG ATTCGCACAT GACCAGTAGC CAACATCGTG CCCAATGCCA GACAGATGGG CATAGTGACG ACGCAAGCCG TACCAGGCGG CGACCCTGGG CGTTTTCCTG GCGGGTCCAC CGACCAAAGG TCGCCCGGCA GCTGCGGGTT GGTACAACAC ATGGTCCGCC GCTGGGACCC GCAAAAGGAC CGCCCAGGTG GCTGGTTTCC AGCGGGCCGT CGACGCCCAA CCATGTTGTG TGTTGCCTTC GAGAAAGCAG CGCTGGAGGC GGGCCTGTAT GCCCGTTCTC TGAACGGCGA CGCTTTTGAT TCCACTACGA ACAACGGAAG CTCTTTCGTC GCGACCTCCG CCCGGACATA CGGGCAAGAG ACTTGCCGCT GCGAAAACTA AGGTGATGCT AAGCGCGCAC TGTTGAAGCT ATCAAACGTG ACCTGGGCAC CGTAGACCTG GTAGTGTACT CTATCGCTGC CCCGAAGCGT TTCGCGCGTG ACAACTTCGA TAGTTTGCAC TGGACCCGTG GCATCTGGAC CATCACATGA GATAGCGACG GGGCTTCGCA ACCGATCCGG CGACCGGCGT TCTGCACAAG GCTTGTCTGA AACCAATCGG CGCGACTTAC ACCAACCGTA CCGTCAACAC TGGCTAGGCC GCTGGCCGCA AGACGTGTTC CGAACAGACT TTGGTTAGCC GCGCTGAATG TGGTTGGCAT GGCAGTTGTG CGACAAAGCG GAGGTGACCG ATGTTAGCAT CGAACCTGCC TCCCCGGAAG AGATCGCGGA CACGGTTAAA GTGATGGGTG GCTGTTTCGC CTCCACTGGC TACAATCGTA GCTTGGACGG AGGGGCCTTC TCTAGCGCCT GTGCCAATTT CACTACCCAC GTGAAGACTG GGAGCTGTGG ATTCAGGCGC TGAGCGAAGC CGGTGTTCTG GCGGAGGGTG CGAAAACCGT GGCGTACTCC CACTTCTGAC CCTCGACACC TAAGTCCGCG ACTCGCTTCG GCCACAAGAC CGCCTCCCAC GCTTTTGGCA CCGCATGAGG TACATTGGCC CTGAGATGAC CTGGCCGGTA TATTGGTCTG GTACTATTGG CGAAGCCAAA AAGGATGTTG AAAAGGCGGC ATGTAACCGG GACTCTACTG GACCGGCCAT ATAACCAGAC CATGATAACC GCTTCGGTTT TTCCTACAAC TTTTCCGCCG TAAACGTATC ACCCAGCAGT ATGGTTGCCC AGCATACCCG GTAGTCGCTA AAGCGCTGGT CACCCAGGCC AGCTCCGCAA ATTTGCATAG TGGGTCGTCA TACCAACGGG TCGTATGGGC CATCAGCGAT TTCGCGACCA GTGGGTCCGG TCGAGGCGTT TTCCGGTAGT TCCACTGTAC ATTTGCCTGC TGTACCGTGT GATGAAAGAA AAAGGTACTC ATGAAGGTTG CATTGAACAG AAGGCCATCA AGGTGACATG TAAACGGACG ACATGGCACA CTACTTTCTT TTTCCATGAG TACTTCCAAC GTAACTTGTC ATGGTTCGTC TGCTGACCAC TAAACTGTAC CCTGAGAACG GTGCTCCGAT CGTGGACGAA GCGGGCCGTG TTCGTGTTGA TACCAAGCAG ACGACTGGTG ATTTGACATG GGACTCTTGC CACGAGGCTA GCACCTGCTT CGCCCGGCAC AAGCACAACT TGACTGGGAA ATGGCTGAAG ACGTGCAGCA AGCTGTTAAA GACCTGTGGT CCCAGGTGTC TACGGCTAAC CTGAAAGACA ACTGACCCTT TACCGACTTC TGCACGTCGT TCGACAATTT CTGGACACCA GGGTCCACAG ATGCCGATTG GACTTTCTGT TCAGCGACTT CGCTGGCTAC CAAACTGAGT TCCTGCGTCT GTTTGGTTTT GGTATCGACG GTGTAGACTA CGACCAGCCG AGTCGCTGAA GCGACCGATG GTTTGACTCA AGGACGCAGA CAAACCAAAA CCATAGCTGC CACATCTGAT GCTGGTCGGC GTTGACGTTG AAGCGGACCT GCCGAGCGCA GCGCAGCAAT AAGAATTCCT CGAGTCATGA CAggccgcaa cccctcgcgg CAACTGCAAC TTCGCCTGGA CGGCTCGCGT CGCGTCGTTA TTCTTAAGGA GCTCAGTACT GTccggcgtt ggggagcgcc ta -3' at -5'

175

V. Ter: Gene name from Treponema denticola ATCC 35405 (Accession #AE017248) 5’- Gtccagcaag ccacGAGATA TACATatgat cgtcaagcca atggtgcgca ataatatctg tctgaacgct cacccgcagg 3’- Caggtcgttc ggtgCTCTAT ATGTAtacta gcagttcggt taccacgcgt tattatagac agacttgcga gtgggcgtcc

gttgtaaaaa gggtgtagaa gaccagattg aatacactaa gaaacgcatc accgcagaag ttaaagcagg tgccaaagca caacattttt cccacatctt ctggtctaac ttatgtgatt ctttgcgtag tggcgtcttc aatttcgtcc acggtttcgt ccgaaaaacg tcctggtgct gggctgcagc aacggctacg gtctggcaag ccgcattacg gctgcattcg gttacggcgc ggctttttgc aggaccacga cccgacgtcg ttgccgatgc cagaccgttc ggcgtaatgc cgacgtaagc caatgccgcg tgctactatt ggtgttagct tcgaaaaggc gggttctgaa accaaatacg gcactccagg ctggtacaac aacctggcat acgatgataa ccacaatcga agcttttccg cccaagactt tggtttatgc cgtgaggtcc gaccatgttg ttggaccgta tcgacgaagc agcgaagcgt gagggtctgt actctgttac catcgacggt gacgcgttct ctgacgagat caaagctcag agctgcttcg tcgcttcgca ctcccagaca tgagacaatg gtagctgcca ctgcgcaaga gactgctcta gtttcgagtc gttatcgagg aagctaaaaa gaaaggtatc aaattcgacc tgattgtgta ctccctggcc tctccggttc gtaccgaccc caatagctcc ttcgattttt ctttccatag tttaagctgg actaacacat gagggaccgg agaggccaag catggctggg ggataccggc atcatgcaca aaagcgtact gaagccgttt ggcaaaacct tcactggtaa aaccgttgat cctttcaccg cctatggccg tagtacgtgt tttcgcatga cttcggcaaa ccgttttgga agtgaccatt ttggcaacta ggaaagtggc gcgagctgaa ggaaatctcc gccgagccag ctaacgatga ggaggctgct gcgaccgtta aagtgatggg tggcgaagac cgctcgactt cctttagagg cggctcggtc gattgctact cctccgacga cgctggcaat ttcactaccc accgcttctg tgggaacgtt ggatcaaaca actgtccaag gaaggtctgc tggaggaggg ctgtattact ctggcatatt cttacatcgg acccttgcaa cctagtttgt tgacaggttc cttccagacg acctcctccc gacataatga gaccgtataa gaatgtagcc cccggaggcg actcaggcac tgtatcgtaa gggcaccatc ggtaaagcga aagaacatct ggaggccacc gctcaccgtc gggcctccgc tgagtccgtg acatagcatt cccgtggtag ccatttcgct ttcttgtaga cctccggtgg cgagtggcag tgaacaagga aaacccgagc atccgtgctt tcgtgtccgt taacaagggc ctggttacgc gcgcttccgc agtaattccg acttgttcct tttgggctcg taggcacgaa agcacaggca attgttcccg gaccaatgcg cgcgaaggcg tcattaaggc gtcattccgc tgtacctggc ttccctgttt aaagtcatga aagaaaaagg caaccacgaa ggttgtatcg aacaaattac cagtaaggcg acatggaccg aagggacaaa tttcagtact ttctttttcc gttggtgctt ccaacatagc ttgtttaatg tcgcctgtat gcggagcgcc tgtaccgtaa ggatggcact atcccggttg atgaagagaa ccgcatccgc attgacgatt agcggacata cgcctcgcgg acatggcatt cctaccgtga tagggccaac tacttctctt ggcgtaggcg taactgctaa gggaactgga agaggatgta cagaaagcgg tttccgcgct gatggaaaaa gtgacgggcg aaaacgcgga atccctgacg cccttgacct tctcctacat gtctttcgcc aaaggcgcga ctaccttttt cactgcccgc ttttgcgcct tagggactgc gatctggcag gttaccgtca cgactttctg gcgtctaatg gtttcgacgt tgagggtatt aactacgagg cagaagttga ctagaccgtc caatggcagt gctgaaagac cgcagattac caaagctgca actcccataa ttgatgctcc gtcttcaact acgtttcgat cgtatttaaT CTAGAACGcc agggcat -3’ tgcaaagcta gcataaattA GATCTTGCgg tcccgta -5’

176

Appendix 2: The complete list of plasmids and strains used in this study

177

Table A2.1. Plasmids and strains that have been previously published. The plasmids and strains used in chapters 2 and 3.

Strain Genotype Source


+) λ- ATCC

BL21(de3) F– ompT gal dcm lon hsdSB(rB

- mB-) λ(DE3 [lacI lacUV5-T7 gene 1

ind1 sam7 nin5]) Novagen

W3110(de3) F– rph-1 INV(rrnD, rrnE) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) [1]

MG1655(de3) F– ilvG- rfb-50 rph-1 λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) [1]

DH10B(de3)

F– endA1 recA1 galE15 galK16 nupG rpsL ∆lacX74 Φ80dlacZ∆M15 araD139 ∆(ara,leu)7697 mcrA ∆(mrr-hsdRMS-mcrBC) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])

[1]

DH5α(de3)

F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZ∆M15 ∆(lacZYA-argF)U169, hsdR17(rK

- mK+), λ(DE3 [lacI

lacUV5-T7 gene 1 ind1 sam7 nin5]) [1]

DH1(de3) endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK

- mK+) λ(DE3 [lacI

lacUV5-T7 gene 1 ind1 sam7 nin5]) [1]


pBAD33-Bu1 phaA.phaB.crt (Ara), araC, Cmr, p15a [1]

pBT33-Bu1 phaA.phaB (Ara), crt (Trc), araC, Cmr, p15a [1]

pTT33-Bu1 phaA.phaB (Trc), crt (Trc), lacIq, Cmr, p15a [1]

pT5T33-Bu1 phaA.phaB (T5), crt (Trc), lacIq, Cmr, p15a [1]


pET-ccr.adhE2 ccr.adhE2 (T7lac), lacIq, Kmr, ColE1 [1]

pBad-ccr.adhE2 ccr.adhE2 (Ara), araC, Cbr, ColE1 [1]

pTrc-ccr.adhE2 ccr.adhE2 (Trc), lacIq, Cbr, ColE1 [1]

pCWOri-ccr.adhE2 ccr.adhE2 (double Tac), lacIq, Cbr, ColE1 [1]

pBBR1 lacIq, Kmr, pBBR1 [2]

pBBR1-aceEF.lpd aceE.aceF.lpd (lac), lacIq, Kmr, pBBR1 [1]

pET16b-His10Ter his10ter (T7lac), lacIq, Cbr, ColE1 [3]

pET23a-His10Tev.Ter his10tev.ter (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(Y240F) his10ter (Y240F) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(I287A) his10ter (I287A) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(L291A) his10ter (L291A) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(F295A) his10ter(F295A) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(Y370A) his10ter (Y370A) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(L276A/V277A) his10ter(L276A/V277A) (T7lac), lacIq, Cbr, ColE1 [3]

pET16b-His10Ter(L276A/V277A/F295A) his10ter(L276A/V277A/F295A) (T7lac), lacIq, Cbr, ColE1 [3]

178

Table A2.2. Plasmids and strains used in unpublished experiments. The plasmids and strains used in chapters 4 and 5.

Strain Genotype Source


+) λ- ATCC

MC1.16 DH1 ∆adhE::FRT Jeff Hanson

MC1.24 DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT Miao Wen

MC1.29 DH1 ∆adhE::FRT ∆atoB::FRT This Study

MC1.36 DH1 ∆adhE::FRT ∆atoB::FRT ∆fabF::FRT This Study

MC1.40 DH1∆adhE::FRT ∆ldhA::FRT ∆ackA-pta::FRT ∆poxB::FRT ∆frdBC::FRT ∆atoB::FRT

This Study

SIMD90 W3110 galKtyr145UAG∆lacU169 [λ cI857 (int-cIII:bet)] mutS:cat [4]

CY242 MG1655 pyrC, fabB15 [5]

CY244 MG1655 fabF1, fabB15 [6]





pBT33-Bu6 nphT7.hbd (Ara), crt (Trc), araC, Cmr, p15a This Study

pTT33-Bu6 nphT7.hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pTT33-(100)Bu6 nphT7.hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pUC19-nphT7 nphT7 (lac), lacIq, Cbr, pUC19 This Study

pCDF2.tac2-gg0 (Tac, Tac), aadH, Spr, pCDF2 This Study

pCDF2.tac2-nphT7 nphT7 (Tac, Tac), aadH, Spr, pCDF2 This Study

pTT33-(100K)hbd-crt hbd (Trc), crt (Trc), araC, Cmr, p15a This Study

pTET-tetR.GBD1_SH3X_PDZY GBD1_SH3X_PDZY (Tet; X = 1, 2, or 4, Y = 1, 2, or 4), TetR, Apr, ColE1 [7]

pCDF.tet-GBD1_SH3X_PDZY GBD1_SH3X_PDZY (Tet; X = 1, 2, or 4, Y = 1, 2, or 4), TetR, Smr, CloDF13

This Study

pBT33-phaA.hbd-SH3_crt. phaA, hbd (Ara), SH3_crt (Trc), araC, Cmr, p15a This Study

pBT33-phaA.hbd-crt_PDZ phaA, hbd (Ara), crt_PDZ (Trc), araC, Cmr, p15a This Study

pCDFDuet.P(Tet) (Tet), TetR, Smr, CloDF13 This Study

pCWOri-ter_PDZ.adhE2 ter_PDZ, adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter.SH3_adhE2 ter, SH3_adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter_PDZ.SH3_adhE2 ter_PDZ, SH3_adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-SH3_ter.adhE2 SH3_ter, adhE2 (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-ter.adhE2_PDZ ter, adhE2_PDZ (double Tac), lacIq, Cbr, ColE1 This Study

pCWOri-SH3_ter.adhE2_PDZ SH3_ter, adhE2_PDZ (double Tac), lacIq, Cbr, ColE1 This Study

pET23a-HisTev_adhE2 HisTev_adhE2 (T7lac), lacIq, Cbr, ColE1 This Study

pET23a-HisTev_SH3_adhE2 HisTev_SH3_adhE2 (T7lac), lacIq, Cbr, ColE1 This Study

pCWOri-ter.adh.aldh46 ter.adh.aldh46 (Tac, Tac), lacIq, Cbr, ColE1 This Study



pBBR2-matB matB (lac), lacIq, Kmr, pBBR1 This Study

pCDF.tet-acs acs (Tet), aadH, Spr, pCDF2 This Study

pCDF.tet-accBC.dtsR1 accBC.dtsR1 (Tet), aadH, Spr, pCDF2 This Study

pCDF.tet-acs.ACC acs.accBC.dtsR1 (Tet), aadH, Spr, pCDF2 This Study

179

References.


2. M. E. Kovach, P. H. Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. Roop, 2nd and K. M. Peterson, Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes, Gene. 1995, 166, 175-176.

3. B. B. Bond-Watts, A. M. Weeks and M. C. Chang, Biochemical and structural characterization of the trans-enoyl-CoA reductase from Treponema denticola, Biochemistry. 2012, 51, 6827-6837.

4. S. Datta, N. Costantino, X. Zhou and D. L. Court, Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages, Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1626-1631.

5. D. Clark and J. E. Cronan, Jr., Further mapping of several membrane lipid biosynthetic genes (fabC, fabB, gpsA, plsB) of Escherichia coli, J. Bacteriol. 1977, 132, 549-554.

6. A. K. Ulrich, D. de Mendoza, J. L. Garwin and J. E. Cronan, Jr., Genetic and biochemical analyses of Escherichia coli mutants altered in the temperature-dependent regulation of membrane lipid composition, J. Bacteriol. 1983, 154, 221-230.


180

Appendix 3: The complete list of plasmids and strains generated

181

T

able

A3.

1. T

he c

ompl

ete

list

of

stra

ins

engi

neer

ed. A

ll s

trai

n en

gine

ered

dur

ing

the

cour

se o

f th

ese

stud

ies,

inc

ludi

ng t

hose

not

use

d in

dis

cuss

ed e

xper

imen

ts, a

nd th

e m

etho

ds u

sed

to g

ener

ate

the

stra

ins.

# S

trai

n

PC

R P

rim

ers

Tem

pla

teM

eth

od

Co

mm

ent

287

DP

10 (

DE

3)

N/A

lyso

geni

zatio

n ki

t

288

DH

10B

(D

E3)

N

/A

ly

soge

niza

tion

kit

28

9 D

H5a

(D

E3)

N

/A

ly

soge

niza

tion

kit

29

0 W

3110

(D

E3

) N

/A

ly

soge

niza

tion

kit

29

1 K

-12

MG

1655

(D

E3)

N

/A

ly

soge

niza

tion

kit

29

2 K

-12

CW

2513

(D

E3)

N

/A

ly

soge

niza

tion

kit

29

3 D

H1

(DE

3)

N/A

lyso

geni

zatio

n ki

t

296

DH

1 ∆

pgi

pg

i KF

1 an

d pg

i KR

1 pK

D13

λ-

red

from

DH

1

DH

1 ∆

pgi

::

319

BL2

1 (d

e3) ∆

ace

BA

K

aceB

AK

KF

2 an

d a

ceB

AK

KR

1 pK

D13

λ-

red

from

BL2

1(de

3)

BL2

1(de

3) ∆

ace

BA

K::

32

0 D

H1 ∆

aceB

AK

ac

eBA

K K

F2

and

ace

BA

K K

R1

pKD

13

λ-re

d fr

om D

H1

D

H1 ∆

aceB

AK

::

343

MC

1.2

fo

cA-p

flB K

F1

and

focA

-pflB

KR

1

pKD

13

λ-re

d fr

om D

H1

D

H1 ∆

focA

-pflB

::

344

MC

1.3

fr

dBC

KF

1 an

d fr

dBC

KR

1

pKD

13

λ-re

d fr

om D

H1

D

H1 ∆

frdB

C::

34

5 M

C1.

4

ldhA

KF

2 an

d ld

hA

KR

4 pK

D13

λ-

red

from

DH

1

DH

1 ∆

ldhA

::

349

MC

1.5

ld

hA K

F2

and

ldh

A K

R4

pKD

13

λ-re

d fr

om M

C1.

4

DH

1 ∆

ldhA

::; ∆

focA

-pflB

:: v

ia M

C1.

4 ∆

focA

-pflB

::

350

MC

1.6

ld

hA K

F2

and

ldh

A K

R4

pKD

13

λ-re

d fr

om M

C1.

4

DH

1 L8

10: v

ia M

C1.

4 ∆

frdB

C::

36

1 M

C1.

7

ldhA

KF

2 an

d ld

hA

KR

4 pK

D13

λ-

red

from

MC

1.4

D

H1 ∆

ldhA

::; ∆

ackA

-pta

via

MC

1.4

∆ac

kA-p

ta::

36

2 M

C1.

8

ackA

KF

2 an

d pt

a K

R2

pK

D13

λ-

red

from

MC

1.5

D

H1 ∆

ldhA

::; ∆

focA

-pflB

::; ∆

ackA

-pta

via

MC

1.5 ∆

ack

A-p

ta

378

MC

1.9

po

xB K

F3

and

pox

B K

R3

pK

D13

λ-

red

from

MC

1.4

D

H1 ∆

ldhA

::; ∆

pox

B::

via

MC

1.4

37

9 M

C1.

10

poxB

KF

3 a

nd p

oxB

KR

3

pKD

13

λ-re

d fr

om M

C1.

5

DH

1 ∆

ldhA

::; ∆

focA

-pflB

::; ∆

poxB

:: v

ia M

C1.

5 38

0 M

C1.

11

poxB

KF

3 a

nd p

oxB

KR

3

pKD

13

λ-re

d fr

om M

C1.

7

DH

1 ∆

ldhA

::; ∆

ackA

-pta

; ∆po

xB::

via

MC

1.7

38

1 M

C1.

12

poxB

KF

3 a

nd p

oxB

KR

3

pKD

13

λ-re

d fr

om M

C1.

8

DH

1 ∆

ldhA

::; ∆

focA

-pflB

::; ∆

ackA

-pta

::; ∆

poxB

:: vi

a M

C1.

8

388

MC

1.13

fr

dBC

KF

1 an

d fr

dBC

KR

1

pKD

13

λ-re

d fr

om M

C1.

11

DH

1 ∆

ldhA

::; ∆

ackA

-pta

; ∆po

xB::,

∆fr

dBC

via

MC

1.11

389

MC

1.14

fr

dBC

KF

1 an

d fr

dBC

KR

1

pKD

13

λ-re

d fr

om M

C1.

12

DH

1 ∆

ldhA

::; ∆

focA

-pflB

::; ∆

ackA

-pta

::; ∆

poxB

::, ∆

frdB

C v

ia

MC

1.12

10

13

MC

1.28

at

oB K

F1

and

atoB

KR

1 pK

D13

λ-

red

from

DH

1

DH

1 ∆

ato

B

1014

M

C1.

29

atoB

KF

1 an

d at

oB K

R1

pKD

13

λ-re

d fr

om M

C1.

16

DH

1 ∆

ato

B::,

∆a

dhE

(vi

a M

C1.

16)

1097

M

C1.

35

fabF

KO

F2

and

fabF

KO

R2

pK

D13

λ-

red

from

MC

1.28

D

H1 ∆

Fab

F::,

∆a

toB

:: (

via

MC

1.2

8)

1098

M

C1.

36

fabF

KO

F2

and

fabF

KO

R2

pK

D13

λ-

red

from

MC

1.29

D

H1 ∆

Fab

F::,

∆a

toB

::, ∆

adhE

:: (v

ia M

C1.

29)

1099

M

C1.

37

mdh

KO

F2

and

mdh

KO

R2

pK

D13

λ-

red

from

MC

1.29

D

H1 ∆

mdh

::, ∆

atoB

::, ∆

adhE

:: (v

ia M

C1.

29)

1100

M

C1.

38

tesB

KO

F2

and

tesB

KO

R2

pK

D13

λ-

red

from

MC

1.29

D

H1 ∆

tesB

::, ∆

atoB

::, ∆

adhE

:: (v

ia M

C1.

29)

1129

M

C1.

40

atoB

KF

1 an

d at

oB K

R1

pKD

13

λ-re

d fr

om M

C1.

24

DH

1 ∆

ackA

-pta

::; ∆

adhE

::; ∆

ldhA

::; ∆

poxB

::;D

frdB

C::;

∆at

oB::

(via

MC

1.24

)

182

T

able

A3.

2. T

he c

ompl

ete

list

of

plas

mid

s cl

oned

. All

pla

smid

s cl

oned

dur

ing

the

cour

se o

f th

ese

stud

ies,

inc

ludi

ng t

hose

not

use

d in

di

scus

sed

expe

rim

ents

, and

the

met

hods

use

d to

clo

ne th

e pl

asm

ids.

# P

lasm

id

PC

R P

rim

ers

T

emp

late

V

ecto

r R

estr

icti

on

E

nzy

mes

M

eth

od

972

pAC

YC

184-

MC

S2

pAC

YC

184

linke

r F

1 an

d pA

CY

C18

4 lin

ker

R1

pA

CY

C18

4

Hin

dIII-

H

incI

I A

nnea

l prim

ers:

S

tand

ard

ligat

ion

596

pAR

O18

1.C

m-

*P(g

ap).

Bu2

.TcR

.T(p

dh)

pUC

19 S

acI e

xpan

der

F1

and

pUC

19 S

acI e

xpan

der

R1

N

o te

mpl

ate

pA

RO

181.

