New strategies to optimize the secretion capacity for ...

New strategies to optimize the secretion capacity

for heterologous proteins in Bacillus subtilis

Dissertation

zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule

Biowissenschaften der Ruhr-Universität Bochum

angefertigt am Institut für Molekulare Enzymtechnologie

vorgelegt von Ulf Brockmeier

aus Münster

Bochum 2006

Neue Strategien zur Optimierung der Sekretionsleistung für heterologe Proteine in Bacillus subtilis

Dissertation

zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule

Biowissenschaften der Ruhr-Universität Bochum

angefertigt am Institut für Molekulare Enzymtechnologie

vorgelegt von Ulf Brockmeier

aus Münster

Bochum 2006

Die vorliegende Arbeit wurde im Rahmen des Europäischen Graduiertenkollegs der

Ruhr-Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems:

Fundamentals and Biotechnological Applications angefertigt.

Referent: Prof. Dr. K.-E. Jäger Korreferent: Prof. Dr. A. J. M. Driessen Tag der mündlichen Prüfung:

Erklärung

Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen

Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel

verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um

fünf in Wort und Bild völlig übereinstimmende Exemplare.

Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten

und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.

Bochum, den

_____________________________________

(Unterschrift)

Danksagungen Herrn Prof. Dr. K.-E. Jäger danke ich für die Überlassung des interessanten wissenschaftlichen Themas, für das Interesse am Fortschritt meiner Arbeit, für zahlreiche konstruktive Diskussionen und die Gelegenheit, diese Arbeit frei und selbständig zu gestalten. I would like to thank Prof. Dr. A. J. M. Driessen for agreeing to co-supervise this thesis. Ich möchte mich für die Finanzierung meiner Forschungsarbeit im Rahmen des Europäischen Graduiertenkollegs EGC 795 und insbesondere bei den Organisatoren Herrn Prof. Dr. M. Rögner und Frau Dr. C. Wüllner für viele interessante Tagungen und Kurse bedanken. Vielen Dank auch an alle Mitglieder des Kollegs für nette Gesprächsrunden und die freundliche Atmosphäre während unserer Treffen. Bei Herrn Prof. Dr. R. Freudl sowie bei Herrn M. Caspers (IBT1, FZ Jülich) möchte ich mich für die erfolgreiche Zusammenarbeit und für die durchgeführten Pulse-Chase Experimente bedanken. Desweiteren danke ich Herrn Prof. Dr. T. Noll und Herrn Dr. A. Jockwer (ehemals IBT2, FZ Jülich) für die Herstellung der polyklonalen Antikörper gegen die Cutinase. Frau E. Knieps-Grünhagen (IBT2, FZ-Jülich) möchte ich für die Aufreinigung der Cutinase danken. Herrn Prof. Dr. J. Büchs und Herrn F. Kensy (Lehrstuhl für Bioverfahrenstechnik, RWTH Aachen) danke ich für die gute Zusammenarbeit und die durchgeführten Wuchs- und Expressionsversuche von B. subtilis in Mikrotiterplatten. Herrn Dr. T. Eggert gilt mein besonderer Dank für die exzellente wissenschaftliche und auch persönliche Unterstützung während meiner gesamten Promotionszeit. Vielen Dank auch für die aufmerksame Durchsicht dieses Manuskriptes. Für ihre große Hilfsbereitschaft sowie für die kritische Durchsicht des Manuskriptes möchte ich mich besonders herzlich bei Herrn Dr. C. Leggewie sowie Herrn M. Puls bedanken. Ich hoffe darauf, dass Freundschaften mit der Entfernung wachsen werden... Meiner langjährigen Arbeitskollegin und „Leidensgenossin“ Frau M. Wendorff möchte ich herzlich danken für die vielen netten Gespräche und aufmunternden Worte, die einen in stressigen Zeiten am Leben halten. Vielen Dank an alle hier nicht namentlich erwähnten Mitglieder meiner Arbeitsgruppe. Ich wünsche Euch für die Zukunft weiterhin ein so gutes Arbeitsklima, wie ich es während meiner Zeit am Forschungszentrum erleben durfte. Ich möchte mich darüber hinaus sehr herzlich bei allen Mitarbeitern und Kollegen des Instituts für Molekulare Enzymtechnologie für die freundschaftliche Atmosphäre bedanken. Ein ganz persönliches Dankeschön geht an meine Familie und meine Freunde. Ihr Rückhalt bedeutet mir sehr viel.

Für Ulla

..wenn der Nahanni fällt, hast Du gelebt.....

Publikationen Brockmeier, U., M. Caspers, R. Freudl, A. Jockwer, T. Noll and T. Eggert. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. Submitted for publication. Brockmeier, U., M. Wendorff and T. Eggert. 2006. Versatile expression and secretion vectors for Bacillus subtilis. Curr. Microbiol. 52:143-148.