Cm

-P

(gap

).B

u2.T

cR.T

(pdh

) S

acI

Prim

er A

nnea

l: S

tand

ard

ligat

ion

971

pAR

O18

1.C

m-B

utan

ol.T

cR

No

PC

R, D

irec

tly c

ut o

ur in

sert

pB

BR

1-B

utan

ol(Z

m)

pAR

O18

1.C

m

Nde

I-B

amH

I D

irect

liga

tion

598

pAR

O18

1.C

m-P

(adh

B).

adhE

2.

P(g

ap).

ccr.

Cm

R.T

(adh

B)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pU

C19

-P

(adh

B).

adhE

2.P

(gap

).cc

r.C

mR

.T(a

dhB

) pA

RO

181.

Cm

N

deI-

Bam

HI

Dire

ct li

gatio

n

N/A

pA

RO

181.

Cm

-P

(gap

).B

u2.T

cR.T

(pdh

) N

o P

CR

, Dir

ectly

cut

our

inse

rt

pUC

19*-

P(g

ap).

Bu2

.TcR

.T(p

dh)

pAR

O18

1.C

m

Sac

I-X

baI

Dire

ct li

gatio

n

596

pAR

O18

1.C

m-P

(pdc

).pd

h.

P(g

ap).

Bu2

.TcR

.T(p

dh)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pR

C2.

1-P

(pdc

).ac

eE.a

ceF

.lpd

pAR

O18

1.C

m-

*P(g

ap).

Bu2

.TcR

.T(p

dh)

Xho

I-S

acI

Dire

ct li

gatio

n

984

pAR

O18

1.C

m-P

dh.B

u1.T

c N

/A

pA

RO

181.

Cm

-P

(pdc

).pd

h.P

(ga

p).B

u1.

TcR

.T(p

dh)

S

ame

plas

mid

985

pAR

O18

1.C

m-P

dh.B

u2.T

c N

/A

pA

RO

181.

Cm

-P

(pdc

).pd

h.P

(ga

p).B

u2.

TcR

.T(p

dh)

S

ame

plas

mid

599

pAR

O18

1.T

c-P

(adh

B).

adhE

2.

P(g

ap).

ter.

Cm

R.T

(adh

B)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pU

C19

-P

(adh

B).

adhE

2.P

(gap

).T

ER

.Cm

R.T

(adh

B)

pAR

O18

1.T

c N

deI-

Bam

HI

Dire

ct li

gatio

n

N/A

pA

RO

181-

P(g

ap).

phaA

.hbd

. P

(eda

).cr

t.T

cR.T

(pdc

)

SO

E: (

P(g

ap)

F1

/SLI

C F

2 an

d H

bd S

OE

R1)

an

d (P

(eda

) S

OE

F

1 an

d P

(eda

) R

1)

Z. m

obili

s G

enom

ic D

NA

; pU

C19

-P

(gap

).B

u2.T

cR.T

(pdh

)

pAR

O18

1.C

m-

P(p

dc).

pdh.

P(g

ap)

.Bu2

.T

cR.T

(pdh

)

Sac

I-N

coI

(par

tial

Nco

I di

gest

for

vect

or)

Sta

ndar

d lig

atio

n

N/A

pB

AD

24-a

ceE

.ace

F

aceE

F1

and

ace

F R

1

Gen

omic

DN

A

pBA

D24

N

heI-

Sal

I S

tand

ard

ligat

ion

98

2 pB

AD

24-a

ceE

.F.lp

d lp

d F

1 an

d lp

d R

1 pB

AD

24-l

pd

pBA

D24

-ace

E.F

X

baI-

Sal

I S

tand

ard

ligat

ion

22

5 pB

AD

24-A

DH

ad

hE2

AD

H F

1 a

nd a

dhE

2 R

1

pBS

K-B

u3

pBA

D24

N

heI-

Kpn

I S

tand

ard

ligat

ion

22

4 pB

AD

24-A

LDH

ad

hE2

F7

and

ad

hE2

AA

DH

R1

pB

SK

-Bu3

pB

AD

24

Nhe

I-K

pnI

Sta

ndar

d lig

atio

n

210

pBA

D24

-ccr

cc

r F

1 an

d cc

r R

2 pB

SK

-Bu

2

pBA

D24

N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

245

pBA

D24

-ccr

.adh

E2

adhE

2 F

1 a

nd a

dhE

2 R

17

pBS

K-B

u3

pBA

D24

-ccr

Eco

RI-

Xho

I in

sert

; E

coR

I-S

alI

vect

or

Sta

ndar

d lig

atio

n

220

pBA

D24

-ccr

-ad

hE

2 cc

r F

1 an

d ad

hE2

R1

pE

T29

a-cc

r.ad

hE

2 pB

AD

24

Nde

I-S

acI

Sta

ndar

d lig

atio

n

N/A

pB

AD

24-lp

d

lpd

F1

and

lpd

R2

DH

10B

gen

om

ic D

NA

pB

BR

2-ac

eE.F

X

baI-

Sac

I S

tand

ard

ligat

ion

N

/A

pBA

D24

-lpd(

E3

54K

) lp

d Q

C F

1 an

d lp

d Q

C R

1

pBA

D24

-lpd

pB

AD

24-lp

d

Q

uick

Cha

nge

N

/A

pBA

D24

-lpd(

H32

2Y

) lp

d Q

C F

2 an

d lp

d Q

C R

2

pBA

D24

-lpd

pB

AD

24-lp

d

Q

uick

Cha

nge

183

N

/A

pBA

D24

-lpd(

H32

2Y

;E35

4K)

lpd

QC

F2

and

lpd

QC

R2

pB

AD

24-lp

d

pBA

D24

-lpd(

E3

54K

)

Qui

ck C

hang

e

231

pBA

D24

-sB

dhB

bd

hB F

101

and

bdh

B R

103

pC

R2.

1-sb

dhB

pB

AD

24

Eco

RI-

Kpn

I S

tand

ard

ligat

ion

21

4 pB

AD

24-s

trep

AL

DH

E2

ad

hE2

F6

and

ad

hE2

R1

pB

SK

-Bu3

pB

AD

24

Nde

I-K

pnI

Sta

ndar

d lig

atio

n

973

pBA

D33

-ato

B(1

0)p

haB

S

OE

: (at

oB F

1 a

nd a

toB

R1)

and

(p

haB

F3

and

phaB

R2)

G

enom

ic D

NA

; pB

AD

33-

phaB

pB

AD

33-p

haA

.pha

B

Nde

I-X

baI

Sta

ndar

d lig

atio

n

208

pBA

D33

-ccr

cc

r F

1 an

d cc

r R

1 pB

SK

-Bu

2

pBA

D33

N

deI-

Xba

I S

tand

ard

ligat

ion

206

pBA

D33

-ccr

-ad

hE

2 cc

r F

1 an

d ad

hE2

R17

pE

T29

a-cc

r.ad

hE

2 pB

AD

33-p

haA

.pha

B

Nde

I-X

hoI

inse

rt;

Nde

I-S

alI

vect

or

Sta

ndar

d lig

atio

n

209

pBA

D33

-ccr

-ph

aA

-pha

B-c

rt

phaA

F1

and

crt

R1

pBS

K-B

u1

pBA

D33

-ccr

X

baI-

Sph

I S

tand

ard

ligat

ion

22

2 pB

AD

33-c

rt

crt F

1 an

d cr

t R

2

pBS

K-B

u1

pBA

D33

S

acI-

Xm

aI

Sta

ndar

d lig

atio

n

601

pBA

D33

-pha

A.h

bd.c

rt

SO

E: (

phaA

F5

and

HB

D/c

rt S

OE

R

1) +

(H

BD

/crt

SO

E F

1 a

nd c

rt

R2)

pB

AD

33-p

haA

.pha

B.c

rt

pBA

D33

X

baI-

Hin

dIII

S

tand

ard

ligat

ion

N/A

pB

AD

33-p

haA

.pha

B

phaA

2 F

2 a

nd p

haB

R2

pC

R2.

1-ph

aA.p

haB

pB

AD

33

Sac

I-X

baI

Sta

ndar

d lig

atio

n

600

pBA

D33

-pha

A.p

haB

.crt

ph

aA F

5 an

d cr

t R

2 pB

SK

-Bu1

pB

AD

33

Xba

I-H

indI

II

Sta

ndar

d lig

atio

n

230

pBA

D33

-sB

dhB

sB

dhB

F1

and

sBdh

B R

103

bh

dB F

100

and

bdh

B R

100

pB

AD

33

Bam

HI-

Kpn

I S

tand

ard

ligat

ion

672

pBB

R1-

But

anol

(Zm

) N

o P

CR

, Dir

ectly

cut

our

inse

rt

pAR

O18

1.T

c-P

(adh

B).

adhE

2.P

(gap

).te

r.C

mR

.T(a

dhB

)

pBB

R1-

P(g

ap).

pha

A.h

bd.P

(eda

).c

rt

Xm

aI-K

pnI

Dire

ct li

gatio

n

534

pBB

R1M

CS

2-pd

hA.p

dhB

.ace

F.lp

dA

pdhA

F2

and

lpd

R2

Gen

omic

DN

A

pB

BR

1MC

S2

K

pnI-

Sac

I S

tand

ard

ligat

ion

N/A

pB

BR

1-P

(gap

).p

haA

.hbd

.P(e

da).

crt

P(g

ap)

F1/

SLI

C F

2 an

d cr

t R

1/S

LIC

R1

pBB

R1-

P(g

ap).

pha

A.h

bd.P

(eda

).c

rt

pBB

R1M

CS

1

Sac

I-X

maI

S

tand

ard

ligat

ion

970

pBB

R1-

P(p

dc).

pdh

P

(pdc

) F

2 an

d P

(pdc

) R

1

Z.

mob

ilis

Gen

om

ic D

NA

pB

BR

2-ac

eE.F

.lpd

(WT

) X

hoI-

Bam

HI

Sta

ndar

d lig

atio

n

673

pBB

R1-

PD

Hc-

Bu

tano

l(Zm

) N

o P

CR

, Dir

ectly

cut

our

inse

rt

pRC

2.1-

P(p

dc).

aceE

.ace

F.lp

d pB

BR

1-B

utan

ol(Z

m)

Xho

I-S

acI

Dire

ct li

gatio

n

555

pBB

R2-

(Bsa

/Nco

)ace

E.a

ceF

.lpd

ac

cE F

3 an

d ac

cE R

2 N

o te

mpl

ate

pB

BR

2-ac

eE.F

.lpd

(WT

) X

hoI-

Bam

HI

Sta

ndar

d lig

atio

n

1188

pB

BR

2.P

(pro

)(g

g)-

0

pBB

R2.

P(P

ro)-

0 gg

F1

and

pBB

R2.

P(P

ro)-

0 gg

R1

N

o te

mpl

ate

pB

BR

2.P

(Pro

)-0

X

maI

-Eco

RI

Ann

eal P

rimer

/Gib

son

Isot

herm

al

1157

pB

BR

2.P

(Pro

)-0

pB

BR

1-P

ro F

1 a

nd p

BB

R1

R1

N

o te

mpl

ate

pB

BR

2.P

(Pro

)-a

ceE

.F.lp

d B

amH

I-K

pnI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

1182

pB

BR

2.P

(pro

)-n

phT

7

nphT

7 G

F2

and

nphT

7 G

R1

pB

T33

-Bu6

pB

BR

2.P

(Pro

)-0

B

amH

I-H

F-

Xba

I G

ibso

n Is

othe

rmal

1158

pB

BR

2.P

(Tac

)-0

pB

BR

1-T

ac F

2 a

nd p

BB

R1

R1

N

o te

mpl

ate

pB

BR

2.P

(Tac

)-a

ceE

.F.lp

d B

amH

I-K

pnI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

1159

pB

BR

2.P

(Trc

)-0

pB

BR

1-T

rc F

2 a

nd p

BB

R1

R1

N

o te

mpl

ate

pB

BR

2.P

(Trc

)-ac

eE.F

.lpd

B

amH

I-K

pnI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

808

pBB

R2-

aceE

(G3

21S

).ac

eF.lp

d

SO

E: (

aceE

F2

and

ace

E S

OE

R

1) a

nd (

aceE

SO

E F

1 a

nd

aceF

R1)

pB

BR

2-ac

eE.a

ceF

.lpd

pBB

R2-

aceE

.ace

F.lp

d X

hoI-

Xba

I S

tand

ard

ligat

ion

810

pBB

R2-

aceE

.ace

F(H

602A

).lp

d

SO

E: (

aceE

F2

and

ace

f SO

E R

2)

and

(ace

F S

OE

F2

and

ace

F

pBB

R2-

aceE

.ace

F.lp

d pB

BR

2-ac

eE.a

ceF

.lpd

Xho

I-X

baI

Sta

ndar

d lig

atio

n

184

R1)

809

pBB

R2-

aceE

.ace

F(H

602C

).lp

d

SO

E: (

aceE

F2

and

ace

f SO

E R

1)

and

(ace

F S

OE

F1

and

ace

F

R1)

pB

BR

2-ac

eE.a

ceF

.lpd

pBB

R2-

aceE

.ace

F.lp

d X

hoI-

Xba

I S

tand

ard

ligat

ion

579

pBB

R2-

aceE

.ace

F.lp

d(E

354K

) lp

d F

1 an

d lp

d R

2 pB

AD

24-lp

d(E

35

4K)

pBB

R2-

aceE

.F

Xba

I-S

acI

Sta

ndar

d lig

atio

n

582

pBB

R2-

aceE

.ace

F.lp

d(H

322

Y)

lpd

F1

and

lpd

R2

pBA

D24

-lpd(

H32

2Y

) pB

BR

2-ac

eE.F

X

baI-

Sac

I S

tand

ard

ligat

ion

581

pBB

R2-

aceE

.ace

F.lp

d(H

322

Y;

E35

4K)

lpd

F1

and

lpd

R2

pBA

D24

-lpd(

H32

2Y

;E35

4K)

pBB

R2-

aceE

.F

Xba

I-S

acI

Sta

ndar

d lig

atio

n

N/A

pB

BR

2-ac

eE.F

ac

eE F

2 an

d ac

eF

R1

D

H10

B g

eno

mic

DN

A

pBB

R1M

CS

2

Xho

I-X

baI

Sta

ndar

d lig

atio

n

339

pBB

R2-

aceE

.F.lp

d (W

T)

lpd

F1

and

lpd

R2

pBA

D24

-lpd

pB

BR

2-ac

eE.F

X

baI-

Sac

I S

tand

ard

ligat

ion

340

pBB

R2-

aceE

.F.lp

d(A

) S

OE

: (lp

d F

1/lp

d S

OE

R3)

+ (

lpd

SO

E F

3/lp

d R

2)

pBB

R2-

aceE

.F.lp

d (W

T)

pBB

R2-

aceE

.F

Xba

I-S

acI

Sta

ndar

d lig

atio

n

962

pBB

R2-

aceE

.F.lp

d(A

2)

SO

E: (

lpd

F1/

lpd

SO

E R

2) +

(lp

d S

OE

F2

/lpd

R2)

pB

BR

2-ac

eE.F

.lpd

(WT

) pB

BR

2-ac

eE.F

X

baI-

Sac

I S

tand

ard

ligat

ion

341

pBB

R2-

aceE

.F.lp

d(C

) S

OE

: (lp

d F

1/lp

d S

OE

R4)

+ (

lpd

SO

E F

4/lp

d R

2)

pBB

R2-

aceE

.F.lp

d(A

) pB

BR

2-ac

eE.F

X

baI-

Sac

I S

tand

ard

ligat

ion

336

pBB

R2-

arcA

ar

cA F

1 an

d a

rcA

R1

DH

10B

gen

om

ic D

NA

pB

BR

1MC

S2

X

hoI-

Sac

I S

tand

ard

ligat

ion

55

6 pB

BR

2-cr

p

crp

F1

and

crp

R1

DH

10B

gen

om

ic D

NA

pB

BR

1MC

S2

X

hoI-

Sac

I S

tand

ard

ligat

ion

33

7 pB

BR

2-fa

dR

fadR

F1

and

fad

R R

1

DH

10B

gen

om

ic D

NA

pB

BR

1MC

S2

X

hoI-

Sac

I S

tand

ard

ligat

ion

33

8 pB

BR

2-ic

lR

iclR

F1

and

iclR

R1

DH

10B

gen

om

ic D

NA

pB

BR

1MC

S2

X

hoI-

Sac

I S

tand

ard

ligat

ion

55

4 pB

BR

2-Y

dbK

Y

dbK

F2

and

Yd

bK R

2

Gen

omic

DN

A

pBB

R1M

CS

2

Kpn

I-S

alI

Sta

ndar

d lig

atio

n

226

pBS

K-s

trep

Bu3

ad

hE2

F6

and

ad

hE2

R3

p

BS

K-B

u3

pBS

K-B

u3

Nde

I-A

scI

Sta

ndar

d lig

atio

n

942

pBT

33-(

100K

-1)B

u6

BT

33-N

phT

7 1

0000

0 F

1 an

d B

T33

-Nph

T7

100

000

R1

N

o te

mpl

ate

pB

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

945

pBT

33-(

100K

-2)B

u6

BT

33-N

phT

7 1

00K

F2

and

BT

33-

Nph

T7

100

K R

2

No

tem

plat

e

pBT

33-B

u6

Nde

I A

nnea

l Prim

ers;

G

ibso

n Is

othe

rmal

941

pBT

33-(

10K

-1)B

u6

BT

33-N

phT

7 1

0000

F1

and

BT

33-N

phT

7 1

0000

R1

N

o te

mpl

ate

pB

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

943

pBT

33-(

10K

-2)B

u6

BT

33-N

phT

7 1

0K

F2

and

BT

33-

Nph

T7

10K

R2

N

o te

mpl

ate

pB

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

944

pBT

33-(

30K

-2)B

u6

BT

33-N

phT

7 3

0K

F2

and

BT

33-

Nph

T7

30K

R2

N

o te

mpl

ate

pB

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

946

pBT

33-(

His

)Bu6

B

T33

-Nph

T7

His

F1,

BT

33-

Nph

T7

His

R1,

and

BT

33-

Nph

T7

His

F2

No

tem

plat

e

pBT

33-B

u6

Nde

I A

nnea

l Prim

ers;

G

ibso

n Is

othe

rmal

1181

pB

T33

-0.h

bd-c

rt

hbd

1209

17 G

F1

and

HB

D S

LIC

R

1 pB

T33

-Bu2

pB

T33

-Bu2

N

deI-

Xho

I G

ibso

n Is

othe

rmal

316

pBT

33-B

u1

pTrc

99a

F4

and

pTrc

99a

R4

pT

rc99

a-c

rt

pBA

D33

-pha

A.p

haB

B

glI

Sta

ndar

d lig

atio

n

318

pBT

33-B

u1(p

haA

*)

SO

E: (

phaA

F2

and

pha

A R

4)

+

(pha

A F

4/ph

aA R

1)

pBT

33-B

u1

pBT

33-B

u1

Nde

I-E

coR

I S

tand

ard

ligat

ion

318

pBT

33-B

u1(p

haA

*,ph

aB*)

S

OE

: (ph

aA F

2 a

nd p

haB

R4

) +

(p

haB

F4/

phaB

R1)

pB

T33

-Bu1

(pha

A*)

pB

T33

-Bu1

N

deI-

Bam

HI

Sta

ndar

d lig

atio

n

318

pBT

33-B

u1(p

haA

*,ph

aB*,

crt*

) S

OE

: (p

Trc

99a

F11

and

crt

R3

) +

(c

rt F

3/cr

t R

2)

pBT

33-B

u1

pBT

33-B

u1(p

haA

*,ph

aB*)

E

agI-

Xm

aI

Sta

ndar

d lig

atio

n

N/A

pB

T33

-Bu1

_2

rrnB

SLI

C F

1 an

d rr

nB S

LIC

R1

pB

AD

33

pBT

33-B

u1

Nde

I and

X

hoI

SLI

C

533

pBT

33-B

u2

phaA

2F2

and

hbd

R10

0

pCR

2.1-

phaA

.hb

d pB

T33

-Bu1

_2

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

772

pBT

33-B

u2 (

hbd

rbs

100K

) N

/A

M

ade

by

Mac

rola

b

185

77

3 pB

T33

-Bu2

(hb

d rb

s 30

0K)

N/A

Mad

e b

y M

acro

lab

771

pBT

33-B

u2 (

hbd

rbs

30K

) N

/A

M

ade

by

Mac

rola

b

N/A

pB

T33

-Bu2

(ph

aA

C37

9A)

SO

E: (

phaA

F2

and

pha

A R

4)

and

(pha

A F

4 an

d hb

d R

100

) pB

T33

-Bu2

pB

T33

-Bu2

N

deI-

Xho

I S

tand

ard

ligat

ion

811

pBT

33-B

u2 (

pha

A C

379A

, hbd

H

138V

, crt

E13

4Q

) S

OE

: (p

Trc

99a

F11

and

crt

R3

) +

(c

rt F

3/cr

t R

2)

pBT

33-B

u2

pBT

33-B

u2 (

pha

A C

379A

, hb

d H

138V

) X

maI

-Eag

I S

tand

ard

ligat

ion

735

pBT

33-B

u2 (

pha

A: C

379A

; hbd

: H

138V

)

SO

E: (

phaA

F2

and

hbd

SO

E R

1)

and

(hbd

SO

E F

1 an

d hb

d R

100)

pB

T33

-Bu2

(ph

aA

C37

9A)

pBT

33-B

u2

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

687

pBT

33-B

u3

SO

E: (

pT

rc99

a F

11 a

nd P

haJ

SO

E R

1) +

(ph

aJ S

OE

F1/

pha

J R

100)

G

ene

synt

hesi

s pB

T33

-Bu1

E

agI-

Xm

aI

Sta

ndar

d lig

atio

n

688

pBT

33-B

u4

SO

E: (

pT

rc99

a F

11 a

nd P

haJ

SO

E R

1) +

(ph

aJ S

OE

F1/

pha

J R

100)

G

ene

synt

hesi

s pB

T33

-Bu2

E

agI-

Xm

aI

Sta

ndar

d lig

atio

n

1149

pB

T33

-hbd

-crt

hb

d G

F1

1302

07 a

nd h

bd G

R1

1302

07

pBT

33-B

u2

pBT

33-B

u2

Nde

I-X

hoI

Gib

son

Isot

herm

al

777

pBT

33-n

phT

7.h

bd-

crt

SO

E: (

Np

hT7

F1

00 a

nd n

phT

7 R

101)

an

d (h

bd

F10

1 an

d hb

d R

100)

pC

R2.

1-np

hT7

pB

T33

-Bu2

N

deI-

Xho

I S

tand

ard

ligat

ion

1127

pB

T33

-PH

A1

S

OE

: (p

Trc

99a

F11

and

Pha

J S

OE

R1)

+ (

phaJ

SO

E F

1/p

haJ

R10

0)

Gen

e sy

nthe

sis

pBT

33-P

haA

BC

P1-

crt

Eag

I-X

maI

674

pBT

33-p

haA

.GB

D_H

BD

-S

H3_

crt

SO

E: (

phaA

F2

and

hbd

aG

BD

-S

OE

F1

) an

d (h

bd

aGB

D-S

OE

R

1 an

d hb

d R

100)

pB

T33

-Bu2

pB

T33

-pha

A.H

BD

-SH

3_cr

t N

deI-

Xho

I S

tand

ard

ligat

ion

818

pBT

33-p

haA

.hbd

_PD

Z-c

rt

phaA

2 F

2 a

nd h

bd

R10

4

pBT

33-B

u2

pBT

33-B

u2

Nde

I-X

hoI

Gib

son

isot

herm

al

911

pBT

33-p

haA

.hbd

-crt

_PD

Z

pTrc

99a

F11

and

crt

R6

pB

T33

-Bu2

pB

T33

-Bu2

X

maI

-Eag

I S

tand

ard

ligat

ion

633

pBT

33-p

haA

.HB

D-S

H3_

crt

SO

E: (

pT

rc99

a F

12 a

nd c

rt S

OE

R

6) a

nd (

crt S

OE

F6

and

crt

R5)

pT

rc99

a-c

rt

pBT

33-B

u2

Xm

aI-E

agI

Sta

ndar

d lig

atio

n

827

pBT

33-p

haA

.hbd

-SH

3_cr

t v2.

0

SO

E: (

pT

rc99

a F

11 a

nd c

rt S

OE

R

7) +

(cr

t SO

E F

7 an

d cr

t R2

) pB

T33

-Bu2

pB

T33

-Bu2

X

maI

-Eag

I S

tand

ard

ligat

ion

N/A

pB

T33

-Pha

AB

CP

1-cr

t sP

haP

1 F

104

and

sPha

P1

R10

4

Gen

e sy

nthe

sis

pBT

33-P

haA

BC

-crt

X

hoI

82

5 pB

T33

-SH

3_ph

aA

.hbd

_PD

Z-c

rt

phaA

F7

and

hbd

R10

4

pBT

33-S

H3_

pha

A.h

bd-c

rt

pBT

33-B

u2

Nde

I-X

hoI

Gib

son

isot

herm

al

817

pBT

33-S

H3_

pha

A.h

bd-c

rt

phaA

F7

and

hbd

R10

0

pBT

33-B

u2

pBT

33-B

u2

Nde

I-X

hoI

Gib

son

isot

herm

al

1187

pB

u6

nphT

7 B

F1

and

hbd

BR

1

pBT

33-B

u6

pBu2

S

acI-

Xho

I G

ibso

n Is

othe

rmal

999

pCD

F.P

(Tet

).P

(Pro

)-0

-0

pCD

F.P

(Tet

).P

(Pro

) F

1 an

d pC

DF

.P(T

et).

P(P

ro)

R1

pP

ro18

pC

DF

.P(T

et)-

0

Nhe

I G

ibso

n Is

othe

rmal

1104

pC

DF

.P(T

et).

P(P

ro)-

Acc

D4

.Acc

E.A

ccA

3-0

Acc

D4:

Acc

D4

F10

3 an

d A

ccD

4 R

102;

Acc

E: A

ccE

F10

2 an

d A

ccE

R10

2; A

ccA

3: A

ccA

3 F

102

and

Acc

A3

R10

3

Acc

D4:

pE

T16

b-H

is.A

cc(M

t)D

4; A

ccE

: pE

T16

b-H

is.A

cc(M

t)E

; A

ccA

3: p

ET

16b-

His

.Acc

(Mt)

A3

pCD

F.P

(Tet

).P

(Pro

)-0

-0

Bgl

II-H

indI

II

Gib

son

Isot

herm

al; 3

in

sert

s

1179

pC

DF

.P(T

et).

P(P

ro)-

Acc

D4.

Acc

E.A

ccA

3-bi

rA

birA

F3

and

birA

R4

DH

10B

Gen

omic

DN

A

pCD

F.P

(Tet

).P

(Pro

)-A

ccD

4.A

ccE

.Acc

A3

-0

Sac

I-B

amH

I G

ibso

n Is

othe

rmal

1105

pC

DF

.P(T

et).

P(P

ro)-

Acc

D6

.Acc

E.A

ccA

3-0

A

ccD

6: A

ccD

6 F

103

and

Acc

D6

R10

2; A

ccE

: Acc

E F

103

and

Acc

D6:

pE

T16

b-H

is.A

cc(M

t)D

6; A

ccE

: pC

DF

.P(T

et).

P(P

ro)-

0-0

B

glII-

Hin

dIII

G

ibso

n Is

othe

rmal

; 3

inse

rts

186

Acc

E R

102;

Acc

A3:

Acc

A3

F10

2 an

d A

ccA

3 R

103

pE

T16

b-H

is.A

cc(M

t)E

; A

ccA

3: p

ET

16b-

His

.Acc

(Mt)

A3

1180

pC

DF

.P(T

et).

P(P

ro)-

Acc

D6.

Acc

E.A

ccA

3-bi

rA

birA

F3

and

birA

R4

DH

10B

Gen

omic

DN

A

pCD

F.P

(Tet

).P

(Pro

)-A

ccD

6.A

ccE

.Acc

A3

-0

Sac

I-B

amH

I G

ibso

n Is

othe

rmal

1000

pC

DF

.P(T

et).

P(P

ro)-

acs.

AC

C-0

pC

DF

.P(T

et).

P(P

ro)

F1

and

pCD

F.P

(Tet

).P

(Pro

) R

1

pPro

18

pCD

F.P

(Tet

)-ac

s.A

CC

N

heI

Gib

son

Isot

herm

al

1178

pC

DF

.P(T

et).

P(P

ro)-

acs.

AC

C-

birA

bi

rA F

3 an

d bi

rA R

4 D

H10

B G

enom

ic D

NA

pC

DF

.P(T

et).

P(P

ro)-

acs

.AC

C-0

S

acI-

Bam

HI

Gib

son

Isot

herm

al

1139

pC

DF

.P(T

et).

P(P

ro)-

btP

haZ

.rrA

BH

.re

Pha

P1-

0

btP

haZ

: btP

haZ

F1

and

bt P

haZ

R

1; r

rAB

H: r

rAB

H F

1 an

d rr

AB

H R

1; r

ePha

P1:

Pha

P1

F1

and

Pha

P R

1

pJ20

4-bt

Pha

Z; p

J204

-rr

AB

H; p

CD

F3

-Pte

t-sP

haZ

1-sP

haP

1

pCD

F.P

(Tet

).P

(Pro

)-M

CS

2-0

B

glII-

Hin

dIII

G

ibso

n Is

othe

rmal

; 3

inse

rts

1145

pC

DF

.P(T

et).

P(P

ro)-

btP

haZ

.rrA

BH

.re

Pha

P1-

Alk

K

alkK

F1

and

alkK

R1

Gen

e sy

nthe

sis

pCD

F.P

(Tet

).P

(Pro

)-bt

Pha

Z.r

rAB

H.r

rAdp

A-0

K

pnI-

Bam

HI

Gib

son

Isot

herm

al

1138

pC

DF

.P(T

et).

P(P

ro)-

btP

haZ

.rrA

BH

.rrA

dpA

-0

btP

haZ

: btP

haZ

F1

and

bt P

haZ

R

1; r

rAB

H: r

rAB

H F

1 an

d rr

AB

H R

2; r

rAd

pA

: rrA

dpA

F1

and

rrA

dpA

R1

pJ20

4-bt

Pha

Z; p

J204

-rr

AB

H; p

J204

-rrA

dpA

pC

DF

.P(T

et).

P(P

ro)-

MC

S2-

0

Bgl

II-H

indI

II

Gib

son

Isot

herm

al; 3

in

sert

s

1147

pC

DF

.P(T

et).

P(P

ro)-

btP

haZ

.rrA

BH

.rrA

dpA

-Alk

K

alkK

F1

and

alkK

R1

Gen

e sy

nthe

sis

pCD

F.P

(Tet

).P

(Pro

)-rr

Pha

Z.r

rAB

H.r

rAdp

A-0

K

pnI-

Bam

HI

Gib

son

Isot

herm

al

1141

pC

DF

.P(T

et).

P(P

ro)-

mat

B

mat

B G

F1

and

mat

B G

R1

pE

T28

-mat

B

pCD

F.P

(Tet

).P

(Pro

)-M

CS

2-0

B

glII

G

ibso

n Is

othe

rmal

1128

pC

DF

.P(T

et).

P(P

ro)-

MC

S2-

0

pCD

F.P

(Tet

) M

CS

2 F

1 an

d pC

DF

.P(T

et)

MC

S2

R1

N

o te

mpl

ate

pC

DF

.P(T

et).

P(P

ro)-

0-0

B

glII

1177

pC

DF

.P(T

et).

P(P

ro)-

MC

S2-

birA

bi

rA F

3 an

d bi

rA R

4 D

H10

B G

enom

ic D

NA

pC

DF

.P(T

et).

P(P

ro)-

MC

S2-

0

Sac

I-B

amH

I G

ibso

n Is

othe

rmal

1142

pC

DF

.P(T

et).

P(P

ro)-

reP

haZ

.rrA

BH

.rrA

dpA

-0

reP

haZ

: Ph

aZ F

4 a

nd P

haZ

R4;

rr

AB

H: r

rAB

H F

1 a

nd r

rAB

H

R2;

rrA

dpA

: rrA

dpA

F1

and

rrA

dpA

R1

pCD

F3

-Pte

t-sP

haZ

1-sP

haP

1; p

J204

-rrA

BH

; pJ

204-

rrA

dpA

pCD

F.P

(Tet

).P

(Pro

)-M

CS

2-0

B

glII-

Hin

dIII

G

ibso

n Is

othe

rmal

1148

pC

DF

.P(T

et).

P(P

ro)-

reP

haZ

.rrA

BH

.rrA

dpA

-Alk

K

alkK

F1

and

alkK

R1

Gen

e sy

nthe

sis

pCD

F.P

(Tet

).P

(Pro

)-re

Pha

Z.r

rAB

H.r

rAdp

A-0

K

pnI-

Bam

HI

Gib

son

Isot

herm

al

1140

pC

DF

.P(T

et).

P(P

ro)-

rrP

haZ

.rrA

BH

.rrA

dpA

-0

rrP

haZ

: rrP

haZ

2 F

1 an

d rr

Pha

Z2

R1;

rrA

BH

: rrA

BH

F1

and

rrA

BH

R2;

rrA

dp

A: r

rAdp

A F

1 an

d rr

Ad

pA R

1

pJ20

4-rr

Pha

Z2;

pJ2

04-

rrA

BH

; pJ2

04-r

rAdp

A

pCD

F.P

(Tet

).P

(Pro

)-M

CS

2-0

B

glII-

Hin

dIII

G

ibso

n Is

othe

rmal

; 3

inse

rts

1146

pC

DF

.P(T

et).

P(P

ro)-

rrP

haZ

.rrA

BH

.rrA

dpA

-Alk

K

alkK

F1

and

alkK

R1

Gen

e sy

nthe

sis

pCD

F.P

(Tet

).P

(Pro

)-bt

Pha

Z.r

rAB

H.r

eP

haP

1-0

Kpn

I-B

amH

I G

ibso

n Is

othe

rmal

805

pCD

F.P

(Tet

)-0

B

glII

into

Xm

a lin

ker

F1

and

Bgl

II in

to X

ma

linke

r R

1

pCD

FD

uet-

tetR

.GB

D_S

H3_

PD

Z

Bgl

II-X

maI

A

nnea

l prim

ers:

S

tand

ard

ligat

ion

95

2 pC

DF

.P(T

et)-

AC

C

Acc

E F

101

and

Acc

E R

101

Gen

e sy

nthe

sis

pCD

F.P

(Tet

)-ac

s.ac

cBC

H

indI

II G

ibso

n Is

othe

rmal

80

7 pC

DF

.P(T

et)-

accB

C

accB

C F

100

and

acc

BC

R10

2

pCR

2.1-

accB

C

pCD

F.P

(Tet

)-0

B

glII-

Bam

HI

Sta

ndar

d lig

atio

n

806

pCD

F.P

(Tet

)-ac

cBC

.dts

R1

S

OE

: (ac

cBC

F1

00 a

nd a

ccB

C

R10

0) a

nd

(dts

R1

F10

0 an

d dt

sR1

R10

0)

accB

C: p

CR

2.1-

accB

C;

dtsR

1: p

CR

2.1

-dts

R1

pCD

F.P

(Tet

)-ac

cBC

B

glII-

Bam

HI

Sta

ndar

d lig

atio

n

953

pCD

F.P

(Tet

)-ac

s.A

CC

A

ccE

F10

1 an

d A

ccE

R10

1 G

ene

synt

hesi

s pC

DF

.P(T

et)-

Hin

dIII

Gib

son

Isot

herm

al

187

acs.

accB

C.d

tsR

1 86

7 pC

DF

.P(T

et)-

acs.

accB

C

Acs

F3

and

Acs

R3

DH

10B

gen

om

ic D

NA

pC

DF

.P(T

et)-

accB

C

Bgl

II G

ibso

n is

othe

rmal

86

8 pC

DF

.P(T

et)-

acs.

accB

C.d

tsR

1 A

cs F

3 an

d A

cs R

3 D

H10

B g

eno

mic

DN

A

pCD

F.P

(Tet

)-ac

cBC

.dts

R1

B

glII

Gib

son

isot

herm

al

1241

pC

DF

2.P

(tac

.tac)

-(gg

) C

DF

2-n

phT

7 (g

g)

F1

and

CD

F2-

nphT

7 (g

g) R

1

No

tem

plat

e

pCD

F2.

P(t

ac.ta

c)-0

B

amH

I-K

pnI

Ann

eal

Prim

er/S

tand

ard

lig

atio

n

1190

pC

DF

2gg-

0

CD

F2

-nph

T7

(gg

) F

1 an

d C

DF

2-np

hT7

(gg)

R1

N

o te

mpl

ate

pC

DF

2

Bam

HI-

Kpn

I

Ann

eal

Prim

er/S

tand

ard

lig

atio

n 12

52

pCD

F2

-nph

T7

np

hT7

gg F

2 an

d n

phT

7 gg

R2

pU

C19

-(gg

)nph

T7

pCD

F2.

P(t

ac.ta

c)-(

gg)

Bsa

I G

olde

n G

ate

664

pCD

FD

uet-

tetR

.GB

D_S

H3

_PD

Z

pTet

Sca

ff F

1 a

nd

pTet

Sca

ff R

2

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

pC

DF

-Due

t-1

X

baI-

Kpn

I S

tand

ard

ligat

ion

711

pCD

FD

uet-

tetR

.GB

D1_

SH

31_

PD

Z2

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

1_P

DZ

2

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

712

pCD

FD

uet-

tetR

.GB

D1_

SH

31_

PD

Z4

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

1_P

DZ

4

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

713

pCD

FD

uet-

tetR

.GB

D1_

SH

32_

PD

Z1

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

2_P

DZ

1

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

714

pCD

FD

uet-

tetR

.GB

D1_

SH

32_

PD

Z2

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

2_P

DZ

2

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

715

pCD

FD

uet-

tetR

.GB

D1_

SH

32_

PD

Z4

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

2_P

DZ

4

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

716

pCD

FD

uet-

tetR

.GB

D1_

SH

34_

PD

Z1

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

4_P

DZ

1

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

717

pCD

FD

uet-

tetR

.GB

D1_

SH

34_

PD

Z2

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

4_P

DZ

2

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

718

pCD

FD

uet-

tetR

.GB

D1_

SH

34_

PD

Z4

D

irect

ly c

ut f

rom

vec

tor

and

gel

purif

y, n

o P

CR

pT

ET

-te

tR.G

BD

1_S

H3

4_P

DZ

4

pTE

T-

tetR

.GB

D1_

SH

31_

PD

Z1

Bgl

II-

Bam

HI-

HF

D

irect

liga

tion

551

pCO

LA.T

7.O

ri-0

-Ydb

K

pCW

Ori

F7

and

pCW

Ori

R7

pC

WO

ri

pCO

LA-0

-Ydb

K

Sal

I-N

deI

Gib

son

isot

herm

al

546

pCO

LA.T

7.T

7-F

pr.

Fd

x.F

ldA

-0

SO

E: (

Fpr

F1

and

Fpr

SO

E R

1)

and

(Fd

x S

OE

F1

and

Fd

x R

1)

pC

OLA

-fld

A-0

N

coI-

Eco

RI

Sta

ndar

d lig

atio

n

547

pCO

LA.T

7.T

7-F

pr.

Fd

x.F

ldB

-0

SO

E: (

Fpr

F1

and

Fpr

SO

E R

1)

and

(Fd

x S

OE

F1

and

Fd

x R

1)

pC

OLA

-fld

B-0

N

coI-

Eco

RI

Sta

ndar

d lig

atio

n

552

pCO

LA.T

rc.O

ri-F

pr.F

dx.

Fld

A-

Ydb

K

pCW

Ori

F7

and

pCW

Ori

R7

pC

WO

ri

pCO

LA.T

rc.T

7-

Fpr

.Fdx

.Fld

A-Y

dbK

S

alI-

Nde

I G

ibso

n is

othe

rmal

553

pCO

LA.T

rc.O

ri-F

pr.F

dx.

Fld

B-

Ydb

K

pCW

Ori

F7

and

pCW

Ori

R7

pC

WO

ri

pCO

LA.T

rc.T

7-

Fpr

.Fdx

.Fld

B-Y

dbK

S

alI-

Nde

I G

ibso

n is

othe

rmal

569

pCO

LA.T

rc.T

7-a

ceE

.ace

F.lp

d-0

la

cIq

F2

and

pTrc

99a

R8

pT

rc99

A

pCO

LAD

uet-

ace

E.F

.lpd(

E.

coli)

-0

Xba

I-N

coI

Gib

son

isot

herm

al

548

pCO

LA.T

rc.T

7-F

pr.F

dx.

Fld

A-0

la

cIq

F2

and

pTrc

99a

R8

pT

rc99

A

pCO

LA.T

7.T

7-

Xba

I-N

coI

Gib

son

isot

herm

al

188

Fpr

.Fdx

.Fld

A-0

550

pCO

LA.T

rc.T

7-F

pr.F

dx.

Fld

A-

Ydb

K

lacI

q F

2 an

d pT

rc99

a R

8

pTrc

99A

pC

OLA

-Fp

r.F

dx.

Fld

A-

Ydb

K

Xba

I-N

coI

Gib

son

isot

herm

al

549

pCO

LA.T

rc.T

7-F

pr.F

dx.

Fld

B-0

la

cIq

F2

and

pTrc

99a

R8

pT

rc99

A

pCO

LA.T

7.T

7-

Fpr

.Fdx

.Fld

B-0

X

baI-

Nco

I G

ibso

n is

othe

rmal

N/A

pC

OLA

.Trc

.T7

-Fpr

.Fd

x.F

ldB

-Y

dbK

la

cIq

F2

and

pTrc

99a

R8

pT

rc99

A

pCO

LA-F

pr.

Fd

x.F

ldB

-Y

dbK

X

baI-

Nco

I G

ibso

n is

othe

rmal

974

pCO

LA-0

-tes

A

tesA

F1

and

tesA

R1

DH

10B

Gen

omic

DN

A

pCO

LAD

uet

Bgl

II-K

pnI

Sta

ndar

d lig

atio

n

960

pCO

LA-0

-Ydb

K

Ydb

K F

1 an

d Y

dbK

R1

pCO

LAD

uet

Nde

I-K

pnI

Sta

ndar

d lig

atio

n

514

pCO

LAD

uet-

0-p

dhA

.pdh

B.

aceF

.lpdA

(E. f

aeca

lis)

pdhA

F1

and

lpdA

R1

Gen

omic

DN

A

pCO

LAD

uet

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

N/A

pC

OLA

Due

t-0

-te

sA

tesA

F1

and

tesA

R1

DH

10B

Gen

omic

DN

A

pCO

LAD

uet

Bgl

II-K

pnI

Sta

ndar

d lig

atio

n

512

pCO

LAD

uet-

ace

E.F

.lpd(

E. c

oli)-

0 N

one

pB

BR

2-(N

co/B

sa).

aceE

.ace

F.lp

d pC

OLA

Due

t

Vec

tor:

N

coI-

Sac

I;

Inse

rt

Bsa

I-S

acI

Dire

ct li

gatio

n

513

pCO

LAD

uet-

pdhA

.pdh

B.a

ceF

.lpdA

(E.

faec

alis

)-0

pdhA

F3

and

lpdA

R2

Gen

omic

DN

A

pCO

LAD

uet

Nco

I-S

acI

Sta

ndar

d lig

atio

n

475

pCO

LAD

uet-

TbA

CS

4-A

tfA.T

esA

A

tfA F

1 an

d A

tfA R

1 G

ene

synt

hesi

s pC

OLA

Due

t-T

bAC

S4-

Tes

A

Nde

I-B

glII

S

tand

ard

ligat

ion

N/A

pC

OLA

Due

t-T

bAC

S4-

Tes

A

TbA

CS

4 F

1 an

d T

bAC

S4

R1

G

ene

synt

hesi

s pC

OLA

Due

t-0

-te

sA

Nco

I-B

amH

I S

tand

ard

ligat

ion

957

pCO

LA-f

ldA

-0

Fld

A F

1 an

d F

ldA

R1

pC

OLA

Due

t E

coR

I-S

acI

Sta

ndar

d lig

atio

n

958

pCO

LA-f

ldB

-0

Fld

B F

1 an

d F

ldB

R1

pC

OLA

Due

t E

coR

I-S

acI

Sta

ndar

d lig

atio

n

495

pCO

LA-F

pr.

Fd

x.F

ldA

-Ydb

K

Ydb

K F

1 an

d Y

dbK

R1

pCO

LA.T

7.T

7-

Fpr

.Fdx

.Fld

A-0

N

deI-

Kpn

I S

tand

ard

ligat

ion

959

pCO

LA-f

pr.fd

x.fld

B-0

S

OE

: (F

pr F

1 an

d F

pr S

OE

R1

) an

d (F

dx

SO

E F

1 an

d F

dx

R1

)

pCO

LA-f

ldB

-0

Nco

I-E

coR

I S

tand

ard

ligat

ion

496

pCO

LA-F

pr.

Fd

x.F

ldB

-Ydb

K

Ydb

K F

1 an

d Y

dbK

R1

pCO

LA.T

7.T

7-

Fpr

.Fdx

.Fld

B-0

N

deI-

Kpn

I S

tand

ard

ligat

ion

N/A

pC

R2.

1-ac

cBC

ac

cBC

F10

0 an

d a

ccB

C R

100

G

ene

synt

hesi

s pC

R2.

1

Non

e

Top

o cl

onin

g N

/A

pCR

2.1-

bcd.

etfB

.etfA

bc

d F

1 an

d et

fA R

2 G

enom

ic D

NA

pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-C

mR

.T(a

dhB

)

SO

E: (

Cm

R F

1/S

LIC

F1

and

Cm

R/a

dhB

(Ter

m)

SO

E R

1)

and

(Cm

R/a

dhB

(Te

rm)

SO

E F

1 an

d ad

hB(T

erm

) R

1/S

LIC

R1

pAR

O18

1Cm

; Z

. m

obili

s G

enom

ic D

NA

pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-dt

sR1

dt

sR1

F10

0 a

nd d

tsR

1 R

100

G

ene

synt

hesi

s pC

R2.

1

Non

e

Top

o cl

onin

g 21

9 pC

R2.

1-la

cUV

5-p

haA

2-ph

aB

phaA

F2

and

pha

B R

1

pBS

K-B

u1

pCR

2.1-

TO

PO

N

one

T

opo

clon

ing

N/A

pC

R2.

1-np

hT7

N

phT

7 F

100

and

Nph

T7

R10

0

Gen

e sy

nthe

sis

pCR

2.1

N

one

T

opo

clon

ing

N/A

pC

R2.

1-P

(adh

B).

adhE

2

SO

E: (

P(a

dhB

) F

2/S

LIC

F2

and

P

(adh

B)/

adhE

2 S

OE

R1)

and

(P

(adh

B)/

adhE

2 S

OE

F1

and

ad

hE2

R2)

Z. m

obili

s G

enom

ic D

NA

; pC

WO

ri-cc

r.ad

hE

2 pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-P

(gap

).cc

r S

OE

: (P

(gap

) F

2 a

nd P

(gap

)/cc

r S

OE

R1)

and

(cc

r S

OE

F1

and

ccr

R3

)

Z. m

obili

s G

enom

ic D

NA

; pC

WO

ri-cc

r.ad

hE

2 pC

R2.

1

Non

e

Top

o cl

onin

g

189

N

/A

pCR

2.1-

P(g

ap).

pha

A.h

bd.c

rt

SO

E: (

(P(g

ap)

F1/

SLI

C F

2 a

nd

P(g

ap)/

pha

A S

OE

R1)

and

(P

(gap

)/ph

aA S

OE

F1

and

HB

D/c

rt S

OE

R1

)) a

nd

(HB

D/c

rt S

OE

F1

and

crt

R

1/S

LIC

R1

)

Z. m

obili

s G

enom

ic D

NA

; pB

BR

2-ac

eE.a

ceF

.lpd;

pB

T33

-Bu2

; pB

T33

-Bu2

pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-P

(gap

).te

r

SO

E: (

P(g

ap)

F2

and

P

(gap

)/T

dTE

R S

OE

R1

) an

d (P

(gap

)/T

dT

ER

SO

E F

1 a

nd

TdT

er

R10

3)

Z. m

obili

s G

enom

ic D

NA

; pC

WO

ri-te

r.ad

hE2

pCR

2.1

N

one

T

opo

clon

ing

N/A

pC

R2.

1-P

(pdc

P).

crt

SO

E: (

PD

C1-

p-f

1 an

d P

DC

1-p

-r1

) an

d (c

rt-f

1 an

d cr

t-r1

) cr

t: pB

T33

-Bu

2; p

dcP

: G

enom

ic D

NA

pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-P

(pgk

1).h

bd.T

(pgi

) S

OE

: ((P

GK

-1-p

-f1

and

PG

K-1

-p-

r1)

and

(hbd

-f1

and

hbd

-r1)

) an

d (P

GI-

t-f1

an

d P

GI-

t-f1

)

hbd:

pB

T33

-Bu2

; pg

k1 a

nd

pgi:

Gen

omic

DN

A

pCR

2.1

N

one

T

opo

clon

ing

N/A

pC

R2.

1-ph

aA.h

bd

hbd

F1

and

hbd

R1

Gen

e sy

nthe

sis

pCR

2.1-

phaA

2.p

haB

E

coR

I-H

indI

II S

tand

ard

ligat

ion

N/A

pC

R2.