Eggert, T., U. Brockmeier, M. J. Dröge, W. J. Quax, and K. E. Jaeger. 2003. Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH. FEMS Microbiol. Lett. 225:319-324.

Posterpräsentationen Brockmeier, U., K.-E. Jaeger and T. Eggert. 2004. Evolution of signal peptides to optimize heterologous secretion in Bacillus subtilis. International Congress on Biocatalysis- Biocat 2004, Hamburg, Germany. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2004. Improvement of the Bacillus

secretion machinery using Directed Evolution. Biospektrum special issue of VAAM Annual conference, Braunschweig, Germany, p. 116. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2003. Secretion of two lipolytic enzymes in Bacillus subtilis. Biospektrum special issue of VAAM Annual conference, Berlin, Germany, p. 77. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2003. Improvement of the Bacillus

secretion machinery using Directed Evolution, 1st combined GBB-EGC Symposium, 11.-12.09. 2003, Groningen, The Netherlands. Vorträge Brockmeier, U., T. Eggert and K.-E. Jaeger. 2002. Secretion pathways for biocatalysts in Bacillus subtilis. EGC-GBB Symposium, 22.11. 2002, Bochum, Germany. Brockmeier, U., K.-E. Jaeger and T. Eggert. 2005. Heterologous protein expression in Bacillus subtilis: Successful manipulation of the Sec pathway by directed evolution. Third combined GBB-EGC Symposium, 15.-16.09. 2005, Groningen, The Netherlands.

Contents

I

Contents

List of Figures……………………………………………………………………………….. III List of Tables………………………………………………………………………………… IV Abbreviations………………………………………………………………………………… V 1. Introduction………………………………………………………………………... 1 1.1 The necessity of protein translocation in cells…………………………………………… 1 1.2 Protein sorting in cellular traffic- The crucial role of signal peptides………….……….. 2 1.3 Signal peptide classification in B. subtillis ……………………………………………….. 4 1.4 The Sec pathway as the major export route……………………………….…………….. 6 1.5 Industrial use of Bacillus species…………………………………………….……………. 8 1.6 Export bottlenecks for foreign recombinant proteins in B. subtilis…………………….. 9 1.7 Strategies to bypass the limitations in protein production…………………….………… 11 1.8 Scope of this thesis…………………………………………………………………………. 14 2. Materials and Methods…………………………………………………………... 16 2.1 Chemicals and enzymes………………………………………………………….………... 16 2.2 Bacterial strains and plasmids…………………………………………………………….. 16 2.3 Oligonucleotides and PCR-primers………………………………………………………. 17 2.4 Culture media and growth conditions…………………………………………………….. 19 2.5 Storage of bacteria…………………………………………………………………………. 20 2.6 Isolation of nucleic acid…………………………………………………………………….. 20 2.7 Agarose gel electrophoresis………………………………………………………………. 20 2.8 In vitro recombination of DNA……………………………………………………………… 20 2.9 Transformations of bacteria………………………………………………………………... 21 2.9.1 Chemical transformation of E. coli………………………………………………………… 21 2.9.2 Electroporation of E.coli …………………………………………………………………… 21 2.9.3 Transformation of B. subtillis………………………………………………………………. 21 2.10 Polymerase chain reaction (PCR)……………………………………………….………... 22

2.10.1 Standard PCR................................................................................................………... 22 2.10.2 Site directed mutagenesis…………………………………………………………………. 22 2.10.3 Saturation mutagenesis……………………………………………………………………. 23 2.10.4 Random mutagenesis…………………………………………………………….………… 23 2.10.5 Cassette mutagenesis of the PPX-domain of secA…………………………………….. 23

2.11 Construction of integration plasmid pMCut………………………………………………. 24 2.12 DNA sequencing……………………………………………………………………………. 24 2.13 Enzyme assays……………………………………………………………………………… 25

2.13.1 Lipolytic plate assay………………………………………………………………………… 25 2.13.2 α-amylase activity plate assay…………………………………………………………….. 25 2.13.3 Lipolytic activity assay……………………………………………………………………… 25 2.13.4 Esterase activity assay……………………………………………………………………... 26 2.13.5 High throughput screening (HTS) assay for cutinase/EstCL1

secretion using a pipetting robot…………………………………………………………...