1-ph

aA.T

(tpi

1)

SO

E: (

phaA

-f2

and

pha

A-r

2) a

nd

(tpi

1-t-

f1 a

nd tp

i1-t

-r1

) ph

aA: p

BT

33-B

u2;

tpi1

: G

enom

ic D

NA

pC

R2.

1

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-sB

dhB

bh

dB F

100

and

bdh

B R

100

G

ene

synt

hesi

s pC

R2.

1-T

OP

O

Non

e

Top

o cl

onin

g

N/A

pC

R2.

1-T

cR.T

(pdh

)

SO

E: (

Tet

R F

2/S

LIC

F2

and

Tet

R/T

(pdc

) S

OE

R2)

and

(T

etR

/T(p

dc)

SO

E F

2 an

d T

(pdc

) R

1/S

LIC

R2)

Z. m

obili

s G

enom

ic D

NA

; pA

RO

181T

c pC

R2.

1

Non

e

Top

o cl

onin

g

559

pCW

ori-*

10gf

p

Kpn

I lin

ker

F3

and

Kpn

I lin

ker

R3

N

o te

mpl

ate

pC

Wor

i-10g

fp

Kpn

I P

rimer

Ann

eal:

Sta

ndar

d lig

atio

n

560

pCW

ori-*

20gf

p

Kpn

I lin

ker

F3

and

Kpn

I lin

ker

R3

N

o te

mpl

ate

pC

Wor

i-20g

fp

Kpn

I P

rimer

Ann

eal:

Sta

ndar

d lig

atio

n

701

pCW

ori-*

gfp

K

pnI l

inke

r F

3 an

d K

pnI l

inke

r R

3

No

tem

plat

e

pCW

ori-g

fp

Kpn

I P

rimer

Ann

eal:

Sta

ndar

d lig

atio

n

821

pCW

Ori-

10C

AT

K

pnI l

inke

r F

1 an

d K

pnI l

inke

r R

1

N/A

pC

WO

ri-C

AT

K

pnI

Ann

eal P

rimer

; S

tand

ard

ligat

ion

N/A

pC

Wor

i-10g

fp

Kpn

I lin

ker

F1

and

Kpn

I lin

ker

R1

N

o te

mpl

ate

pC

Wor

i-gfp

K

pnI

Prim

er A

nnea

l: S

tand

ard

ligat

ion

822

pCW

Ori-

20C

AT

K

pnI l

inke

r F

2 an

d K

pnI l

inke

r R

2

N/A

pC

WO

ri-C

AT

K

pnI

Ann

eal P

rimer

; S

tand

ard

ligat

ion

N/A

pC

Wor

i-20g

fp

Kpn

I lin

ker

F2

and

Kpn

I lin

ker

R2

N

o te

mpl

ate

pC

Wor

i-gfp

K

pnI

Prim

er A

nnea

l: S

tand

ard

ligat

ion

96

9 pC

WO

ri-A

DH

(ad

hE2)

ad

hE2

F13

and

adh

E2

R1

pC

WO

ri-cc

r.ad

hE

2 pC

WO

ri

Nde

I-H

indI

II

Sta

ndar

d lig

atio

n

N/A

pC

WO

ri-bc

d.et

fB.e

tfA

SO

E: (

bcd

F1

and

etfB

SO

E R

1)

and

(etfB

SO

E F

1 an

d et

fA R

2)

pCR

2.1-

bcd.

etfB

.etfA

pC

WO

ri

Nde

I-X

baI

Sta

ndar

d lig

atio

n

820

pCW

Ori-

CA

T

CA

T F

1 a

nd C

AT

R1

pBA

D33

pC

WO

ri

Nde

I and

X

baI

Sta

ndar

d lig

atio

n

249

pCW

ori-c

cr

N/A

pCW

ori-c

cr.a

dhE

2 E

coR

I-K

pnI

Pfu

to g

ive

blun

t end

s;

Sta

ndar

d lig

atio

n

375

pCW

Ori-

ccr(

S. c

inna

mon

ensi

s)

CC

R (

S. c

inna

) F

0 an

d C

CR

(S

. ci

nna)

R0

G

ene

synt

hesi

s pC

WO

ri

Nde

I-X

baI

376

pCW

Ori-

ccr(

S.

CC

R (

S. c

inna

) F

0 an

d C

CR

(S

. pC

WO

ri-cc

r(S

. pC

WO

ri-cc

r.ad

hE

2 N

deI-

Eco

RI

190

cinn

amon

ensi

s).a

dhE

2

cinn

a) R

0

cinn

amon

ensi

s)

979

pCW

Ori-

ccr(

x).a

dhE

2 cc

r F

1 an

d cc

r R

2 pT

rc99

a-c

cr(x

) pC

WO

ri-cc

r.ad

hE

2 N

de-E

coR

I S

tand

ard

ligat

ion

966

pCW

Ori-

ccr(

x).a

dhE

2(A

LDH

x)

SO

E: (

adhE

2 F

1 an

d ad

hE2

R8)

an

d (a

dhE

2 F

8 a

nd a

dhE

2 R

1)

pCW

Ori-

ccr.

adh

E2

pCW

Ori-

ccr(

x).a

dhE

2 E

coR

I-K

pnI

Sta

ndar

d lig

atio

n

965

pCW

Ori-

ccr(

x).a

dhE

2(xx

x)

SO

E: (

adhE

2 F

1 an

d ad

hE2

R8)

an

d (a

dhE

2 F

8 a

nd a

dhE

2 R

1)

pCW

Ori-

stre

p.ad

hE2(

AD

Hxx

) pC

WO

ri-cc

r(x)

.adh

E2

Eco

RI-

Kpn

I S

tand

ard

ligat

ion

250

pCW

ori-c

cr.a

dhE

2 cc

r F

1 an

d ad

hE2

R1

pE

T29

a-cc

r.ad

hE

2 pC

WO

ri

Nde

I-H

indI

II

Sta

ndar

d lig

atio

n

347

pCW

Ori-

ccr.

adh

E2.

AD

H

adhE

2 F

14 a

nd a

dhE

2 R

12

pCW

ori-c

cr.a

dhE

2 pC

Wor

i-ccr

.adh

E2

Kpn

I-H

indI

II

Sta

ndar

d lig

atio

n

251

pCW

ori-c

cr.a

dhE

2.sB

dhB

bd

hB F

100

and

bdh

B R

103

p

CR

2.1-

sBdh

B

pCW

Ori-

ccr.

adh

E2

Kpn

I S

tand

ard

ligat

ion

56

4 pC

Wor

i-ccr

.adh

E2-

S

ccr

F1

and

adhE

2 R

10

pCW

Ori

-ccr

.ad

hE

2 pC

WO

ri-S

N

deI-

Kpn

I S

tand

ard

ligat

ion

55

7 pC

Wor

i-ccr

.His

cc

r F

1 an

d cc

r R

11

pCW

Ori

-ccr

.ad

hE

2 pC

WO

ri

Nde

I-X

baI

Sta

ndar

d lig

atio

n

N/A

pC

WO

ri-cc

r-S

cc

r F

1 an

d cc

r R

10

pCW

Ori-

ccr.

adh

E2

pCW

Ori-

S

Nde

I-S

alI

Sta

ndar

d lig

atio

n

561

pCW

ori-c

cr-S

.ad

hE2

ccr

F1

and

pCW

Ori-

Sta

g R

4

pCW

Ori

-ccr

.Sta

g

pCW

Ori-

ccr.

adh

E2

Nde

I-E

coR

I S

tand

ard

ligat

ion

N

/A

pCW

ori-g

fp

GF

P Q

C F

1 an

d G

FP

QC

R1

pCW

ori-g

fp(~

)

Qui

ck C

hang

e

N/A

pC

Wor

i-gfp

(~)

GF

P F

1 an

d G

FP

R1

pET

31b+

-GF

P

pCW

Ori-

CA

T

Kpn

I-X

baI

Sta

ndar

d lig

atio

n

500

pCW

Ori-

JFA

R

Jfar

F1

and

Jfa

r R

1 G

ene

synt

hesi

s pC

WO

ri

Nde

I-X

baI

Sta

ndar

d lig

atio

n

N/A

pC

WO

ri-S

S

-Tag

F1

and

S-T

ag R

1

N/A

pC

WO

ri

Sal

I-H

indI

II A

nnea

l P

rimer

s/S

tand

ard

ligat

ion

964

pCW

Ori-

sALD

H5

93

sALD

H59

3 F

1 an

d sA

LDH

593

R1

G

ene

synt

hesi

s pC

WO

ri

Nde

I-X

baI

Sta

ndar

d lig

atio

n

934

pCW

Ori-

SH

3_H

is_t

er.a

dhE

2

TdT

er

F10

6 an

d T

dTe

r R

106

pC

WO

ri-te

r.ad

hE2

pCW

Ori-

ter.

adhE

2 N

deI-

Eco

RI

Gib

son

Isot

herm

al

897

pCW

Ori-

SH

3_te

r.ad

hE2

T

dTe

r F

104

and

TdT

er

R10

6

pCW

Ori

-ter

.adh

E2

pCW

Ori-

ter.

adhE

2 N

deI-

Eco

RI

Gib

son

Isot

herm

al

899

pCW

Ori-

SH

3_te

r.ad

hE2_

PD

Z

Ter

: T

dTe

r F

104

and

Td

Ter

R10

6;

Adh

E2:

adh

E2

F1

and

adhE

2 R

19

pCW

Ori-

ter.

adhE

2; p

CW

Ori-

ter.

adhE

2

pCW

Ori-

ter.

adhE

2 N

deI-

Kpn

I G

ibso

n Is

othe

rmal

; 2

inse

rts

N/A

pC

WO

ri-st

rep.

adhE

2(A

DH

x)

SO

E (

adhE

2 F

6 an

d ad

hE2

R9)

an

d (a

dhE

2 F

9 a

nd a

dhE

2 R

1)

pCW

Ori-

ccr.

adh

E2

pCW

Ori

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

967

pCW

Ori-

stre

p.ad

hE2(

AD

Hxx

) S

OE

(ad

hE2

F6

and

adhE

2 R

11)

and

(adh

E2

F11

and

adh

E2

R1)

pC

WO

ri-st

rep.

adhE

2(A

DH

x)

pCW

Ori

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

295

pCW

ori-s

trep

.bd

hA

bdhA

F10

1 an

d b

dhA

R1

G

ene

synt

hesi

s pC

WO

ri

Nde

I-H

indI

II

Sta

ndar

d lig

atio

n

348

pCW

Ori-

stre

pAD

H

adhE

2 A

DH

F2

and

adh

E2

R1

pC

Wor

i-cc

r.ad

hE2

pCW

Ori

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

21

5 pC

Wor

i-str

epA

LD

HE

2

adhE

2 F

6 a

nd a

dhE

2 R

1

pBS

K-B

u3

pBA

D24

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

23

3 pC

Wor

i-str

ep-s

Bdh

B

bdhB

F10

3 an

d b

dhB

R10

0

pB

AD

24-s

Bdh

B

pCW

Ori

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

49

0 pC

WO

ri-T

er.a

dhE

2 T

dTe

r F

1 a

nd T

dT

er R

102

G

ene

synt

hesi

s pC

WO

ri-cc

r.ad

hE

2 N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

812

pCW

Ori-

ter.

adhE

2 (T

er

Y24

0F,

adhE

2 C

244A

H73

5A,

H72

1A)

TdT

er

F1

and

Td

Ter

R1

02

pET

16b-

His

.Ter

(Y24

0F

) pC

WO

ri-cc

r(x)

.adh

E2(

xxx)

N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

898

pCW

Ori-

ter.

adhE

2_P

DZ

ad

hE2

F1

and

ad

hE2

R19

pC

WO

ri-te

r.ad

hE2

pCW

Ori-

ter.

adhE

2 E

coR

I-K

pnI

Gib

son

Isot

herm

al

N/A

pC

WO

ri-te

r.ad

lh46

al

d46

1209

25 F

1 a

nd a

ld46

12

0925

R2

pC

DF

3-t

er.a

ldh

46

pCW

Ori-

ter.

adhE

2 E

coR

I-H

F-

Hin

dIII

Gib

son

Isot

herm

al

775

pCW

Ori-

Ter

.SH

3_a

dhE

2

adhE

2 F

25 a

nd a

dhE

2 R

1

pCW

Ori-

ter.

adhE

2 pC

WO

ri-te

r.ad

hE2

Eco

RI-

Kpn

I S

tand

ard

ligat

ion

63

4 pC

WO

ri-T

ER

_PD

Z.a

dhE

2

TdT

er

F1

and

ter

R10

5

pCW

Ori

-ter

.adh

E2

pCW

Ori-

ter.

adhE

2 N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

816

pCW

Ori-

TE

R_P

DZ

.adh

E2

v2.0

T

dTe

r F

1 a

nd te

r R

105

pC

WO

ri-te

r.ad

hE2

pCW

Ori-

ter.

adhE

2 N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

902

pCW

Ori-

ter_

pdz.

adhE

2_S

H3

T

dTe

r F

108

and

TdT

er

R10

7/8

pC

WO

ri-T

ER

_PD

Z.a

dhE

2

pCW

Ori-

TE

R_P

DZ

.adh

E2

E

coR

I-K

pnI

Gib

son

Isot

herm

al

826

pCW

Ori-

TE

R_P

DZ

.SH

3_a

dhE

2

TdT

er

F1

and

ter

R10

5

pCW

Ori

-ter

.adh

E2

pCW

Ori-

Ter

.SH

3_a

dhE

2

Nde

I-E

coR

I S

tand

ard

ligat

ion

31

5 pC

Wor

iX

pCW

Ori

F2

and

pCW

Ori

R4

N

/A

pCW

Ori

N

deI-

Hin

dIII

A

nnea

l prim

ers

with

191

Sta

ndar

d lig

atio

n

800

pES

C.H

is-B

u2

Par

t 1: p

haA

F1

and

tpi1

-t-r

1; P

art

2: P

GK

1-p-

f1 a

nd

PG

I-t-

r1; P

art

3:

PD

C1-

p-f

1 an

d cr

t-r1

pCR

2.1-

phaA

.T(t

pi1)

; pC

R2.

1-P

(pgk

1).h

bd.T

(pgi

);

pCR

2.1-

P(p

dcP

).cr

t

pES

C.H

is

Eco

RI

SLI

C (

Seq

uenc

ing

and

Liga

tion

Inde

pen

dent

cl

onin

g)

801

pES

C.L

eu-H

is.U

ra.H

is-B

u2

N/A

pN

KY

51

pE

SC

Leu-

-Bu2

B

glII-

Hin

dIII

D

irect

liga

tion

802

pES

C.L

eu-U

ra-B

u2

Ura

F1

and

Ura

R1

pES

C.U

ra

pES

CLe

u--B

u2

Bgl

II S

tand

ard

ligat

ion

795

pES

CLe

u2d-

ter-

adhE

2

No

PC

R, D

irec

tly c

ut o

ur in

sert

pE

SC

Leu-

ter-

adhE

2 pE

SC

Leu2

d

Sal

I-S

peI

Dire

ct c

ut; S

tand

ard

ligat

ion

813

pES

CLe

u--B

u2

Par

t 1:P

(Tef

) Y

eas

t F1

and

phaA

Y

east

R4;

Par

t 2:

P(p

gk1)

Y

east

F1

and

T(p

gi1)

Yea

st R

1;

Par

t 3: P

(pdc

) Y

est

F1

and

crt

Yea

st R

4

pCR

2.1-

phaA

.T(t

pi1)

; pC

R2.

1-P

(pgk

1).h

bd.T

(pgi

);

pCR

2.1-

P(p

dcP

).cr

t

pES

C.L

eu-B

u2

Bam

HI-

Xho

I G

ibso

n cl

onin

g

N/A

pE

SC

Leu-

ter-

0

Not

I-T

er F

1 an

d T

er-S

peI

R1

pC

WO

ri-te

r.ad

hE2

pES

CLe

u2

Not

I-S

peI

Sta

ndar

d lig

atio

n

794

pES

CLe

u-te

r-ad

hE2

Xm

aI-a

dhE

2 F

1 an

d ad

hE2-

Sal

I R

1 pC

WO

ri-te

r.ad

hE2

pES

CLe

u-te

r-0

X

maI

-Sal

I S

tand

ard

ligat

ion

N/A

pE

T.O

99a

-0-a

dhE

2 S

OE

: (p

T09

9a F

1 an

d pT

O99

a S

OE

R1)

and

(p

ET

Due

t F1

and

pET

Due

t R1)

Par

t 1: p

Trc

.Ori9

9a-0

-0; P

art

2: p

ET

Due

t pT

rc.O

ri99a

-0-a

dhE

2 N

siI-

Nco

I S

tand

ard

ligat

ion

N/A

pE

T.O

ri16b

-0-a

dhE

2 pC

WO

ri F

6 an

d pC

WO

ri R

6

pTrc

.Ori9

9a-0

-ad

hE2

pET

16b

E

agI

Sta

ndar

d lig

atio

n

532

pET

.Ori1

6b-H

is.T

dTe

r-ad

hE2

pC

WO

ri F

6 an

d pC

WO

ri R

6

pTrc

.Ori9

9a-0

-ad

hE2

pET

16b-

His

.Ter

E

agI

Sta

ndar

d lig

atio

n

531

pET

.Ori1

6b-T

dTer

-adh

E2

pC

WO

ri F

6 an

d pC

WO

ri R

6

pTrc

.Ori9

9a-0

-ad

hE2

pET

16b-

TdT

er

Eag

I S

tand

ard

ligat

ion

954

pET

.Ori9

9a-0

-ad

hE2

SO

E: (

pT

O9

9a F

1 an

d pT

O99

a S

OE

R1)

and

(p

ET

Due

t F1

and

pET

Due

t R1)

pT

rc.O

ri99a

; pE

TD

uet

pTrc

.Ori9

9a-0

-ad

hE2

Nsi

I-N

coI

Sta

ndar

d lig

atio

n

530

pET

.Ori9

9a-T

dTer

-adh

E2

td

Ter

F10

1 an

d td

Ter

R10

3

pCW

Ori-

ter.

adh

e2

pET

.O9

9a-0

-adh

E2

Bsa

I ins

ert

(Nco

I ve

ctor

)-K

pnI

Sta

ndar

d lig

atio

n

1016

pE

T16

b-H

is.A

cc(M

t)A

3

Acc

A3

F10

1 an

d A

ccA

3 R

101

Gen

e sy

nthe

sis

pET

16b

N

deI-

Xho

I G

ibso

n Is

othe

rmal

10

17

pET

16b-

His

.Acc

(Mt)

D4

A

ccD

4 F

101

and

Acc

D4

R10

1

Gen

e sy

nthe

sis

pET

16b

N

deI-

Xho

I G

ibso

n Is

othe

rmal

10

18

pET

16b-

His

.Acc

(Mt)

D5

A

ccD

5 F

101

and

Acc

D5

R10

1

Gen

e sy

nthe

sis

pET

16b

N

deI-

Xho

I G

ibso

n Is

othe

rmal

10

19

pET

16b-

His

.Acc

(Mt)

D6

A

ccD

6 F

101

and

Acc

D6

R10

1

Gen

e sy

nthe

sis

pET

16b

N

deI-

Xho

I G

ibso

n Is

othe

rmal

10

20

pET

16b-

His

.Acc

(Mt)

E

Acc

E F

101

and

Acc

E R

101

Gen

e sy

nthe

sis

pET

16b

N

deI-

Xho

I G

ibso

n Is

othe

rmal

631

pET

16b-

His

.hbd

hb

d F

100

and

hbd

R10

2

pBT

33-B

u2

pET

16b

N

deI-

Bam

HI

Sta

ndar

d lig

atio

n

643

pET

16b-

His

.hbd

(H13

8E)

SO

E: (

hbd

F10

0 an

d hb

d S

OE

R

3) a

nd (

hbd

SO

E F

3 an

d hb

d R

102)

pB

T33

-Bu2

pE

T16

b

Nde

I-B

amH

I S

tand

ard

ligat

ion

641

pET

16b-

His

.hbd

(H13

8V)

SO

E: (

hbd

F10

0 an

d hb

d S

OE

R

1) a

nd (

hbd

SO

E F

1 an

d hb

d R

102)

pB

T33

-Bu2

pE

T16

b

Nde

I-B

amH

I S

tand

ard

ligat

ion

642

pET

16b-

His

.hbd

(H13

8Y

) S

OE

: (hb

d F

100

and

hbd

SO

E

R2)

and

(hb

d S

OE

F2

and

hbd

R10

2)

pBT

33-B

u2

pET

16b

N

deI-

Bam

HI

Sta

ndar

d lig

atio

n

N/A

pE

T16

b-H

is.P

haB

ph

aB F

1 an

d ph

aB

R1

pBT

33-B

u1

pET

16b

N

deI-

Bam

HI

Sta

ndar

d lig

atio

n

192

97

5 pE

T16

b-H

is.p

haB

(Q

C1)

ph

aB Q

C F

1 an

d p

haB

QC

R1

pE

T16

b-H

is.p

haB

Q

uick

Cha

nge

97

8 pE

T16

b-H

is.p

haB

(Q

C1,

3)

phaB

QC

F3

and

pha

B Q

C R

3

pET

16b-

His

.pha

B (

QC

1)

Qui

ck C

hang

e

976

pET

16b-

His

.pha

B (

QC

2)

phaB

QC

F2

and

pha

B Q

C R

2

pET

16b-

His

.pha

B

Qui

ck C

hang

e

977

pET

16b-

His

.pha

B (

QC

3)

phaB

QC

F3

and

pha

B Q

C R

3

pET

16b-

His

.pha

B

Qui

ck C

hang

e

528

pET

16b-

His

.Ter

T

dTe

r F

1 a

nd T

dT

er R

101

pE

T16

b-T

dTer

pE

T16

b

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

639

pET

16b-

His

.Ter

(Y24

0F

) S

OE

: (T

dTe

r F

1 an

d T

dT

er S

OE

R

1) a

nd (

Td

Ter

SO

E F

1 a

nd

TdT

er

R10

1)

pCW

Ori-

ter.

adhE

2 pE

T16

b

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

640

pET

16b-

His

.Ter

(Y29

0F

) S

OE

: (T

dTe

r F

1 an

d T

dT

er S

OE

R

2) a

nd (

Td

Ter

SO

E F

2 a

nd

TdT

er

R10

1)

pCW

Ori-

ter.

adhE

2 pE

T16

b

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

1080

pE

T16

b-H

isT

er(F

295A

) T

er Q

C F

5 an

d T

er Q

C R

5

pET

16b-

His

.Ter

Q

uick

Cha

nge

10

78

pET

16b-

His

Ter

(I28

7A)

Ter

QC

F3

and

Ter

QC

R3

pE

T16

b-H

is.T

er

Qui

ck C

hang

e

1083

pE

T16

b-H

isT

er(K

247A

) T

er Q

C F

8 an

d T

er Q

C R

8

pET

16b-

His

.Ter

Q

uick

Cha

nge

10

82

pET

16b-

His

Ter

(K24

9A)

Ter

QC

F7

and

Ter

QC

R7

pE

T16

b-H

is.T

er

Qui

ck C

hang

e

1077

pE

T16

b-H

isT

er(L

276A

; V27

7A)

Ter

QC

F2

and

Ter

QC

R2

pE

T16

b-H

is.T

er

Qui

ck C

hang

e

1085

pE

T16

b-H

isT

er(L

276A

; V27

7A;

F29

5A)

Ter

QC

F5

and

Ter

QC

R5

pE

T16

b-H

isT

er(L

276A

; V

277A

)

Q

uick

Cha

nge

1079

pE

T16

b-H

isT

er(L

291A

) T

er Q

C F

4 an

d T

er Q

C R

4

pET

16b-

His

.Ter

Q

uick

Cha

nge

10

84

pET

16b-

His

Ter

(Q23

7A)

Ter

QC

F9

and

Ter

QC

R9

pE

T16

b-H

is.T

er

Qui

ck C

hang

e

1076

pE

T16

b-H

isT

er(Y

230A

) T

er Q

C F

1 an

d T

er Q

C R

1

pET

16b-

His

.Ter

Q

uick

Cha

nge

10

81

pET

16b-

His

Ter

(Y37

0A)

Ter

QC

F6

and

Ter

QC

R6

pE

T16

b-H

is.T

er

Qui

ck C

hang

e

N/A

pE

T16

b-st

rep.

crt

crt F

4 an

d cr

t R

2

pBS

K-B

u1

pET

16b

N

deI-

Hin

dIII

S

tand

ard

ligat

ion

527

pET

16b-

TdT

er

tdT

er F

101

and

tdT

er R

101

G

ene

synt

hesi

s pE

T16

b

Bsa

I ins

ert

(Nco

I ve

ctor

)-X

hoI

Sta

ndar

d lig

atio

n

635

pET

23a-

His

.Tev

.Ter

T

dTe

r F

102

and

TdT

er

R10

1

pCW

Ori-

ter.

adhE

2 pE

T23

a

Sfo

I-X

hoI

Sta

ndar

d lig

atio

n

895

pET

23a-

His

Tev

_adh

E2

ad

hE2

F26

and

adh

E2

R20

pC

WO

ri-te

r.ad

hE2

pET

23a

E

coR

I-K

pnI

Gib

son

Isot

herm

al

896

pET

23a-

His

Tev

_SH

3_ad

hE2

S

H3

F1

and

adh

E2

R20

pC

WO

ri-T

er.S

H3

_adh

E2

pE

T23

a

Eco

RI-

Kpn

I G

ibso

n Is

othe

rmal

21

7 pE

T29

a(+

)-cc

r cc

r F

1 an

d cc

r R

2 pB

SK

-Bu2

pE

T29

a

Nde

I-E

coR

I S

tand

ard

ligat

ion

566

pET

29a-

ccr.

adh

E2-

S

adhE

2 F

1 a

nd p

CW

Ori

Sta

g R

5

pCW

Ori-

ccr.

adh

E2-

S

pET

29a

E

coR

I-H

indI

II S

tand

ard

ligat

ion

563

pET

29a-

ccr-

S.a

dhE

2 cc

r F

1 an

d pC

WO

ri-S

tag

R4

pC

WO

ri-c

cr.S

tag

pE

T29

a-cc

r.ad

hE

2 N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

963

pET

29a-

stre

p.A

LD

H59

3

sALD

H59

3 F

101

and

sA

LDH

593

R1

pCW

Ori-

sALD

H5

93

pET

29a

N

deI-

Xba

I S

tand

ard

ligat

ion

246

pET

29-c

cr

ccr

F1

and

ccr

R2

pBS

K-B

u2

pE

T29

a

Nde

I-E

coR

I S

tand

ard

ligat

ion

24

7 pE

T29

-ccr

.adh

E2

adhE

2 F

1 a

nd a

dhE

2 R

1

pBS

K-B

u3

pET

29-c

cr

Eco

RI-

Kpn

I S

tand

ard

ligat

ion

24

8 pE

T29

-ccr

.sB

dhB

bd

hB F

101

and

bdh

B R

101

pC

R2.

1-sb

dhB

pE

T29

a-cc

r.ad

hE

2 E

coR

I-S

acI

Sta

ndar

d lig

atio

n

956

pET

Due

t-0-

ter

Ter

F1

and

Ter

R10

3 pC

WO

ri-te

r.ad

hE2

pET

Due

t N

deI-

Kpn

I S

tand

ard

ligat

ion

1096

pJ

204-

btP

haZ

N

/A

P

urch

ased

from

D

NA

2.0

1093

pJ

204-

rrA

BH

N

/A

P

urch

ased

from

D

NA

2.0

1094

pJ

204-

rrA

dpA

N

/A

P

urch

ased

from

D

NA

2.0

10

95

pJ20

4-rr

Pha

Z2

N

/A

P

urch

ased

from

193

DN

A2.

0

951

pOriT

33-

Bu6

N

phT

7-O

ri F

1 an

d N

phT

7-O

ri R

1

pCW

Ori

pT

5T33

-Bu6

N

siI-

Nde

I G

ibso

n Is

othe

rmal

22

3 pP

ro33

-crt

cr

t F1

and

crt

R2

pB

SK

-Bu

1

pBA

D33

S

acI-

Xm

aI

Sta

ndar

d lig

atio

n

602

pPro

33-p

haA

.hb

d.cr

t ph

aA F

5 an

d cr

t R

2 pB

AD

33-p

haA

.hbd

.crt

pP

ro33

X

baI-

Hin

dIII

S

tand

ard

ligat

ion

81

9 pQ

E30

Xb

-lacI

q la

cIq

F1

and

lacI

q R

1

pTrc

99a

-0

pQE

-30X

a

Bgl

I S

tand

ard

ligat

ion

N/A

pR

C2.

1-P

(pdc

).a

ceE

.ace

F.lp

d S

OE

: (P

(pdc

) F

1 an

d P

(pdc

)/P

dh

SO

E R

1) a

nd (

P(p

dc/P

dh S

OE

F

1 an

d P

dh R

1/S

LIC

R1)

Z. m

obili

s G

enom

ic D

NA

; pB

BR

2-ac

eE.a

ceF

.lpd

pCR

2.1

Top

o cl

onin

g

815

pRS

FD

uet-

0-te

sA

tesA

F1

and

tesA

R1

DH

10B

Gen

omic

DN

A

pRS

FD

uet

Bgl

II-K

pnI

Sta

ndar

d lig

atio

n

497

pT.O

99a

-bcd

.etf

B.e

tfA-a

dhE

2

bcd

F3

and

etfA

R4

pCW

Ori-

bcd.

etfB

.etfA

pT

rc.O

ri99a

-0-a

dhE

2

Bsa

I ins

ert

(Nco

I ve

ctor

)-K

pnI

Sta

ndar

d lig

atio

n

797

pT5T

33-B

u6

lacI

q R

2 an

d T

5 R

1 pQ

E30

Xb

-lacI

q pB

T33

-nph

T7.

hb

d-cr

t N

siI-

Sac

I S

tand

ard

ligat

ion

49

9 pT

5T33

-pha

A.H

BD

-crt

la

cIq

R2

and

T5

R1

pQE

30X

b-la

cIq

pBT

33-B

u2

Nsi

I-S

acI

Sta

ndar

d lig

atio

n

491

pT5T

33-p

haA

.ph

aB-c

rt

laqI

q R

2 an

d T

5 R

1 pQ

E30

Xb

-lacI

q pB

T33

-Bu1

N

siI-

Sac

I S

tand

ard

ligat

ion

869

pT7T

33-B

u6

lacI

q R

2 an

d T

7 S

R1

pET

16b

pB

T33

-Bu6

M

goM

IV-

Nde

I G

ibso

n Is

othe

rmal

583

pTE

T-t

etR

.GB

D1

_SH

31_P

DZ

1

N/A

Fro

m th

e D

uebe

r la

b 58

4 pT

ET

-tet

R.G

BD

1_S

H31

_PD

Z2

N

/A

F

rom

the

Due

ber

lab

585

pTE

T-t

etR

.GB

D1

_SH

31_P

DZ

4

N/A

Fro

m th

e D

uebe

r la

b 58

6 pT

ET

-tet

R.G

BD

1_S

H32

_PD

Z1

N

/A

F

rom

the

Due

ber

lab

587

pTE

T-t

etR

.GB

D1

_SH

32_P

DZ

2

N/A

Fro

m th

e D

uebe

r la

b 58

8 pT

ET

-tet

R.G

BD

1_S

H32

_PD

Z4

N

/A

F

rom

the

Due

ber

lab

589

pTE

T-t

etR

.GB

D1

_SH

34_P

DZ

1

N/A

Fro

m th

e D

uebe

r la

b 59

0 pT

ET

-tet

R.G

BD

1_S

H34

_PD

Z2

N

/A

F

rom

the

Due

ber

lab

591

pTE

T-t

etR

.GB

D1

_SH

34_P

DZ

4

N/A

Fro

m th

e D

uebe

r la

b 40

5 pT

rc.O

ri99a

pC

WO

ri F

1 an

d pC

WO

ri R

1

pCW

Ori

pT

rc99

a

Ngo

MIV

S

tand

ard

ligat

ion

N

/A

pTrc

.Ori9

9a-0

-ad

hE2

adhE

2 F

17 a

nd a

dhE

2 R

17

pET

29a-

ccr.

adh

E2

pTrc

.Ori9

9a

Nde

I-X

hoI

Sta

ndar

d lig

atio

n

955

pTrc

.Ori9

9a-0

-ter

T

er F

1 an

d T

er R

101

pCW

Ori-

ter.

adhE

2 pT

rc.O

ri99a

N

deI-

Xho

I S

tand

ard

ligat

ion

529

pTrc

.Ori9

9a-T

dTer

-adh

E2

td

Ter

F10

1 an

d td

Ter

R10

3

pCW

Ori-

ter.

adh

e2

pT.O

99a

-bcd

.etf

B.e

tfA-

adhE

2

Bsa

I in

sert

(Nco

I v

ecto

r)-

Kpn

I

Sta

ndar

d lig

atio

n

252

pTrc

99a

-ccr

N

/A

pT

rc99

a-c

cr.a

dhE

2 E

coR

I-K

pnI

Pfu

to g

ive

blun

t end

s;

Sta

ndar

d lig

atio

n

968

pTrc

99a

-ccr

(x)

SO

E: (

ccr

F1

and

ccr

R8)

and

(cc

r F

8 an

d cc

r R

2)

pTrc

99a

-ccr

pT

rc99

a-c

cr

Nde

-Eco

RI

Sta

ndar

d lig

atio

n

253

pTrc

99a

-ccr

.adh

E2

ccr

F2

and

adhE

2 R

2

pET

29a-

ccr.

adh

E2

pTrc

99a

Sac

I and

B

saI

inse

rt;

Nco

I ve

ctor

Sta

ndar

d lig

atio

n

254

pTrc

99a

-ccr

.adh

E2.

sBdh

B

bdhB

F10

0 an

d b

dhB

R10

3

pC

R2.

1-sB

dhB

pC

WO

ri-cc

r.ad

hE

2 K

pnI

Sta

ndar

d lig

atio

n

565

pTrc

99a

-ccr

.adh

E2-

S

ccr

F2

and

pCW

Ori

-Sta

g R

6

pCW

Ori-

ccr.

adh

E2-

S

pTrc

99a

Sac

I and

B

saI

inse

rt;

Nco

I

Sta

ndar

d lig

atio

n

194

vect

or

562

pTrc

99a

-ccr

-S.a

dhE

2 cc

r F

2 an

d pC

WO

ri-S

tag

R4

pC

WO

ri-cc

r.S

tag

pT

rc99

a-c

cr.a

dhE

2 N

deI-

Eco

RI

Sta

ndar

d lig

atio

n

980

pTrc

99a

-crt

cr

t F2

and

crt

R2

pB

SK

-Bu1

pT

rc99

a

Nco

I-X

maI

S

tand

ard

ligat

ion

981

pTrc

99a

-crt

(x)

SO

E: (

crt

F2

and

crt

R3)

an

d (c

rt

F3

and

crt

R2)

pT

rc99

a-c

rt

pTrc

99a

N

coI-

Xm

aI

Sta

ndar

d lig

atio

n

983

pTrc

99a

-pha

A2.

phaB

ph

aA2

F1

and

M13

SR

1

pCR

2.1-

phaA

2.p

haB

pT

rc99

a

Bsa

I in

sert

(Nco

I v

ecto

r)-

Kpn

I

Sta

ndar

d lig

atio

n

232

pTrc

99a

-sB

dhB

bd

hB F

101

and

bdh

B R

103

pC

R2.

1-s

bdhB

pT

rc99

a

Eco

RI-

Kpn

I S

tand

ard

ligat

ion

949

pTT

33-(

100K

-2)B

u6

BT

33-N

phT

7 1

00K

F2

and

TT

33-

Nph

T7

100

K R

2

No

tem

plat

e

pTT

33-B

u6

Nde

I A

nnea

l Prim

ers;

G

ibso

n Is

othe

rmal

947

pTT

33-(

10K

-2)B

u6

BT

33-N

phT

7 1

0K

F2

and

TT

33-

Nph

T7

10K

R2

N

o te

mpl

ate

pT

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

948

pTT

33-(

30K

-2)B

u6

BT

33-N

phT

7 3

0K

F2

and

TT

33-

Nph

T7

30K

R2

N

o te

mpl

ate

pT

T33

-Bu6

N

deI

Ann

eal P

rimer

s;

Gib

son

Isot

herm

al

950

pTT

33-(

His

)Bu6

T

T33

-Nph

T7

His

F1,

BT

33-N

phT

7

His

R1,

and

BT

33-

Nph

T7

His

F

2

No

tem

plat

e

pTT

33-B

u6

Nde

I A

nnea

l Prim

ers;

G

ibso

n Is

othe

rmal

796

pTT

33-B

u6

pTrc

99a

F7

and

pTrc

99a

R6

pT

rc99

a

pBT

33-n

phT

7.h

bd-

crt

Nsi

I-S

acI

Sta

ndar

d lig

atio

n

498

pTT

33-p

haA

.HB

D-c

rt

pTrc

99a

F7

and

pTrc

99a

R6

pT

rc99

a

pBT

33-B

u2

Nsi

I-S

acI

Sta

ndar

d lig

atio

n

492

pTT

33-p

haA

.ph

aB

-crt

pT

rc99

a F

7 an

d pT

rc99

a R

6

pB

T33

-Bu1

N

siI-

Sac

I S

tand

ard

ligat

ion

11

89

pUC

19-(

gg)n

phT

7 np

hT7

gg F

1 an

d n

phT

7 gg

R1

pB

T33

-Bu6

pU

C19

E

coR

I-K

pnI

Gib

son

Isot

herm

al

N/A

pU

C19

*-P

(gap

).B

u1.T

cR.T

(pdh

) pU

C19

Sac

I exp

ande

r F

1 an

d pU

C19

Sac

I exp

ande

r R

1

No

tem

plat

e

pUC

19-

P(g

ap).

Bu1

.TcR

.T(p

dh)

Sac

I P

rimer

Ann

eal:

Sta

ndar

d lig

atio

n

595

pUC

19*-

P(g

ap).

Bu2

.TcR

.T(p

dh)

pUC

19 S

acI e

xpan

der

F1

and

pUC

19 S

acI e

xpan

der

R1

N

o te

mpl

ate

pU

C19

-P

(gap

).B

u2.T

cR.T

(pdh

) S

acI

Prim

er A

nnea

l: S

tand

ard

ligat

ion

N/A

pU

C19

-Cm

R.T

(adh

B)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pC

R2.

1-C

mR

.T(a

dhB

) pU

C19

K

pnI-

Bam

HI

Dire

ct li

gatio

n

N/A

pU

C19

-P(a

dhB

).ad

hE2

N

o P

CR

, Dir

ectly

cut

our

inse

rt

pCR

2.1-

P(a

dhB

).ad

hE2

pU

C19

E

coR

I-S

acI

Dire

ct li

gatio

n

N/A

pU

C19

-P(a

dhB

).ad

hE2.

P

(gap

).cc

r N

o P

CR

, Dir

ectly

cut

our

inse

rt

pCR

2.1-

P(g

ap).

ccr

pCR

2.1-

P(a

dhB

).ad

hE2

S

acI-

Kpn

I D

irect

liga

tion

597

pUC

19-P

(adh

B).

adhE

2.

P(g

ap).

ccr.

Cm

R.T

(adh

B)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pC

R2.

1-C

mR

.T(a

dhB

) pU

C19

-P

(adh

B).

adhE

2.P

(gap

).c

cr

Kpn

I-B

amH

I D

irect

liga

tion

961

pUC

19-P

(adh

B).

adhE

2.

P(g

ap).

TE

R.C

mR

.T(a

dhB

) N

o P

CR

, Dir

ectly

cut

our

inse

rt

pCR

2.1-

P(g

ap).

ter

pUC

19-

P(a

dhB

).ad

hE2.

P(g

ap).

ccr

.Cm

R.T

(adh

B)

Sac

I-K

pnI

Dire

ct li

gatio

n

N/A

pU

C19

-P(g

ap).

Bu2

.TcR

.T(p

dh)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pU

C19

-P(g

ap).

pha

A.h

bd.c

rt

pUC

19-

P(g

ap).

pha

A.h

bd.c

rt

Xm

aI-X

baI

Dire

ct li

gatio

n

N/A

pU

C19

-P(g

ap).

pha

A.h

bd.c

rt

No

PC

R, D

irec

tly c

ut o

ur in

sert

pC

R2.

1-P

(gap

).p

haA

.hbd

.crt

pU

C1

S

acI-

Xm

aI

Dire

ct li

gatio

n

594

pUC

19-P

(pdc

).p

dh.P

(gap

).

Bu1

.TcR

.T(p

dh)

No

PC

R, D

irec

tly c

ut o

ur in

sert

pR

C2.

1-P

(pdc

).ac

eE.a

ceF

.lpd

pUC

19*-

P(g

ap).

Bu1

.TcR

.T(p

dh)

Xho

I-S

acI

Dire

ct li

gatio

n

N/A

pU

C19

-P(p

dc).

pdh

.P(g

ap).

B

u2.T

cR.T

(pdh

) N

o P

CR

, Dir

ectly

cut

our

inse

rt

pRC

2.1-

P(p

dc).

aceE

.ace

F.lp

d pU

C19

*-P

(gap

).B

u2.T

cR.T

(pdh

) X

hoI-

Sac

I D

irect

liga

tion

195

Appendix 4: Oligonucleotides used for molecular cloning and strain engineering

196

Table A4.1. Oligonucleotides used for molecular cloning and strain engineering. Name Sequence AccA3 F101 cacagcagcggccatatcgaaggtcgtcatATGGCAAGCCACGCAGGTAGCCGTATCGCCC AccA3 F102 GCCGCTAAaaactttctaaggaggtcccacATGGCAAGCCACGCAGGTAGCCGTATCGCC AccA3 R101 tcgggctttgttagcagccggatcctcgagTTATTTAATCTCCGCCAGGACCGTGCCTTG AccA3 R103 tgagatgagtttttgttcgggcccaagcttagatctTTATTTAATCTCCGCCAGGACCGTGCCTTG accBC F100 gaaaagatctgggctgccgaagccac accBC R100 gtcctccttatagttgtcggtatcgctaccttacttaatctccagcagaacgacacctttattaacac accBC R102 ggatccTTACTTAATCTCCAGCAGAACGACACCT AccD4 F101 cacagcagcggccatatcgaaggtcgtcatATGACGGTTACGGAGCCGGTTCTGCACACG

AccD4 F103 tttcgtcttcacgctagcaaagaggagaaAacacaattttattcttaggaggtcccttATGACGGTTACGGAGCCGGTTC

TGCACACG AccD4 R101 tcgggctttgttagcagccggatcctcgagTTAGACCGGGATCAGACCATGTTTACGACC AccD4 R102 agtgctcctccttattaattctcgtctaagaaTTAGACCGGGATCAGACCATGTTTACGACC AccD5 F101 cacagcagcggccatatcgaaggtcgtcatATGACCAGCGTGACCGATCGTAGCGCTCAC AccD5 R101 tcgggctttgttagcagccggatcctcgagTTACAGCGGGACGTTACCATGCTTTTTCGG AccD6 F101 cacagcagcggccatatcgaaggtcgtcatATGACGATTATGGCACCGGAAGCAGTCGGC

AccD6 F103 tttcgtcttcacgctagcaaagaggagaaAtaaaacaaaaatacaggtaaggagtacacggagaATGACGATTATGGCA

CCGGAAGCAGT AccD6 R101 tcgggctttgttagcagccggatcctcgagTTACAGCGGGATGTTTTTATGACGACCACG AccD6 R102 agtgctcctccttattaattctcgtctaagaaTTACAGCGGGATGTTTTTATGACGACCACG AccE F101 agcatggtaatatgccgctgtaaTTAaatagttagctataaagggaggggatcaagcatg AccE F102 ttcttagacgagaattaataaggaggagcactATGGGTACGTGTCCGTGCGAGAGCAGCG AccE F103 ttcttagacgagaattaataaggaggagcactATGGGTACGTGTCCGTGCGAGAGCAGCG accE F3 GACGGACCAGGAGGTGGCCGCTCTGACCGTTG AccE R101 tgagtttttgttcgggcccaagcttTTAgaaaaagttcacgttctgaaacgcgctc AccE R102 CTTGCCATgtgggacctccttagaaagtttTTAGCGGCGCATGTGGGTCATTTCTTGCAA accE R2 CGTGGTCTCTTCGGACATGCTTGATCCCCTCCCTTTAT aceBAK KF2 ATGACTGAACAGGCAACAACAACCGATGAACTGGCgtgtaggctggagctgcttc

aceBAK KR1 GGCTTTACGGATAACCCTCGATACATTGCGGAGAAAAATTATATGGAAGCTTTACattccgggg

atccgtcgacc aceE F1 CTGATgctagcaaggagatattatATGtcagaacgtttcccaaatgacgtggatccgatcgaaactcgcg aceE F2 acgcCTCGAGgctagcaaggagatattatATGtcagaacgtttcccaaatgacgtggatccgatcgaaactcgcg aceE SOE F1 GAACGAAACCGTTGACagcGACTACCAGACCTTCAAATCGAAAGATGGT aceE SOE R1 ctttgatttcgatagccatggtatatctccttcttatggtctagaTTAcgccagacgcgggttaactttatctgcatcgatgttg aceF R1 atatctagaTTAcatcaccagacggcgaatgtcagacagcgtgttg aceF SOE F1 cccgcgtctggcgtaatctagaccataagaaggagatataccATGgctatcgaaatcaaagtaccggacatcggggc aceF SOE F2 TCTCTCTCCTTCGACgccCGCGTGATCGACGGTGCTG acef SOE R1 gagttttgatttcagtactcataggatatctccttaaaccaggtaTTAcatcaccagacggcgaatgtcagacagcgtgttg acef SOE R2 GTCGATCACGCGggcGTCGAAGGAGAGAGAAATCGGCAGCATCAG ackA KF2 ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCAgtgtaggctggagctgcttc Acs F3 ctttcgtcttcacgctagcaaagaggagaaaagatctATGagccaaattcacaaacacac Acs R3 ccttctattagccgtggcttcggcagccctTTAcgatggcatcgcgatagcc adhB(Term) R1/SLIC R1 gcatgcctgcaggtcgactctagaggatccATTTCGTCGCCTTCATAAGGCAAACGCGGC adhE2 AADH R1 ATCaagcttggtaccTTAgttttcacggcgttctgcaacagatttgatgttcagcagatgc

adhE2 ADH F1 CTGATgctagcAAGAAGGAGATATACCatgCTGTGGTTCAAAGTCCCACAGAAAATTTACTTCA

AATACGGCTGCC

adhE2 ADH F2 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatgctgtggttcaaagtc

ccacagaaaatttacttcaaatacggctgcc adhE2 F1 ATgaattcAAGAAGGAGATATACCatgaaagtcacgaaccagaaggaactgaagcagaaactgaacgaactgcgcg adhE2 F11 cctgggcgtgtgcGCGtctatggctcacaaactgggtgctatgcaccacgtgcc adhE2 F13 gatataCATatgCTGTGGTTCAAAGTCCCACAGAAAATTTACTTCAAATACGGCTGCC

adhE2 F14 ATCggtaccAAGAAGGAGATATACCatgCTGTGGTTCAAAGTCCCACAGAAAATTTACTTCAAAT

ACGGCTGCC adhE2 F17 tcgatatacatATGAAAGTCACGAACCAGAAGGAACTGAAGCAGAAACTG

adhE2 F25 ATgaattcaagaaggagatataccATGCCACCGCCAGCTCTGCCACCGAAACGTCGTCGTGGTTCTG

GTTCTGGCAGCGGCAGCatgaaagtcacgaaccagaaggaactga adhE2 F26 tggctgtcgaacgcaaataaggtaccactagtgcggccgcagatctaagaaggagatataccatgaaagtcacgaaccag

adhE2 F6 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatgaaagtcacgaacc

agaaggaactgaagcagaaactgaacgaactgc

adhE2 F7 CTGATgctagcAAGAAGGAGATATACCatgaaagtcacgaaccagaaggaactgaagcagaaactgaacgaactg

cgcg adhE2 F8 cgataacggtgttatcGCAgcaagcgaacagtccatcctggttatgaactccatctacg adhE2 F9 cacgtgccgGCGggtatcgcgtgtgctgtcctgatcgaagaagtaattaagtacaacgc