26 2.14 Determination of protein concentration…………………………………………………… 27 2.15 TCA precipitation of proteins………………………………………………………………. 27 2.16 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)………………………………… 27 2.17 Cell culture and production of cutinase-specific antibodies by hybridoma cells……… 28 2.18 Immunodetection of proteins………………………………………………………………. 28 2.19 Purification of overexpressed cutinase in E. coli………………………………………… 29 2.20 Pulse-chase protein labelling……………………………………………………………… 30 2.21 Cell fractioning of B. subtilis……………………………………………………………….. 31 2.22 Computational methods……………………………………………………………………. 31 3. Results……………………………………………………………………………… 32

3.1 New expression and secretion vectors for Bacillus subtilis……………………………. 32 3.1.1 Choice of expression host: B. subtillis versus E. coli……………………………………. 32 3.1.2 Construction of vector pBSMuL1 and pBSMuL2………………………………………… 33

Contents

II

3.1.3 Overexpression, secretion and purification of His-tagged cutinase in pBSMuL vectors………………………………………………………………………………………...

34

3.1.4 Stability of pBSMuL vectors in B. subtilis under non selective conditions……………. 36 3.2 In search of the most functional signal peptide for heterologous protein

secretion in B. subtillis………………………………………………………………………

38 3.2.1 Establishment of a high-throughput screening (HTS-) system to evaluate

secretion efficiency for heterologous proteins in B. subtillis…………………………….

38 3.2.1.1 Construction of a Bacillus vector system using cutinase as a secretion reporter……. 39 3.2.1.2 Analysis of growth differences in microtiter plates………………………………………. 41 3.2.1.3 Development of a HTS-process for detection of export efficiency using a

pipetting robot………………………………………………………………………………..

42 3.2.1.4 Validation of the automated HTS process in B. subtillis………………………………… 44

3.2.2 Library construction and high-throughput screening of all Sec-type signal sequences fused to cutinase……………………………………………………………….

45

3.2.3 The highest D-score does not necessarily predict the optimal signal peptide for secretion of the heterologous cutinase………………………………………………..

47

3.2.4 Translocation efficiency is not solely responsible for the varying export amounts of cutinase……………………………………………………………………………………

53

3.2.5 High level production of cutinase in B. subtilis using the best identified signal peptide………………………………………………………………………………………..

55

3.3 One-step optimization of the heterologous esterase EstCL1 for secretion using the SP-library………………………………………………………………………….

57

3.4 Directed Evolution of SP-AmyE to achieve high yield of cutinase export…………….. 59 3.4.1 Random mutagenesis of SP-AmyE did not lead to increased amount of exported

cutinase ………………………………………………………………………………………

60 3.4.2 Saturation mutagenesis of SP-AmyE revealed improved variants…………………….. 61

3.5 Coexpression and mutagenesis of B. subtilis secA to increase cutinase export…….. 67 3.5.1 Construction and characterization of marker strain B. subtilis Marc1…………………. 67 3.5.2 Co-overexpression of SecA improves cutinase secretion……………………………… 69 3.5.3 The precursor form of cutinase fused to SP-LipA is processed rapidly under

condition of SecA-overexpression…………………………………………………………

70 3.5.4 A SecA variant leading to eight-fold enhanced protein export………………………… 71 3.5.5 The improved export effect of SecA variant A23T, K809D was neutralised

by a further amino acid exchange H289Y in the PPX-domain………………………….

72 4. Discussion…………………………………………………………………………. 73 4.1 A new Bacillus vector series……………………………………………………………….. 74 4.2 A new secretion marker system for HTS processes in B. subtillis…………………….. 76 4.3 Improvement of heterologous protein secretion in B. subtilis by signal sequence

screening……………………………………………………………………………………..

77 4.4 Saturation mutagenesis as a useful tool for the characterization and optimization

of a signal peptide…………………………………………………………………………...

81 4.5 Improvement of heterologous protein secretion in B. subtilis by

co-overexpression of SecA…………………………………………………………………

85 4.6 High level secretion for any foreign protein in B. subtilis- prospects for a

general strategy……………………………………………………………………………...

88 5. Summary…………………………………………………………………………… 92 6. Zusammenfassung……………………………………………………………….. 94 7. References…………………………………………………………………………. 97 8. Appendix…………………………………………………………………………… 111

Contents

III

List of Figures

Fig. 1 Simplified model for the interaction of a signal peptide with a cell

membrane……………………………………………………………………………….

3 Fig 2 Classification of cleavable N-terminal signal peptides in B. subtillis……………… 5 Fig. 3 Schematic overview of the targeting of secretory proteins containing

a Sec-type signal peptide in B. subtilis……………………………………………….

7 Fig. 4 Plasmid map of the pBSMuL series………………………………………………….. 34 Fig. 5 Purification of His-Tag cutinase from B. subtillis culture supernatant…………….. 35 Fig. 6 Expression and secretion efficiency of cutinase in B. subtillis using

vectors pBSMuL1,2……………………………………………………………………..