197

adhE2 R1 atcAAGCTTGGTACCttaaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacgcggg adhE2 R10 acgcGTCGACaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacgcggg adhE2 R11 gtgagccatagaCGCgcacacgcccaggaacgcgttgg adhE2 R12 atcAAGCTTttaaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacgcggg adhE2 R17 agcCTCGAGttaaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacg

adhE2 R19 TTTGACAGCTTATCATCGATAAGCTTGGTACCTTAGACCAGGCTCTCTTTGACACCACCGG

AACCGCTACCGGAACCAGAGCCAAAAGATTTGATATAAATGTCTTTCAGCTCAGAGATCAGCGGGTAACGCGGG

adhE2 R2 cgGAGCTCttaaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacgcggg adhE2 R20 ctcatccgccaaaacagccaagcttGCATGCttaaaaagatttgatataaatgtctttcagctcagagatcagcg adhE2 R3 aataGGCGCGCCttaaaaagatttgatataaatgtctttcagctcagagatcagcgggtaacgcggg adhE2 R8 tggactgttcgcttgcTGCgataacaccgttatcgtaagtcttggacagaataatggaagatactgccatatcg adhE2 R9 cgcgataccCGCcggcacgtggtgcatagcacccag adhE2-SalI R1 cagGTCGACttaaaaagatttgatataaatgtctttcagctcagagatcagc ald46 120925 F1 gcagaagttgaacgtttcgatcgtatttaa ald46 120925 R2 ctcatgtttgacagcttatcatcgataagcttggtaccTtagccggccagaacacaacga alkK F1 taacaagctagcgaattcgagctcggtaccCaaaaaatattaaataaggaggtctacgATGctgggtcagatgatgcgtaatc alkK R1 gcttgcatgcctgcaggtcgactctagaggatccTTAttcgcagacgctgctgctagaac

arcA F1 agaTCTAGACTCGAGaaggagatatcatATGCAGACCCCGCACATTCTTATCGTTGAAGACGAGT

TGG arcA R1 acgcGTCGACGAGCTCttaatcttccagatcaccgcagaagcgataaccttcaccgtgaatgg AtfA F1 aaggagatataCATatgcgcccactgcacccg AtfA R1 aagtacgtAGATCTttattagttcgcggttttaatgtcttcctgtttggcg atoB F1 cgGAGCTCaaggagatatacatATGaaaaattgtgtcatcgtcagtgcggtacgtactgctatcgg atoB KF1 GACGGCACCCCTACAAACAGAAGGAATATAAgtgtaggctggagctgcttc atoB KR1 CCCGATAACTTTCGCTATCGGGTGTTTTTATTGAcatatgaatatcctccttag atoB R1 gcgatgcgctgggtcattatatatctccttttagttactagaattcTTAattcaaccgttcaatcaccatcgcaattccctgaccgccgcc

bcd F1 gagatatacatATGGATTTTAATTTAACAAGAGAACAAGAATTAGTAAGACAGATGGTTAGAGAA

TTTGC

bcd F1 gagatatacatATGGATTTTAATTTAACAAGAGAACAAGAATTAGTAAGACAGATGGTTAGAGAA

TTTGC bcd F3 atGGTCTCccATGgattttaatttaacaagagaacaagaattagtaagacagatggttagag

bdhA F101 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatgctgtccttcgattattc

tatcccgaccaaagtattcttcgg bdhA R1 GATAAGCTTTTAATAGCTTTTCTTGAAAATTTCCAGAACGTCCTCGGCGTTGA BglII into Xma linker F1 ccggGTATGGCATAGATCTGCTGCACTTCGTC BglII into Xma linker R1 ccggGACGAAGTGCAGCAGATCTATGCCATAC

birA F3 agttgataacaagctagcgaattcgagctCttatataagctttaaggaggtcgaagATGAAGGATAACACCGTGCCAC

TGAAATTG birA R4 tgcctgcaggtcgactctagaggatccccgTTATTTTTCTGCACTACGCAGGGATATTTCACCGCCC BT33-NphT7 10000 F1 TTTTCACATCAGTCTCATTCTACAGGTTTTAAGCatgaccgacgttcgttttcgtatcat BT33-NphT7 10000 R1 GCTTAAAACCTGTAGAATGAGACTGATGTGAAAAacagtagagagttgcgataaaaagcg BT33-NphT7 100000 F1 GTAGAAAGATAATACTCTACATTAAGGAGGAGAGatgaccgacgttcgttttcgtatcat BT33-NphT7 100000 R1 CTCTCCTCCTTAATGTAGAGTATTATCTTTCTACacagtagagagttgcgataaaaagcg BT33-NphT7 100K F2 ATCGGGCTTTTTCACAACAGGAGGATTTATCatgaccgacgttcgttttcgtatcattg BT33-NphT7 100K R2 GATAAATCCTCCTGTTGTGAAAAAGCCCGATgagctcgaattcgctagcccaaaaaaac BT33-NphT7 10K F2 AACAACAAAGTTTTCTCTCAAGGAATAGGTTTCAGatgaccgacgttcgttttcgtatca BT33-NphT7 10K R2 CTGAAACCTATTCCTTGAGAGAAAACTTTGTTGTTgagctcgaattcgctagcccaaaaa BT33-NphT7 30K F2 CATCCTTATCGCTCATAATTCGTAGGAGGAGAATatgaccgacgttcgttttcgtatcat BT33-NphT7 30K R2 ATTCTCCTCCTACGAATTATGAGCGATAAGGATGgagctcgaattcgctagcccaaaaaa BT33-NphT7 His F1 tagcgaattcgagctcaaggagatatacatATGggccatcatcatcatcatcatcatcat BT33-NphT7 His F2 tcacagcagcggccatatcgaaggtcgtcatATGaccgacgttcgttttcgtatcattgg BT33-NphT7 His R1 acgaccttcgatatggccgctgctgtgatgatgatgatgatgatgatgatgatggcccat btPhaZ F1 ttcgtcttcacgctagcaaagaggagaaaagatctAgcattttttaaggaggtaccaaATGattaaaccggcgacgatgg btPhaZ R1 tatctacctccttaatctgcttgtcgtctctgTTAtttttgttccagccagtcttcaacg CAT F1 gagatataCATATGGGTACCgagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaac CAT R1 agaTCTAGAttacgccccgccctgccactcatcg CCR (S. cinna) F0 gagatataCATatgaaagaaatcctggacgctattcaggcacaaacggc CCR (S. cinna) R0 TCTCCtctagaGAATTCttagacgttgcggaaacggttaatagcatcgatgtg ccr F1 gagatatacatATGacggtaaaagatattctggacgcgattcagtccaaagacgctacc ccr F2 atGGTCTCccatgAcggtaaaagatattctggacgcgattcagtccaaagacgctacc ccr F8 cgaccgtaatgccgaaggcTTCaaattttggaaagatgaacatacccaagaccctaaggaatggaaacg ccr R1 ATcttaagTTACACATTACGAAAACGGTTGATCGCGTCGATATGCTGGGC ccr R10 ACGCgtcgacCACATTACGAAAACGGTTGATCGCGTCGATATGCTGGGC

ccr R11 atcTCTAGAttaGTGATGATGATGATGATGATGATGATGATGgccgctgctcacattacgaaaacggttgatcg

cgtcgatatgctgggc

198

ccr R2 ATgaattcTTACACATTACGAAAACGGTTGATCGCGTCGATATGCTGGGC ccr R3 cgggtaccTTACACATTACGAAAACGGTTGATCGCGTCGATATGCTGGGC ccr R8 gttcatctttccaaaatttGAAgccttcggcattacggtcgatgatagcctcggc ccr SOE F1 gctacttaataagttaggagaataaacATGACGGTAAAAGATATTCTGGACGCGATTCAG

CDF2-nphT7 (gg) F1 gatccGCAATCAGGCATTTAATAAGGAGGTAATAAatgtgagaccgagctcaactagtcccgcggtggtctctgct

tGGTAC CDF2-nphT7 (gg) R1 CaagcagagaccaccgcgggactagttgagctcggtctcacatTTATTACCTCCTTATTAAATGCCTGATTGCg CmR F1/SLIC F1 TCAACCGTTTTCGTAATGTGTAAggtacccgACGTTGATCGGCACGTAAGAGGTTCCAAC CmR/adhB(Term) SOE F1 tttcaaaacaggaaaacggttttccgtcctgtcttgattttcaagc CmR/adhB(Term) SOE R1 aggacggaaaaccgttttcctgttttgaaaTTACGCCCCGCCCTGCCACT crp F1 aaggaCTCGAGaaggagatatcatATGGTGCTTGGCAAACCGCAAACAGACCCG crp R1 agtcGAGCTCttataaCAATTgTTAACGAGTGCCGTAAACGACGATGGTTTTACCGTGTGC crt F1 cgGAGCTCaaggagatatcatatggaactgaacaacgtgatcctggaaaaagaggg crt F2 ataCCatggaactgaacaacgtgatcctggaaaaagagggtaaagtagcagttgtcacc crt F3 tttggtcaaccgCAAgttggcctgggcattacgccggg

crt F4 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatggaactgaacaacgt

gatcctggaaaaagagggtaaagtagcagt crt R1 aataGCATGCttaacgatttttgaagccttcgattttacgtttttcgatgaaagcggtcatggc crt R1/SLIC R1 gatgataagctgtcaaacatgagaattcttCCCGGGttaacgatttttgaagccttcg crt R2 aataAAGCTTCCCGGGttaacgatttttgaagccttcgattttacgtttttcgatgaaagcggtcatggc crt R3 gcccaggccaacTTGcggttgaccaaagcgagcgttagaagatgcg crt R5 caggtcgactctagaggatcCCCGGGttaacgatttttgaagccttcgattttacgtttttcgatgaaagcggtcatggc

crt R6 caggtcgactctagaggatcCCCGGGttagaccaggctctctttgacaccaccggaaccgctaccggaaccagagccACGA

TTTTTGAAGCCTTCGATTTTACGTTTTTCGATGAAAGCGGTCATGGC

crt SOE F6 gcgctgccgccgaaacgccgtcgcggctccggtagcggtagcggcagcggcATGGAACTGAACAACGTGATCCT

GG crt SOE F7 gccgtcgcggctccggtagcggtagcggcagcggtatggaactgaacaacgtgatcctgg crt SOE R6 cgcgacggcgtttcggcggcagcgccggcggcggcatggtctgtttcctgtgtgaaattgttatccgctca crt SOE R7 accgctaccggagccgcgacggcgtttcggcggcagcgccggcggcggcatggtctgtttcctgtgtgaaattgttatccgc

crt Yeast-R4 CTTTTCGGTTAGAGCGGATCTTAGCTAGCCGCGGTACCAAGCTTACTCGAGttaacgatttttgaag

ccttcgattttacgtttttcgatg crt-f1 aataacacagtcaaatcaatcaaaATGgaactgaacaacgtgatcctgg crt-r1 accttttttataacttatttaataataaaaatcataaatcataagaaattcgcTTAacgatttttgaagccttcgattttacgtttttcg dtsR1 F100 gtgttaataaaggtgtcgttctgctggagattaagtaaggtagcgataccgacaactataaggaggac dtsR1 R100 cgagtcaggatccttacagcggcatattaccatgcttgc

etfA R2 ATAtctagaCTTAATTATTAGCAGCTTTAACTTGAGCTATTAATTCTGGTACAACTTTATTTACAT

CACC etfA R4 tcgGGTACCttaattattagcagctttaacttgagctattaattctggtacaactttatt etfB SOE F1 AGCAGCTGCtTATGTTGTCTCAAAATTAAAAGAAGAACACTATATTTAAGTTAGGAGGG etfB SOE R1 TTTGAGACAACATAaGCAGCTGCTTCCTTAACAGGCTTATCAATAACTTCTCCCTGTCC fabF KO F2 tctttttgtcccactagaatcattttttccctccctggaggacaaacGTGgtgtaggctggagctgcttc fabF KO R2 aaaaaaggcccgcaagcggaccttttataagggtggaaaatgacaacTTAcatatgaatatcctccttag fadR F1 aaggaCTCGAGaaggagatatcatATGGTCATTAAGGCGCAAAGCCCGGCGG fadR R1 aataAAGCTTGAGCTCttatcgcccctgaatggctaaatcacccgg Fdx R1 tattacgGAATTCttaatgctcacgcgcatggttgatagtg Fdx SOE F1 cggagcattactggtaaggatccaaggagatatcatgatgccaaagattgttattttgcctcatcagg FldA F1 TTAgaattcAAGGAGATTCATCGatggctatcactggcatctttttcggc FldA R1 tagtacatGAGCTCtcaggcattgagaatttcgtcgagatgcaac FldB F1 TTAgaattcAAGGAGATTCATCGatgaatatgggtcttttttacggttccagcacctg FldB R1 tagtacatGAGCTCtcaggcgtaatgctctgccatttcg focA-pflB KF1 TTACTCCGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAgtgtaggctggagctgcttc focA-pflB KR1 ATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGCTGTTCTcatatgaatatcctccttag Fpr F1 tcagtctaCCatggctgattgggtaacaggcaaagtcac Fpr SOE R1 ctccttggatccttaccagtaatgctccgctgtcatgtggcccggtcggcg frdBC KF1 ATGGCTGAGATGAAAAACCTGAAAATTGAGGTGGTGCGCTATAACgtgtaggctggagctgcttc frdBC KR1 TTACCAGTACAGGGCAACAAACAGGATTACGATGGTGGCAACCACcatatgaatatcctccttag GFP F1 ATCggtaccATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTG GFP QC F1 ctcttatggtgttcaatgcttttcccgttatccggatcaCatgaaacggcatgactttttcaagagtgccatgcccg

GFP QC R1 cgggcatggcactcttgaaaaagtcatgccgtttcatGtgatccggataacgggaaaagcattgaacaccataagag GFP R1 agaTCTAGAttatttgtagagctcatccatgccatgtgtaatcccagcagc

hbd 120917 GF1 CTAGCGAATTCGAGCTCAAGGAGATATACATATGTTAGATCTCGGGTACCGAATTCAAGGA

GATATATAATGAAAAAGGTTTGCG

hbd aGBD-SOE F1 GCAATACGTCTTTGCATCGGCACGCTAGGTAAGGTCGCTGCTTCCACTTCTGGTCCGCCC

GCTACTTCTGCTTCTGCCATCGCCGTCGCCGAGACCAAGACCAtactttttccaaacgcaataaccacgcccatgataccc

199

hbd aGBD-SOE R1 ACCTTACCTAGCGTGCCGATGCAAAGACGTATTGCACGTAGTCTCGCGGATGGTCGTAatat

atagaggaacttaagaataaacgcaagctgtcggtcac hbd BR1 gaaaatcttctctcatccgccaaaacagccctcgagttacttggagtaat hbd F1 ATgaattcaaggagatatataatgaaaaaggtttgcgttattggtgcgggtactatggg hbd F100 tgcgtatcgCATatgaaaaaggtttgcgttattggtgcgggtac

hbd F101 agcactggctgtcgaacgcaaataaCGACTACGGGTAATTCGGAACGACAGTCGCCCCAAAatgaaaaag

gtttgcgttattggtgcgg HBD GF1-130207 aatttcacacgagctcaaggagatataCATatgaaaaaggtttgcgttattggtgcgggt HBD GR1-130207 aatcttctctcatccgccaaaacagccctcgagctcttacttggagtaatcgtaaaaacc hbd R1 ATAaagcttggtaccgacgtcctcgagttacttggagtaatcgtaaaaacc hbd R100 attgtagcCTCGAGCTCttacttggagtaatcgtaaaaacctttgccagatttacgacc hbd R102 gtcatataGGATCCttacttggagtaatcgtaaaaacctttgccagatttacgacc

hbd R104 cgactcgagTTACACCAGGCTTTCTTTCACGCCACCCGAACCCGAACCCGAACCCGAACCcttgg

agtaatcgtaaaaacctttgccagatttacgacccagc HBD SLIC R1 cccgtttagaggccccaaggggttatgctaCTCGAGCTCttacttggagtaatcgtaaaaacctttgccagatttacgacc hbd SOE F1 ggtgattggtatgGTTttctttaacccggcaccagttatgaaactg hbd SOE F2 ggtgattggtatgTACttctttaacccggcaccagttatgaaactg hbd SOE F3 ggtgattggtatgGAAttctttaacccggcaccagttatgaaactg Hbd SOE R1 TCTGTTTCCTGTGTGAAAttacttggagtaatcgtaaaaacctttgccagatttacgacc hbd SOE R1 TCTGTTTCCTGTGTGAAAttacttggagtaatcgtaaaaacctttgccagatttacgacc hbd SOE R2 gccgggttaaagaaGTAcataccaatcaccttatccggacgtttgg hbd SOE R3 gccgggttaaagaaTTCcataccaatcaccttatccggacgtttgg HBD/crt SOE F1 CTCCAAGTAAtttcacacaggaaacagaccATGGAACTGAACAACGTGATCCTGGAAAAA HBD/crt SOE R1 ggtctgtttcctgtgtgaaaTTACTTGGAGTAATCGTAAAAACCTTTGCCAGATTTACGA hbd-f1 tactttttacaacaaatataaaacaATGaaaaaggtttgcgttattggtgcggg hbd-r1 tctttaggtatatatttaagagcgatttgtTTActtggagtaatcgtaaaaacctttgcc iclR F1 aataAAGCTTCTCGAGaaggagatatcatATGGTCGCACCCATTCCCGCGAAACGC iclR R1 aataTCTAGAGAGCTCtcagcgcattccaccgtacgccagcg Jfar F1 catgtaatCATatggaagaaatgggcagcatcctggagttcc Jfar R1 aaaaaaaaTCTAGAttagttcaggacatgctccacaacacccgg Kpn linker F1 cggcggtggctctggtggtggctccggtggccgtac Kpn linker F2 cggcggtggctctggtggtggctccggtggcggctctggcggtggctccggcggtggttctcgtac Kpn linker F3 taattgagtaaggtac Kpn linker R1 ggccaccggagccaccaccagagccaccgccggtac Kpn linker R2 gagaaccaccgccggagccaccgccagagccgccaccggagccaccaccagagccaccgccggtac Kpn linker R3 cttactcaattagtac lac Iq F1 atacaGACGTCgacaccatcgaatggtgcaaaacctttcgcggtatggc lac Iq R1 atacaGACGTCctaactcacattaattgcgttgcgctcactgcccgc lacIq F2 tacggattcccgacaccatcacTCTAGAtgtgaaaccagtaacgttatacgatgtcgcag lacIq R2 aattATGCATctaactcacattaattgcgttgcgctcactgcccgc lacIq R2 aattATGCATctaactcacattaattgcgttgcgctcactgcccgc ldhA KF2 ATGAACTCGCCGTTTTATAGCACAAAACAGTACGACAAGAAGTACgtgtaggctggagctgcttc ldhA KR4 TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTcatatgaatatcctccttag lpd F1 ataTCTAGAggtttaaggagatatcctATGagtactgaaatcaaaactcaggtcgtggtacttggggcaggc lpd F1 ataTCTAGAggtttaaggagatatcctATGagtactgaaatcaaaactcaggtcgtggtacttggggcaggc lpd QC F1 cgaaagttatcccgtccatcgcctataccAaaccagaagttgcatgggtgggtctg lpd QC F2 cgtcggtcaaccgatgctggcaTacaaaggtgttcacgaaggtcacgttgcc lpd QC R1 cagacccacccatgcaacttctggttTggtataggcgatggacgggataactttcg lpd QC R2 ggcaacgtgaccttcgtgaacacctttgtAtgccagcatcggttgaccgacg lpd R1 gtaGTCGACttacttcttcttcgctttcgggttcggcaggtcgg lpd R2 agtcGAGCTCCCGCGGttacttcttcttcgctttcgggttcggcaggtcgg lpd R2 agtcGAGCTCCCGCGGttacttcttcttcgctttcgggttcggcaggtcgg lpd SOE F2 GTGGTTGCTCGCAAGCACCAGGTTATCCGTgcagctgacaaagacatcgttaaagtcttcaccaagcg lpd SOE F3 GTGGTTGTGCGCAAGCACCAGGTTATCCGTgcagctgacaaagacatcgttaaagtcttcaccaagcg lpd SOE F4 ggtaatgggtggcggtatcatcgctctggaaatggcgaccgtttaccacgcgctgggttcacagattgacgtggttg lpd SOE R2 ACGGATAACCTGGTGCTTGCGAGCAACCACGTCAATCTGTGAACCCAGCGCGTGG lpd SOE R3 ACGGATAACCTGGTGCTTGCGCACAACCACGTCAATCTGTGAACCCAGCGCGTGG lpd SOE R4 cgccatttccagagcgatgataccgccacccattaccagcaggcg lpdA R1 agcCTCGAGttaaatatgaattggtaaacctaaagccaattcagctgtatcc lpdA R2 taacgGAGCTCttaaatatgaattggtaaacctaaagccaattcagctgtatcc M13 SR1 CAGGAAACAGCTATGAC matB GF1 cgctagcaaagaggagaaaagatctCgcacataaggaggttaagcATGtcctctctcttcccggc matB GR1 agatgagtttttgttcgggcccaagcttcTCAgtcacggttcagcgcccg mdh KO F2 cagcggagcaacatatcttagtttatcaatataataaggagtttaggATGgtgtaggctggagctgcttc mdh KO R2 caaaaaaccggagtctgtgctccggttttttattatccgctaatcaaTTAcatatgaatatcctccttag NotI-Ter F1 gacGCGGCCGCatgatcgtcaagccaatggtgcg

200

nphT7 BF1 tacccgtttttttgggctagcgaattcgagctcaaggagatatacatatgaccgac NphT7 F100 cttgatcgtCATatgaccgacgttcgttttcgtatcattggcac

nphT7 GF2 gctttgtctttatcaacgcaaataacaagttgataacaaCtcgagAATTTAAGGAGGTAAAAACatgaccgacgttcgttttc

gtatcat nphT7 gg F1 cgacgttgtaaaacgacggccagtgaattcggtctctaATGaccgacgttcgttttcgtatcattggc nphT7 gg F2 cgacgttgtaaaacgacggccagtgaattcggtctctaATG nphT7 gg R1 tgcctgcaggtcgactctagaggatccccgggtctctaagcttaTTAccactcgatcagcgcgaag nphT7 gg R2 CGACTCTAGAGGATCCCCGGGTCTCTAAGCTTATTA nphT7 GR1 gtgagcgcgcgtaatacgactcactatagggcgaattgTTAccactcgatcagcgcgaag NphT7 R100 AACGCAAACCTTTTTCATTATATATCTCCTTGAATTCttaCCACTCGATCAGCGCGAAGCTC nphT7 R101 ttaCCACTCGATCAGCGCGAAGCTC NphT7-Ori F1 tgtttgacagcttatcatcgatgcatCGAcggccagtgaatccgtaatcatggtcatagc NphT7-Ori R1 tgatacgaaaacgaacgtcggtcatATGacctcctaagcatcgatggatcctgtttcctg

P(adhB) F2/SLIC F2 aggaaacagctatgacatgattacgaattcGGCAAAATCGGTAACCACATCTCAATTATTAAACAATACTT

C P(adhB)/adhE2 SOE F1 gtatgtagggtgaggttatagctATGAAAGTCACGAACCAGAAGGAACTGAAGCAGAAAC P(adhB)/adhE2 SOE R1 tggttcgtgactttCATagctataacctcaccctacatactagtttaaagcaacaacccg P(eda) R1 gttgttcagttCCATGGtagactcctgctttatttcccttaggggttc P(eda) SOE F1 GATTACTCCAAGTAATTTCACACAGGAAACAGAttgcaagtgagtgacatgaaaccgg P(gap) F1/SLIC F2 GAAAGCGAAGAAGAAGTAAgagctcTTTGTTCGATCAACAACCCGAATCCTATCGTAATG P(gap) F2 GAGCTCagCTCGAGtcGTCGACtacTTTGTTCGATCAACAACCCGAATCCTATCGTAATG

P(gap)/ccr SOE R1 GAATATCTTTTACCGTcatGTTTATTCTCCTAACTTATTAAGTAGCTACTATATTCCATAGCTAT

TTTTTAACGTG P(gap)/phaA SOE F1 gtagctacttaataagttaggagaataaacATGACTGATGTTGTGATTGTAAGCGCTGCA

P(gap)/phaA SOE R1 aatCACAACATCAGTcatGTTTATTCTCCTAACTTATTAAGTAGCTACTATATTCCATAGCTATT

TTTTAACGTG P(gap)/TdTER SOE F1 gctacttaataagttaggagaataaacATGATCGTCAAGCCAATGGTGCGCAATAATATC

P(gap)/TdTER SOE R1 CCATTGGCTTGACGATcatGTTTATTCTCCTAACTTATTAAGTAGCTACTATATTCCATAGCTA

TTTTTTAACGTG P(pdc) F1 atGGTCTCaaattGGCGTATTCGCCATGCTTGTCCTCGAT P(pdc) F2 agcCTCGAGatGGTCTCaaattCCAGAGGGGCAGACCGGTTACGG

P(pdc) R1 ATAggatccacgtcatttgggaaacgttctgacatTGCTTACTCCATATATTCAAAACACTATGTCTGAATCA

GG P(Pdc) Yeast-F1 CTCATGAAGCCTCCAGTATACCCCCGGGcatgcgactgggtgagcatatgttccgc P(pdc)/Pdh SOE F1 agtttatttaaaaaATGtcagaacgtttcccaaatgacgtggatccgatcgaaactcgcg P(pdc)/Pdh SOE R1 tttgggaaacgttctgaCATtttttaaataaacttagagcttaaggcgaaaagcccgtcc P(Pgk1) Yeast-F1 AAAAAGGAGTAGAAACATTTTGGGATCCacgcacagatattataacatctgcataataggc P(Tef) Yeast-F1 AGATGTTATAATATCTGTGCGTGGATCCcaaaatgtttctactccttttttactcttccagattttc pACYC184 linker F AGCTTctcgagGGTACCcccgggTTAATTAActtaagCTGCAGggcgcgccCCTAGGgtc pACYC184 linker R gacCCTAGGggcgcgccCTGCAGcttaagTTAATTAAcccgggGGTACCctcgagA pBBR1 R1 gcgtaatacgactcactatagggcgaattgTtctagacgaattcgtcccgggaggatcC pBBR1-Pro F1 ttatcaacgcaaataacaagttgataacaaGgatcctcccgggacgaattcgtctagaA pBBR1-tac F2 ggataacaatttcacacaggaaacaggatcGgatcctcccgggacgaattcgtctagaA pBBR1-trc F2 gagcggataacaatttcacacaggaaacagGgatcctcccgggacgaattcgtctagaA pBBR2.Pro-0gg F1 aagaaggagatatacaatgtgagaccgagctcaactagtcccgcggtggtctctgctttcgaattcgtctagaacaattcgccctatagt

pCDF.P(Tet) MCS2 F1 cctttcgtcttcacgctagcaaagaggagaaaagatctTacggccgtccatgggAagcttgggcccgaacaaaaactcatctcaga

agag pCDF.P(Tet) MCS2 R1 ctcttctgagatgagtttttgttcgggcccaagcttcccatggacggccgtaagatcttttctcctctttgctagcgtgaagacgaaagg pCDF.P(Tet).P(Pro) F1 cggatacatatttgaatgtatttagaaaaataaacaaataCgccagcaaccgcacctgtg pCDF.P(Tet).P(Pro) R1 gccgcagtctcacgcccggagcgtagcgaccgagtgaAaaaggccatccgtcaggatggc pCDF.P(Tet).P(Pro) R1 gccgcagtctcacgcccggagcgtagcgaccgagtgaAaaaggccatccgtcaggatggc pCWOri F1 ATTATgccggcGGAAACAGGATCGATCCATCGATG pCWOri F2 TATGatgcatgagctcactagtgctagcaggaggaattcaccatggtacccggggatcctctagagtcgacctgcaggcatgcA pCWOri F6 ATTGCTAAcggccgGGCTTTACACTTTATGCTTCCGGCTCG pCWOri F7 cctgagagctcggcgcgcctgcagGTCGACgagttagctcactcattaggcacccc pCWOri R1 ttaataGCCGGCtgaaaaaaaagcccgctcattaggcgggctcag pCWOri R4 AGCTTgcatgcctgcaggtcgactctagaggatccccgggtaccatggtgaattcctcctgctagcactagtgagctcatgcatCA pCWOri R6 tatgatcaCGGCCGcaaaaaacccctcaagacccgtttagaggc pCWOri R7 cgcgccattaccgtcaatagtaatCATATGacctcctaagcatcgatggatcctgtttcc pCWOri Stag R5 atcAAGCTTGGTACCttagctgtccatgtgctggcgttcgaat pCWori Stag R6 atcGAGCTCttagctgtccatgtgctggcgttcgaat pCWOri-Stag R4 gcgcgGAATTCttagctgtccatgtgctggcgttcgaat PDC1-p-f1 gagtaaagaccatgagcttcaataccctgattgactggaacagcggatccCATgcgactgggtgagcatatg PDC1-p-r1 tccaggatcacgttgttcagttcCATtttgattgatttgactgtgttattttgcgtgagg Pdh R1/SLIC R1 ttcgggttgttgatcgaacaaaGAGCTCTTACTTCTTCTTCGCTTTCGGGTTCGGCAG pdhA F1 attagtcagCATatggcaaaggctaagaaacaaaaacctattgactttaaagagc

201

pdhA F2 cgGGTACCaaggagatataCATatggcaaaggctaagaaacaaaaacctattgactttaaagagc pdhA F3 ataCCatggcaaaggctaagaaacaaaaacctattgactttaaagagc pETDuet F1 GCGCGAATTGATCTGccggcgtagaggatcgagatcgatctcg pETDuet R1 gctgctgcccatggtatatctccttcttaaag

pgi KF1 TACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCTAatgATTCCGGGGATCC

GTCGACC

pgi KR1 GCCTTATCCGGCCTACATATCGACGATGATTAACCGCGCCACGCTTTataTGTAGGCTGGAG

CTGCTTCG PGI-t-f1 tttacgattactccaagTAAacaaatcgctcttaaatatatacctaaagaacattaaagc PGI-t-r1 gatgatcttccgggggctttctcatgcgttcatgcaccactggaagatctGGTatactggaggcttcatgagttatgtcc PGK1-p-f1 taactcatgaagcctccagtataccACGCACAGATATTATAACATC PGK1-p-f1 taactcatgaagcctccagtataccACGCACAGATATTATAACATC PGK1-p-r1 acaatcaactatctcatatacaatgactgatgttgtgattgtaagcgctgc phaA F1 ataTCTAGAaaggagatatacatATGactgatgttgttatcgtttctgcggcccg phaA F2 aaggagatatacatATGactgatgttgtGATTGTAAGCGCTGCAcgtactgctgttggtaagttcggtggctccctggc phaA F4 gcctggcaagcctgGCAattggtggtggtatgggtgtagcactggctgtcg phaA F5 tcaataTCTAGAaaggagatatacatATGactgatgttgtGATTGTAAGCGCTGC

phaA F7 gatatacatatgCCACCGCCAGCTCTGCCACCGAAACGTCGTCGTGGCAGCGGCAGCGGCAGC

GGCAGCGGCatgactgatgttgtgattgtaagcgctg phaA R1 ctccttGAATTCttatttgcgttcgacagccagtgctacacccataccaccacca phaA R4 ataccaccaccaatTGCcaggcttgccaggccctttttagcatcacgg phaA Yeast-R4 gattggagacttgaccaaacctctggcgaagaattgttaattaagagctcTTATTTGCGTTCGACAGCCAGTGC phaA2 F1 atGGTCTCccatgActgatgttgtgattgtaagcgctgcacgtactgctgttggtaagttcgg phaA2 F2 cgGAGCTCaaggagatatacatatgactgatgttgtgattgtaagcgctgcacgtactgc phaA2 F2 cgGAGCTCaaggagatatacatatgactgatgttgtgattgtaagcgctgcacgtactgc

phaA-f2 tcgaGAATTCgcacaatatttcaagctataccaagcatacaatcaactatctcatatacaATGACTGATGTTGTGATTG

TAAGCGCTGCACG phaA-r2 GATAATATTTTTATATAATTATATTAATCttatttgcgttcgacagccagtgctacaccc phaB F1 gagatatacatATGacccagcgcatcgcttacgtaaccggtggcatggg phaB F3 gaattcTAGTAACTAAaaggagatatataatgacccagcgcatcgcttacgtaaccggtgg phaB F4 ttcggccagactaacTTTtctaccgcgaaagcaggtctgcacgg phaB QC F1 gcatcgcttacgtaaccggtggcatgGCTggtattggcaccgcaatctgtcagcgtctg phaB QC F2 gggttgcggtccgaactctccgGACcgtgaaaaatggctggaacagcaaaaagcgctgg phaB QC F3 gggttgcggtccgaactctccgGAAcgtgaaaaatggctggaacagcaaaaagcgctgg phaB QC R1 cagacgctgacagattgcggtgccaataccAGCcatgccaccggttacgtaagcgatgc phaB QC R2 ccagcgctttttgctgttccagccatttttcacgGTCcggagagttcggaccgcaaccc phaB QC R3 ccagcgctttttgctgttccagccatttttcacgTTCcggagagttcggaccgcaaccc phaB R1 aataGGCGCGCCGGATCCttagcccatgtgcaggccaccgttcaggg phaB R2 agaTCTAGAttagcccatgtgcaggccaccgttcaggg phaB R4 tgctttcgcggtagaAAAgttagtctggccgaattgacctttttggccgttcac phaJ R100 ATCTAGTTCCCGGGTTACGGCAGTTTGACCACGGCC phaJ SOE F1 GGAATTGTGAGCGATTCCACCAATAGGTCACTAATGAGCGCGCAGAGCCTGG phaJ SOE R1 TAGTGACCTATTGGTGGAATCGCTCACAATTCCACACATTATACGAGCCG PhaP1 F1 ttgtggacttcctggttaaataaatgaggaggtaaaaaATGattctgaccccggaacagg PhaP1 R1 atgagtttttgttcgggcccaagcttcccatggagTTAagctgccgtggtcttctttgcg PhaZ F4 cgtcttcacgctagcaaagaggagaaaagatctAattttagggaggtacccaATGctgtatcaattgcacgagtttcaac PhaZ R4 gcttcgtcattatctacctccttaatctgcttgtcgtctctgTTAgcgcgtcgcggacgc

poxB KF3 ATGAAACAAACGGTTGCAGCTTATATCGCCAAAACACTCGAATCGGCAGGgtgtaggctggagctg

cttc

poxB KR3 TTACCTTAGCCAGTTTGTTTTCGCCAGTTCGATCACTTCATCACCGCGTCcatatgaatatcctcctta

g pta KR2 TGTGCAGACTGAATCGCAGTCAGCGCGATGGTGTAGACGAcatatgaatatcctccttag pTet Scaff F1 GATTACAcctaggACTAGTGATCCGTTTCCATTTAGGTGGGTA pTet Scaff R2 ccatgggcagcagagatcttttctcctctttgctag pTO99a F1 ggttttttgatgcatttacgttgacaccatcgaatggtgc pTO99a F1 ggttttttgatgcatttacgttgacaccatcgaatggtgc pTO99a SOE R1 cctctacgccggCAGATCAATTCGCGCTAACTTACATTAATTGCGTTG pTrc99a F11 CCCTTCCGGCCGACTGCACGG pTrc99a F12 acttctgcgctcggcccttcCGGCCGACTGCACGG pTrc99a F4 atcGCCCTTCCGGCcgactgcacggtgcaccaatgcttctggcg pTrc99a F7 aattatgcATGCATttacgttgacaccatcgaatggtgcaaaacctttcgcgg pTrc99a R4 atcGCCCTTCCGGCaagagtttgtagaaacgcaaaaaggccatccgtcaggatggcc pTrc99a R6 cgGAGCTCgtgtgaaattgttatccgctcacaattccacacattatacgagccgg pTrc99a R8 ttagtgactttgcctgttacccaatcagCCATGGtctgtttcctgtgtgaaattgttatc pUC19 SacI expander F1 tACTAGTtgcGCGGCCGCttcCCTAGGtacCTCGAGtagtGAGCT pUC19 SacI expander R1 CactaCTCGAGgtaCCTAGGgaaGCGGCCGCgcaACTAGTaagct

202

rrABH F1 taacagagacgacaagcagattaaggaggtagataATGacgaagccgctgctgc rrABH R1 tccggggtcagaatcattttttacctcctcatTTAtttaaccaggaagtccacaatcggc rrABH R2 ttaacctgccttagggttatagatgaggttttTTAtttaaccaggaagtccacaatcggc rrAdpA F1 aaataaaaaacctcatctataaccctaaggcaggttaaaATGgccaaacaaccggaaacg rrAdpA R1 gatgagtttttgttcgggcccaagcttcccatggagTTActtctgggtcgtcgcagcacg rrnB SLIC F1 TAcatatgAGATCTctcgagCTCGAGGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCC rrnB SLIC R1 ttgaagcatttatcagggttattgtctcatgagcggatac rrPhaZ2 F1 cgtcttcacgctagcaaagaggagaaaagatctTtctttaataaggaggtttataATGctgtacaatctgcacgagatcc rrPhaZ2 R1 cttcgtcattatctacctccttaatctgcttgtcgtctctgTTAggcaactgccgcaccg sALDH593 F1 gagatatacatatgaacaaagataccctgatcccgactacgaaagacctgaaactg

sALDH593 F101 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatgaacaaagataccct

gatcccgactacgaaagacctgaaactg sALDH593 R1 atctctagatcagccggccagtacgcaacgacgttgac sBdhB F1 gagctcggcgcgccgaattcggatccaagg sBdhB F100 cgGGTACCaaggagatataccatggtggacttcgagtattctatcccaacccg sBdhB F101 atGAATTCaaggagatataccatggtggacttcgagtattctatcccaacccg

sBdhB F103 gatatacatATGGCAAGCTGGAGCCACCCGCAGTTCGAAAAGGGTGCAGGTatggtggacttcgagtatt

ctatcccaacccgtatctttttcggtaaag sBdhB R100 atcAAGCTTttaaacggatttcttgaaaatttgcagtacctcggaagcgttaaccggg sBdhB R101 cgGAGCTCttaaacggatttcttgaaaatttgcagtacctcggaagcgttaaccggg sBdhB R103 cgGGTACCttaaacggatttcttgaaaatttgcagtacctcggaagcgttaaccggg sBdhB R103 cgGGTACCttaaacggatttcttgaaaatttgcagtacctcggaagcgttaaccggg SH3 F1 CATATGCCACCGCCAGCTCTGC sPhaP1F104 ctgggcgatacgtcaaagccaaggcatgactcgagaggtaattaatgattctgaccccggaacaggtggc sPhaP1R104 aaaatcttctctcatccgccaaaacagccctcgagttaagctgccgtggtcttctttgcgg

STag F1 tcgacTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTC

GAACGCCAGCACATGGACAGCTAAa

STag R1 agcttTTAGCTGTCCATGTGCTGGCGTTCGAATTTAGCAGCAGCGGTTTCTTTCATACCAGAA

CCGCGTGGCACCAGACCAGAAGAg T(pdc) R1/SLIC R2 aagcttgcatgcctgcaggtcgactctagaGCGAGCGCAGCGACGCCTTTTG T(Pgi1) Yeast-R1 CATATGCTCACCCAGTCGCATGCCCGGGggtatactggaggcttcatgagttatgtcc T5 R1 cgGAGCTCtgtgtgaaattgttatccgctcacaattgaatctattataattgttatccgc T7 SR1 TATGCTAGTTATTGCTCAG TbACS4 F1 gtcagataCCatgggtggctgcgtaatctctgtg TbACS4 R1 gtatgctaGGATCCttattagcattcttctacgaacagagacacaatggtgtcgg TdTer F1 gagatatacatatgatcgtcaagccaatggtgcgc TdTER F1 gagatatacatatgatcgtcaagccaatggtgcgc tdTer F101 atGGTCTCccATGATCGTCAAGCCAATGGTGCGCAATAATATCTGTCTG TdTer F102 GGTCTGCAggcgccATGATCGTCAAGCCAATGGTGCGCAATAATATCTGTCTG

TdTer F104 ggatccatcgatgcttaggaggtcatATGCCACCGCCAGCTCTGCCACCGAAACGTCGTCGTGGTTCTG

GTTCTGGCAGCGGCAGCGGCatcgtcaagccaatggtgcgc TdTer F106 gatccatcgatgcttaggaggtcatATGccaccgccagctctgccaccgaaacgtcgtcgtATGggccatcatcatcatcatcat TdTer F108 gttccggtggtgtcaaagagagcctggtctaaGAAttcaagaaggagatataccatgaa tdTer R101 agcCTCGAGttaAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTer R101 agcCTCGAGttaAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTER R101 agcCTCGAGttaAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTer R102 ctccttgaattcTTAAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC tdTer R103 TAGTATCGggtaccTTAAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTer R103 TAGTATCGggtaccTTAAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTER R103 TAGTATCGggtaccTTAAATACGATCGAAACGTTCAACTTCTGCCTCGTAGTTAATACCC TdTer R106 actttcatggtatatctccttcttgaattcTTAaatacgatcgaaacgttcaacttctgc

TdTer R107/8 acagcttatcatcgataagcttggtaccTTAacgacgacgtttcggtggcagagctggcggtgggccggaaccgctgccagaacc

agaaccaaaagatttgatataaatgtctttcagct Ter QC F1 aggagggctgtattactctggcatattctGCcatcggcccggaggcgactcaggcactgt Ter QC F2 cgtgctttcgtgtccgttaacaagggcGCggCtacgcgcgcttccgcagtaattccggtc Ter QC F3 ttacgcgcgcttccgcagtaattccggtcGCtccgctgtacctggcttccctgtttaaag Ter QC F4 tccgcagtaattccggtcattccgctgtacGCggcttccctgtttaaagtcatgaaagaa Ter QC F5 cggtcattccgctgtacctggcttccctgGCtaaagtcatgaaagaaaaaggcaaccacg Ter QC F6 acgcggaatccctgacggatctggcaggtGCccgtcacgactttctggcgtctaatggtt Ter QC F7 tgtatcgtaagggcaccatcggtaaagcgGCagaacatctggaggccaccgctcaccgtc Ter QC F8 aggcactgtatcgtaagggcaccatcggtGCagcgaaagaacatctggaggccaccgctc Ter QC F9 catattcttacatcggcccggaggcgactGCggcactgtatcgtaagggcaccatcggta Ter QC R1 acagtgcctgagtcgcctccgggccgatgGCagaatatgccagagtaatacagccctcct Ter QC R2 gaccggaattactgcggaagcgcgcgtaGccGCgcccttgttaacggacacgaaagcacg Ter QC R3 ctttaaacagggaagccaggtacagcggaGCgaccggaattactgcggaagcgcgcgtaa Ter QC R4 ttctttcatgactttaaacagggaagccGCgtacagcggaatgaccggaattactgcgga

203

Ter QC R5 cgtggttgcctttttctttcatgactttaGCcagggaagccaggtacagcggaatgaccg Ter QC R6 aaccattagacgccagaaagtcgtgacggGCacctgccagatccgtcagggattccgcgt Ter QC R7 gacggtgagcggtggcctccagatgttctGCcgctttaccgatggtgcccttacgataca Ter QC R8 gagcggtggcctccagatgttctttcgctGCaccgatggtgcccttacgatacagtgcct Ter QC R9 taccgatggtgcccttacgatacagtgccGCagtcgcctccgggccgatgtaagaatatg

ter R105 ttcttgaattcttagaccaggctctctttgacaccaccggaaccgctaccggaaccagagccaatacgatcgaaacgttcaacttctgc

c ter SOE F1 caggcactgTTCcgtaagggcaccatcggtaaagcg ter SOE F2 ccgctgTTCctggcttccctgtttaaagtcatgaaagaaaaaggc ter SOE R1 gcccttacgGAAcagtgcctgagtcgcctcc ter SOE R2 cagggaagccagGAAcagcggaatgaccggaattactgcg Ter-SpeI R1 cgtACTAGTttaaatacgatcgaaacgttcaacttctgcc tesA F1 tacttctaAGATCTaaggagATATACTatggcagcggacacgttattgattctggg tesA R1 tacttctaGGTACCttattatgagtcatgatttactaaaggctgcaactgcttcg tesB KO F2 tcaactcactttggcttgctgcggcagctttgttactggagagttatATGgtgtaggctggagctgcttc tesB KO R2 aagcactgcaaaaaacagccggacggttttcacctccggctatttttTTAcatatgaatatcctccttag TetR F2/SLIC F2 aaatcgttaaCCCGGGaagaattctcatgtttgacagcttatcatcgataagctttaatg TetR/T(pdc) SOE F2 GGGCCACCTCGACCTGAtttttaaataaacttagagcttaaggcgaaaagcccg TetR/T(pdc) SOE R2 gctctaagtttatttaaaaaTCAGGTCGAGGTGGCCCGGCTCC tpi1-t-f1 CTGGCTGTCGAACGCAAATAAgattaatataattatataaaaatattatcttcttttctttatatctagtgttatg tpi1-t-r1 cagatgttataatatctgtgcgtCTATATAACAGTTGAAATTTGGATAAGAACATCTTCTCAACGCG TT33-NphT7 100K R2 GATAAATCCTCCTGTTGTGAAAAAGCCCGATgagctcgtgtgaaattgttatccgctcac TT33-NphT7 10K R2 CTGAAACCTATTCCTTGAGAGAAAACTTTGTTGTTgagctcgtgtgaaattgttatccgc TT33-NphT7 30K R2 ATTCTCCTCCTACGAATTATGAGCGATAAGGATGgagctcgtgtgaaattgttatccgct TT33-NphT7 His F1 aatttcacacgagctcaaggagatatacatATGggccatcatcatcatcatcatcatcat Ura F1 tcgtcagtaAGATCTtcatgtttgacagcttatcatcgataagcttttcaattc Ura R1 ttagtacgtAGATCTcgagattcccgggtaataactgatataattaaattgaagctc XmaI-adhE2 F1 gtaCCCGGGatgaaagtcacgaaccagaaggaactgaa YdbK F1 tagtgtcaaCATatgattactattgacggtaatggcgcggttgc YdbK F2 aagggaacaaaagctgGGTACCgtataagaaggagatatacatatgattactattgacgg YdbK R1 tactgtaGGTACCttaatcggtgttgcttttttccgcttttccgg YdbK R2 ctgtcaaacaagcttatcgataccGTCGACttaatcggtgttgcttttttccgcttttcc

204

Appendix 5: Oligonucleotides used for sequencing

205

Table A5.1. Oligonucleotides used for sequencing. Name Sequence 16S Eub F gcacaagcggtggagcatgtgg 16S F ccagcagccgcggtaatacg aadA SF1 cgaaggatgtcgctgccgactgggcaatggag AccA3 F101 cacagcagcggccatatcgaaggtcgtcatatggcaagccacgcaggtagccgtatcgccc AccA3 F19 atgctacgttgaacgttatttggataagccgcgccacg AccA3 F40 caataccatcgagccgttcaccgatggtgagccgctg AccA3 R101 tcgggctttgttagcagccggatcctcgagttatttaatctccgccaggaccgtgccttg AccA3 R40 gtgaacggctcgatggtattgttccactcggtctcgatcc accBC R15 cgctgctttaattgcgatcggcaggccgaactcctc accBC R33 ctaatctcgctgccctcgaccacgccgctgtcca accBC R49 agaacgacacctttattaacaccctcacccgccgcaaccg AccD4 F101 cacagcagcggccatatcgaaggtcgtcatatgacggttacggagccggttctgcacacg AccD4 F11 gtcgtaaagttgcacgtctgatggagtggtgtgcgat AccD4 F36 ttacgattcgcaagagctatggcggcgcgtacgctgt AccD4 R101 tcgggctttgttagcagccggatcctcgagttagaccgggatcagaccatgtttacgacc AccD6 F10 gccaggtgttcgaggcgatgatccgcgcttctggc AccD6 F101 cacagcagcggccatatcgaaggtcgtcatatgacgattatggcaccggaagcagtcggc AccD6 R101 tcgggctttgttagcagccggatcctcgagttacagcgggatgtttttatgacgaccacg accE F101 agcatggtaatatgccgctgtaattaaatagttagctataaagggaggggatcaagcatg AccE F101 agcatggtaatatgccgctgtaattaaatagttagctataaagggaggggatcaagcatg AccE R101 tgagtttttgttcgggcccaagcttttagaaaaagttcacgttctgaaacgcgctc aceBAK F1 tatttttaattaaaatggaaattgtttttgattttgcattttaaatgagtagtcttagtt aceBAK R1 ggctttacggataaccctcgatacattgcggagaaaaattatatggaagctttac aceE SF1 atgtcagaacgtttcccaaatgacgtgg aceE SF2 ctggataacctggtcttcgttatcaactg aceE SF3 cgtgctctgaacgtgatgctgaag aceE SF4 ggcgtaggttctgacgtttatagcg aceE SR1 cgatcagatactgagcacgctcaacaccttc aceF SF1 atggctatcgaaatcaaagtaccggac aceF SF2 tttgcaggcgtcgtgaaggaactg aceF SF3 aacggtctggttgttccggtattc aceF SR1 catttggtacatataagccatgatctgtatcagtagc ackA F1 atacccactatcaggtatcctttagcagcctgaaggcc Acs SF1 ctgaagcgtactggcgggaaaattg Acs SF2 cggcaacgagaaatgtccggtg adhB SF1 gaggaaagcctgatctgccattttgagcag adhB SF2 gtctttattttaatgttgtgcaatttatacagtatatttcgccatatacg adhB SF3 cctcctcaagctaccgcgacc adhB SF4 ctatttcggcagagcgggtggcggtag adhB SF5 gatgcagcgtttgagacaattgattagatc adhB SR1 cgataaggcacccaccggcggtgaaatcg adhB SR2 gaaatcaaactctgcattaggcatttacccgataaaacg adhB SR5 gcagaaatagaagtcgaactatgcgctgctcc adhB(Term) R1/SLIC R1 gcatgcctgcaggtcgactctagaggatccatttcgtcgccttcataaggcaaacgcggc adhE2 SF1 atgaaagtcacgaaccagaaggaac adhE2 SF1b atgaaagtcacgaaccagaaggaactgaagc adhE2 SF2 ccatcctggttatgaactccatctacga adhE2 SF2b gcgaacagtccatcctggttatgaactccatctacg adhE2 SF3 aagttctggatgaaattgacatcaag adhE2 SF3b ccaaagttctggatgaaattgacatcaagtactcc adhE2 SF4 acaacgctactgattgcccgac adhE2 SF4b cgctactgattgcccgactaaacagaccgcc adhE2 SR1 ttgcgcgttttcagggaaatcaggg adhE2 SR1b gcgttgcgcgttttcagggaaatcaggg adhE2 SR2 ccaccgatgctgatgatggtgtccgg aldh46 SF1 gaaaaggcgggtcgttccatc aldh46 SR1 caacagcaaaggcaacacac ALDH593 R23 gttgccagcacccgcgccaattgcctttttaccg ALDH593 R31 gattctacgtcgatttcgtcggagaacagtttagcgtc alkK F1 taacaagctagcgaattcgagctcggtacccaaaaaatattaaataaggaggtctacgatgctgggtcagatgatgcgtaatc alkK SF1 aggtgatgataactatatctggccggatg alkK SF2 gatctgagcccggacgaaaaactg

206

alkK SF3 catctatggtgttgaggtcatccatgc alkK SR1 gttcgataaacagacgcgggttg arcA F1 agatctagactcgagaaggagatatcatatgcagaccccgcacattcttatcgttgaagacgagttgg arcA R1 acgcgtcgacgagctcttaatcttccagatcaccgcagaagcgataaccttcaccgtgaatgg AtfA F18 cgttattcaggaactgtctcagacggtattcaaagacatcgg AtfA R22 gatgttacggaagcgatccaggtcgaagctctgagc atoB F1 cggagctcaaggagatatacatatgaaaaattgtgtcatcgtcagtgcggtacgtactgctatcgg atoB R1 gcgatgcgctgggtcattatatatctccttttagttactagaattcttaattcaaccgttcaatcaccatcgcaattccctgaccgccgcc atoB SF1 gcgatgtacttcagcgaacatggc atoB SR1 ctgcggcgggtaaagttcagcagg bcd F1 gagatatacatatggattttaatttaacaagagaacaagaattagtaagacagatggttagagaatttgc bcd SF1 gatggaggaagaattggtatagcagctcaagctttagg bcd SOE F1 gctacttaataagttaggagaataaacatggattttaatttaacaagagaacaagaattagtaagacagatgg bcd SR1 ccatatctgccatcatccatgcaagaccttgg bdhA R1 gataagcttttaatagcttttcttgaaaatttccagaacgtcctcggcgttga bdhA R15 cgttggtttccatattgctaataaccgcgatctgatcca birA F3 agttgataacaagctagcgaattcgagctcttatataagctttaaggaggtcgaagatgaaggataacaccgtgccactgaaattg birA R1 caggtcgactctagaggatccccgggtaccttatttttctgcactacgcagggatatttc birA R3 tctccgtgtactccttacctgtatttttgttttattatttttctgcactacgcagggatatttcaccgccc birA SF1 cgtgttaaatggcctaatgacctctatctgcagg birA SR1 ctctccgatacgatcaagaaggtactgattc btPhaZ R1 tatctacctccttaatctgcttgtcgtctctgttatttttgttccagccagtcttcaacg Bu2 SF1 atgacggtaaaagatattctggacgcg Bu2 SF2 ggtggcgctaacccgatctgcgtgg Bu2 SR1 tgtagttcacgctagaggccataac CaAdhE2 SF1 caaagacttatgacaatggagtaatatgcgcttctgaac CaAdhE2 SF2 ggatgtcttagatttgcattaaaagaattaaaagatatgaataagaaaagagcc CAT F1 gagatatacatatgggtaccgagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaac CCR (S. cinna) R20 accccaaatcagtacgttgtcgccctgtttcatgcctg CCR (S. cinna) R31 ttcgcgaagtgggagccgataatacgcttcagggacat ccr SF1 atgacggtaaaagatattctggacgcgattcagtcc ccr SF1b atgacggtaaaagatattctggacgcgattcagtcc ccr SF2 ggccggtggcgctaacccgatctgcgtgg ccr SF2b ggccggtggcgctaacccgatctgcgtgg ccr SR1 tgtagttcacgctagaggccataaccgcaacc ccr SR1b tgtagttcacgctagaggccataaccgcaacc CmR F1 ctgatgctagcgatgtccggcggtgcttttgccg CmR R1 ataccatgggaatttgctttcgaatttctgccattcatccgcttattatcac CmR SF1 atggagaaaaaaatcactggatataccaccgttgatatatccc CmR SF2 gttctttacgatgccattgggatatatcaacggtggtatatccagtg CmR/adhB(Term) SOE F1 tttcaaaacaggaaaacggttttccgtcctgtcttgattttcaagc CmR/adhB(Term) SOE R1 aggacggaaaaccgttttcctgttttgaaattacgccccgccctgccact Cra F1 aaggactcgagaaggagatatcatatgaaactggatgaaatcgctcggctgg Cra R1 acgcgtcgacgagctcttagctacggctgagcacgccg crt SF1 atggaactgaacaacgtgatcctggaaaaagaggg crt SF1b atggaactgaacaacgtgatcctggaaaaagaggg crt SF2 cgcgatgagctgcgacatccgtatcgc crt SF2b cgcgatgagctgcgacatccgtatcgc crt SOE F7 gccgtcgcggctccggtagcggtagcggcagcggtatggaactgaacaacgtgatcctgg crt SOE R7 accgctaccggagccgcgacggcgtttcggcggcagcgccggcggcggcatggtctgtttcctgtgtgaaattgttatccgc crt SR1 gcgatacggatgtcgcagctcat crt SR1b gcgatacggatgtcgcagctcatcgcg crt SR2 gcgatacggatgtcgcagctcatcgcg dtsR1 R100 cgagtcaggatccttacagcggcatattaccatgcttgc dtsR1 R16 gcacccataatcacggaaatctgcgggataacaccgctc dtsR1 R34 ccgtaggcgtacagcagtttcgcgccacgacgca etfB SF1 gcacatgtaacagttataagtatgggacctccacaagc etfB SOE F1 agcagctgcttatgttgtctcaaaattaaaagaagaacactatatttaagttaggaggg etfB SOE R1 tttgagacaacataagcagctgcttccttaacaggcttatcaataacttctccctgtcc EUB F933 (16S) gcacaagcggtggagcatgtgg eutESF1 gccgtcagtgaaaccggcatg eutESR1 caatcgcctggttgagcagc FabB SF2 ttggaaaaatagacatcgtcaaaatctcgggaaacaggtgtaccctcagc FabB SR2 tgcagcgcaaggcgaggagtatccccgtctcatctctctgg fadR F1 aaggactcgagaaggagatatcatatggtcattaaggcgcaaagcccggcgg fadR R1 aataaagcttgagctcttatcgcccctgaatggctaaatcacccgg

207

Fdx SOE F1 cggagcattactggtaaggatccaaggagatatcatgatgccaaagattgttattttgcctcatcagg FldA F1 ttagaattcaaggagattcatcgatggctatcactggcatctttttcggc FldB F1 ttagaattcaaggagattcatcgatgaatatgggtcttttttacggttccagcacctg focA SF1 cgcctgatgctcccttttaattaactgttttagcggaggat Fpr SOE R1 ctccttggatccttaccagtaatgctccgctgtcatgtggcccggtcggcg Fpr SR1 gcagaatcgataaataagggccaatcgc Fpr SR2 ccacctgcacttcatcgcctgg frdB SF1 gtatggcgcactccgcaatggcacgtaaagag frdC SR1 cgtcagaacgctttggatttggattaatcatctcaggctcc frdC SR1 cgtcagaacgctttggatttggattaatcatctcaggctcc Gal1 F1 agtacggattagaagccgccgagcgggtgacag Gal1 R agctagccgcggtaccaagc Gal10 F ggatggacgcaaagaagttta Gal10 R ggcgaagaattgttaattaag GFP SR1 gtagtgacaagtgttggccatggaacaggtagttttcc GFP SR2 ctgctagttgaacggatccatcttcaatgttgtggcg GND F1 gccttcagatggcacttaaaaagtacttttagatatttagccagaaaacg GND R1 atttcatgctctcagaattaacttaactgtgaatcatgatgtttttagcatcc GND SF1 gccttcagatggcacttaaaaagtacttttagatatttagccagaaaacg hbd F1 atgaattcaaggagatatataatgaaaaaggtttgcgttattggtgcgggtactatggg hbd F1 atgaattcaaggagatatataatgaaaaaggtttgcgttattggtgcgggtactatggg HBD F100 tgcgtatcgcatatgaaaaaggtttgcgttattggtgcgggtac hbd F100 tgcgtatcgcatatgaaaaaggtttgcgttattggtgcgggtac HBD F100 tgcgtatcgcatatgaaaaaggtttgcgttattggtgcgggtac hbd F100 tgcgtatcgcatatgaaaaaggtttgcgttattggtgcgggtac

hbd F101 agcactggctgtcgaacgcaaataacgactacgggtaattcggaacgacagtcgccccaaaatgaaaaaggtttgcgttattggtgcg

g HBD F14 tccggataaggtgattggtatgcacttctttaaccc HBD F18 atccggtagaagttgctgaagcaccgggtt hbd R101 ggcgtcgacccccatttgataatggggattcttg hbd SF1 gcacacgctgctgaaaaag hbd SOE F1 ggtgattggtatggttttctttaacccggcaccagttatgaaactg HBD SOE F2 ggtgattggtatgtacttctttaacccggcaccagttatgaaactg hbd SOE F2 ggtgattggtatgtacttctttaacccggcaccagttatgaaactg hbd SOE R1 gccgggttaaagaaaaccataccaatcaccttatccggacgtttgg hbd SOE R1 tctgtttcctgtgtgaaattacttggagtaatcgtaaaaacctttgccagatttacgacc HBD SOE R2 gccgggttaaagaagtacataccaatcaccttatccggacgtttgg hbd SR1 ataaagcttggtaccgacgtcctcgagttacttggagtaatcgtaaaaacc iclR F1 aataaagcttctcgagaaggagatatcatatggtcgcacccattcccgcgaaacgc iclR R1 aatatctagagagctctcagcgcattccaccgtacgccagcg Jfar F37 agccaaagaatccatcgtggaagctgatatgttctatttcgacc Jfar R5 acgtttggctgagaacgcagcactttttcaacgaagattt lac Iq F1 atacagacgtcgacaccatcgaatggtgcaaaacctttcgcggtatggc lac Iq R1 atacagacgtcctaactcacattaattgcgttgcgctcactgcccgc lac Iq R1 atacagacgtcctaactcacattaattgcgttgcgctcactgcccgc lacIq SF1 cgcctgctggggcaaaccagcg lacIq SR1 agcaatggcatcctggtcatccagcgg lacIq SR2 ccagtaacgttatacgatgtcgcagagtatgccgg lacIq SR3 cgggaaacggtctgataagagacaccggc lacUV5 SF1 gtgagcggataacaagagct ldhA F1 cagtaataacagcgcgagaacggctttatatttacccagc ldhA R1 ctggtcacgggcttaccgtttacgctttccagcac ldhA SF1 cagtaataacagcgcgagaacggctttatatttacccagc ldhA SR1 ctggtcacgggcttaccgtttacgctttccagcac lpd SF1 caaagacatcgttaaagtcttcaccaagcg lpd SR1 gccgtcttctttcgcttcaacgg lpdA SF1 ggcggcgttggcatgttattg lpdA SF2 gctgaagcaatttctggtaagaaagttgcagttgattac lysC SR1 cgttccagaatggcaaactggatgttgcggatagcgtcga M13 SF1 gttttcccagtcacgac M13 SR1 caggaaacagctatgac M13 SR2 aaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc M13 SR3 ccgctacagggcgcgtaaa matB SF1 ggaccggcgaggacgtgc matB SF2 accgaggacggcttcttccgc mdh F2 tgaacggtagggtatattgtc

208

NphT7 F100 cttgatcgtcatatgaccgacgttcgttttcgtatcattggcac

nphT7 GF1 aagcgcgcaattaaccctcactaaagggaacaaaagctgctcgagaatttaaggaggtaaaaacatgaccgacgttcgttttcgtatca

t nphT7 GR1 gtgagcgcgcgtaatacgactcactatagggcgaattgttaccactcgatcagcgcgaag NphT7 R10 tgcacacagcgttaacatcaaatgccgcggtgccggttg nphT7 SF1 ggttacgcactggtcattggtgc nphT7 SR1 cccgccggcacacgaat NphT7F9 gtgcaacatcatctgggcgcaaccggcaccgcggc NphT7-Ori F1 tgtttgacagcttatcatcgatgcatcgacggccagtgaatccgtaatcatggtcatagc P(adhB) F2/SLIC F2 aggaaacagctatgacatgattacgaattcggcaaaatcggtaaccacatctcaattattaaacaatacttc P(adhB)/adhE2 SOE R1 tggttcgtgactttcatagctataacctcaccctacatactagtttaaagcaacaacccg P(gap)/phaA SOE R1 aatcacaacatcagtcatgtttattctcctaacttattaagtagctactatattccatagctattttttaacgtg P(pdc) R1 ataggatccacgtcatttgggaaacgttctgacattgcttactccatatattcaaaacactatgtctgaatcagg P(pdc)/Pdh SOE R1 tttgggaaacgttctgacattttttaaataaacttagagcttaaggcgaaaagcccgtcc pACYC184 SF1 cgcgcagaccaaaacgatctcaaga pACYC184 SR1 agaagacagtcataagtgcggcga pACYCduet-1 SF1 cgactcactataggggaattgtgagcggataacaattcccctg pARO181 SR3 ggctacgtcttgctggcgttcg pBAD33 F1 gctatgccatagcatttttatccataagatta pBAD33 R1 caggcaaattctgttttatcagaccg pBAD33 SF1 gctatgccatagcatttttatccataagatta pBAD33 SR1 caggcaaattctgttttatcagaccg pCWOri F2 tatgatgcatgagctcactagtgctagcaggaggaattcaccatggtacccggggatcctctagagtcgacctgcaggcatgca pCWOri F7 cctgagagctcggcgcgcctgcaggtcgacgagttagctcactcattaggcacccc pCWOri SF1 cacacaggaaacaggatcgatccatcgatg pCWori SF1 cacacaggaaacaggatcgatccatcgatg pCWOri SR1 cgtcttcaagcagatctgaaaaaaaagcccgc pdc.ZMF5 aaattcgaattccccgccgccaccatggagatgagttatactgtcggtacc PDC1-p-r1 tccaggatcacgttgttcagttccattttgattgatttgactgtgttattttgcgtgagg pdc-ZM SF2 cgatttacaccccggaagaagctccggctaaaatcg pdhA SF1 atggcaaaggctaagaaacaaaaacctattgac pdhA SF2 gcaggaattcctggtattcaagttgatggtatgg pdhA SR1 cgtagtagttccctgctacgtgaccacg pdhB SF1 gtatttgacgaaatcgttggtcaaatggctcg pdhB SF2 gaaagatatcgaagcaaaagctagagaaatcgtcg pESCLeu F1 gtaccgaattctagagctcggtcgaccgctgcgctcggtcgttc pETDuet1-1 SF atgcgtccggcgtaga pETDuet1-1 SR tatatctccttcttatactta pETDuet1-2 SF tcgaacagaaagtaatcgtattgt pETDuet1-2 SR caaaaaacccctcaagacccg pflB SR1 cccactttcgtggagcctttattgtacgctttttactgtacgatttcagtc pflB SR1 cccactttcgtggagcctttattgtacgctttttactgtacgatttcagtc PGI-t-f1 tttacgattactccaagtaaacaaatcgctcttaaatatatacctaaagaacattaaagc PGK1-p-r1 acaatcaactatctcatatacaatgactgatgttgtgattgtaagcgctgc phaA SF1 atgactgatgttgttatcgtttctgc phaA SF1b atgactgatgttgttatcgtttctgc phaA SF2 cgatgaggagattgttccggttctg phaA SF2b cgatgaggagattgttccggttctgattccac phaA SR1 tcacgggaacccggcagaacatgcgg phaA SR1b tcacgggaacccggcagaacatgcggtgccgc phaA2 SF1 atgactgatgttgtgattgtaagcgctgc phaA2 SF1b atgactgatgttgtgattgtaagcgctgc phaB SF1 atgacccagcgcatcgcttacgtaaccg phaB SF1b atgacccagcgcatcgcttacgtaaccgg phaB SR1 ttagcccatgtgcaggccaccg phaB SR1b ccttggatccttagcccatgtgcaggccaccg phaC F1 gttaagtataagaaggagatatatatatggcgaccggcaaaggcgcggcag phaC R1 atgctactcgagtcatgccttggctttgacgtatcgc phaC SF2 gcatggcgcaccaacctcccatatcgctt phaC SF3 cggccaagaacaagcgcagccactggactaa phaCSF1 acgagagcgcgtttgaggtc PhaP1 F1 ttgtggacttcctggttaaataaatgaggaggtaaaaaatgattctgaccccggaacagg PhaP1 R1 atgagtttttgttcgggcccaagcttcccatggagttaagctgccgtggtcttctttgcg phaP1 SF1 atgattctgaccccggaacaggtggcggc PhaZ R1 gtgcaattgatacagcat PhaZ SF1 gcgtttcatctgagcgactacatctactatattcaag

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poxB F1 agtggtttccggtgaatatacggtgagcagcac poxB R1 gttcgcagtgactgagcagagcgaccaggt poxB SF1 agtggtttccggtgaatatacggtgagcagcac poxB SF2 gaactaaacttgttaccgttatcacattcaggagatggagaacc poxB SR1 gttcgcagtgactgagcagagcgaccaggt poxB SR2 ggcatgtccttattatgacgggaaatgccacccttt pPro SF2 caatgaaacgcggtgaaacattgc pPro18 SF3 ggcaattgtggcacacccc pPro18 SR2 ctgttttatcagaccgcttctgcg Pro SF1 gcgcaccgcaaagttaagaaaccgaatattg prp SF3 gggattacaggcaagcgaaaccgtg prpR SF1 gaaggggcgtttaccggctcg prpR SF2 gacgccatcatcgccgctggatc prpR SR1 cttccagcacccgcagcagcc prpR SR2 ggtaatgtagctacgttgatcgaggcgc pRSF Ori R1 agaccgattgattcatcatctcata pRSFOri F1 cggtaatacggttatccacagaat pta R1 tcaccaacgtatcgggcattgcccatcttc pTet SF1 gtagatcctctagagtcgactaagaaaccattattatcatgac pTet SR1 gatgatgatggtcgacggcgc pTrc99a R1 ccgagctcgaattcatatgtctgtttcctgtgtgaaattgttatccgctcacaattccacacattatacg pTrc99a SF1 gcgccgacatcataacggttctggc pTrc99a SF2 gcgccgacatcataacggttctggc pTrc99a SR1 aggtgggaccaccgcgctactgccgcc R-ECH F100 ggtctgcaggcgccatgagcgcgcagagcctgg R-ECH R100 atctagttcccgggttacggcagtttgaccacggcc red c2 gatcttccgtcacaggtagg red K1 cagtcatagccgaatagcct red k1 cagtcatagccgaatagcct red K2 cggtgccctgaatgaactgc red Kt cggccacagtcgatgaatcc rrABH F1 taacagagacgacaagcagattaaggaggtagataatgacgaagccgctgctgc rrABH R1 tccggggtcagaatcattttttacctcctcatttatttaaccaggaagtccacaatcggc rrAdpA F1 aaataaaaaacctcatctataaccctaaggcaggttaaaatggccaaacaaccggaaacg rrAdpA R1 gatgagtttttgttcgggcccaagcttcccatggagttacttctgggtcgtcgcagcacg rrnB SF1 tgttttggcggatgagagaagattttcagcc rrnB SF2 ctgttgtttgtcggtgaacgctct rrnB1 SF1 tgttttggcggatgagagaagattttcagcc rrPhaZ R1 cttcgtcattatctacctccttaatctgcttgtcgtctctgttaggcaactgccgcaccg rrPhaZ SF1 cgtcttcacgctagcaaagaggagaaaagatctttctttaataaggaggtttataatgctgtacaatctgcacgagatcc sALDH593 F28 tgttatcatcaacgaagaccaggtatctaaactgattgatctgg sALDH593 F8 ttaccgaaatccgtaaagctgctctggaaaacaaaga sALDH593 R11 ggtatatttggcaaccagttcgtgtttcagaatcttatcc sALDH593 R16 caacagcattgcccgctgcaatcataccgatggaa sALDH593 R17 acacatttcttcgcgccagggtgaccgttaaaca sALDH593 R30 tttgcccacccatttcttgttgatgaagtattcttgagt sALDH593 R31 gattctacgtcgatttcgtcggagaacagtttagcgtc sBdhB F3 cggatccaaggagatataccatggtggacttcgagtattc sBdhB R8 cagccagttcgtagaatttaatggagttcttttccagaatagac SH3 SF1 ccgccagctctgccaccgaaacgtcgt T(pdc)/TcR SOE F1 gggccacctcgacctgatgcttactccatatattcaaaacactatgtctgaatcaggatg t0 R1 ccgagcgttctgaacaaatccagatgg t0 SR1 ccgagcgttctgaacaaatccagatgg t5 SF1 ctcgagaaatcataaaaaatttatttgctttgtgagcgg T7 pol SF1 atgaacacgattaacatcgctaagaacgac T7 pol SF3 gtgtcgataaggttccgttccctgag T7 pol SF4 ctgctggctgctgaggtcaaagataagaa T7 pol SR1 caatcatcttagggagtagggtagtgatgagagg T7 SF1 taatacgactcactataggg T7 SR1 tatgctagttattgctcag T7pol SF2 ctggcgtagtaggtcaagactctgagacta TbACS4 F17 tattatctgcaacgctaaaaacgtgtccgtggttatcaaattcatgt TbACS4 F30 aatacaacccgtctctgctgattggtgttccgcgtatttatg TbACS4 F38 accgaaaccgtttgcgtaggcggcgttcagctgac TbACS4 R20 tcttcccaagaaaccaggtgacaggcttcctggtcaac TcR SR1 tcctgcattaggaagcagcc

210

TdTer F1 gagatatacatatgatcgtcaagccaatggtgcgc tdter F10 aaatacggcactccaggctggtacaacaacctggc TdTER F101 atggtctcccatgatcgtcaagccaatggtgcgcaataatatctgtctg TdTER F18 agctgaaggaaatctccgccgagccagctaacg TdTER F28 ttccctgtttaaagtcatgaaagaaaaaggcaaccacg TdTER F33 gctgatggaaaaagtgacgggcgaaaacgcggaa TdTer R101 agcctcgagttaaatacgatcgaaacgttcaacttctgcctcgtagttaataccc TdTER R2 tcgtggcttgctggac TdTer R21 ccagcagaccttccttggacagttgtttgatccaac TdTer R8 ccgaatgcagccgtaatgcggcttgccaga ter F1 gagatatacatatgatcgtcaagccaatggtgcgc Ter R103 tagtatcgggtaccttaaatacgatcgaaacgttcaacttctgcctcgtagttaataccc tesB F1 cgccgcactcaacagaatacgc tesB R1 ggactgccattctctatatcggttggtttg tesB SF1 cgccgcactcaacagaatacgc tesB SR1 ggactgccattctctatatcggttggtttg TetR/T(pdc) SOE F2 gggccacctcgacctgatttttaaataaacttagagcttaaggcgaaaagcccg TetR/T(Pdc) SOE F2 gggccacctcgacctgatttttaaataaacttagagcttaaggcgaaaagcccg TetR/T(pdc) SOE R2 gctctaagtttatttaaaaatcaggtcgaggtggcccggctcc U (16S) F1 ccagcagccgcggtaatacg U F ccagcagccgcggtaatacg U F1 16S ccagcagccgcggtaatacg U1 F ccagcagccgcggtaatacg U1 F (16S) ccagcagccgcggtaatacg YdbK SF1 cggtctatgaccatgttgaacaggcgatgaatg YdbK SF2 ccttcgagtgagcgaacagccg YdbK SF3 cactcagctattcgcgcaaaagtgg YdbK SR1 gcaccgccatgacgtcggaatg YdbK SR2 gccagtttgtacagcgtcgggatc yjbF R1 gaacaggcctgtaataacaggcagataagaatgagtgcaggtcgcttcac

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Date post:	25-Jul-2018
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Construction and characterization of an n-butanol...

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