36 Fig. 7 Stability of pBSMuL vectors in B. subtillis …………………………………………... 37 Fig. 8 Schematic overview about the hydrolysis-reaction which the assay

is based on…………………………………………...................................................

39 Fig. 9 Plasmid map of shuttle vector pBSMuL3……………………………………………. 40 Fig. 10 Growth of B. subtillis in DeepWell microtiter plates………………………………… 41 Fig. 11 The pipetting robot “TECAN workstation Genesis 200 Freedom”………………… 42 Fig. 12 Two different Software scripts providing the automated pipetting process………. 43 Fig. 13 Reproducibility and stability study of cutinase activity of B. subtillis

supernatant in the HTS …………………………………………………....................

44 Fig. 14 Fluctuations in cutinase activity due to transformation events…………………….. 45 Fig. 15 Schematic overview of amplification, cloning and screening of all

Sec-type signal peptides of B. subtillis ………………………………………………

46 Fig. 16 Overall composition of signal peptides fused to mature cutinase

and its computer analysis exercising SignalP 3.0…………………………………..

48 Fig. 17 Comparison of all 148 screened signal peptides used for export

of the heterologous cutinase in B. subtillis ………………………………………….. 49

Fig. 18 Immunodetection of cutinase in B. subtillis expression strain……………………... 50 Fig. 19 Analysis of the processing kinetics of cutinase precursor proteins

with different SPs in B. subtillis via pulse-chase experiment………………………

54 Fig. 20 Cutinase production by B. subtillis via secretion into the medium… 56 Fig. 21 Identification of the most efficient signal peptide in secretion

of the heterologous esterase EstCL1 in B. subtillis ………………………………...

58 Fig. 22 Composition of hybrid protein SP-AmyE-cutinase………………………………….. 60 Fig. 23 Screening results after saturation mutagenesis generated

in the C-region and +1 position of SP-AmyE fused to cutinase…………………..

63 Fig. 24 Processing kinetics of cutinase precursor protein with SP-AmyE

in B. subtillis …………………………………………………………………………….

64 Fig. 25 Screening results of saturation mutagenesis libraries generated in

the N-region of the SP-AmyE fused to cutinase……………………………………..

65 Fig. 26 Comparative analysis of export efficiency and calculated D-score for

chosen variants of SP-AmyE with amino acid exchanges in the N-region……….

66 Fig. 27 Construction and characterization of B. subtillis secretion

reporter strain MArc1…………………………………………………………………...

68 Fig. 28 The effect of overexpressed SecA for cutinase export in B.subtillis

marker strain Marc1…………………………………………………………………….

69 Fig. 29 Processing of precursor protein cutinase fused to SP-LipA in different

B. subtilis Marc1 strains………………………………………………………………..

70 Fig. 30 The effect of two B. subtilis SecA variants for the export level of cutinase………. 71 Fig. 31 Localisation of amino acid substitutions in the protein structure of

B. subtilis SecA that effected cutinase secretion …………………………………...

87 Fig. 32 Comparison of cutinase expressing B. subtillis strains in LB- and TB-medium

using the on-line light scattering measurement technique for shaken MTPs…….

89 Fig. 33 Idealized model of a general strategy to optimize secretion yields of

heterologous proteins…………………………………………………………………..

91 Fig. 34 Cassette mutagenesis to generate a library of the PPX-domain of SecA……….. 116 Fig. 35 Overview of the complete Gemini pipetting

Script Ulf_PnPP1_10_tiptouch_bis_10PL……………………………………………

116

Contents

IV

List of Tables Tab. 1 Various destinations of exoproteins and their export pathways

in B. subtilis……………………………………………………………………………...

4 Tab. 2 Protein production of commercial interest in different Bacillus species………….. 9 Tab. 3 Performed or planned strategies to overcome export bottlenecks

in B. subtilis……………………………………………………………………………...

14 Tab. 4 Bacterial strains and plasmids used in this study…………………………………… 16 Tab. 5 Oligonucleotides used in this study…………………………………………………... 18 Tab. 6 Relevant antibiotics used for growth selection……………………………………… 19 Tab. 7 Comparison of chosen screened signal peptides used for cutinase export……… 51 Tab. 8 Signal peptides not tolerated in B. subtillis when constitutively

expressed in front of heterologous cutinase………………………………………....

52 Tab. 9 Positions of SP-AmyE chosen for saturation mutagenesis………………………... 61 Tab. 10 Base composition in oligonucleotides mix to saturate the C-region

of SP-AmyE……………………………………………………………………………...

62 Tab. 11 Properties and applications of the pBSMuL vector series…………………………. 74 Tab. 12 Oligonucleotides used for PCR-amplification of all Sec-dependent

signal sequences from genomic DNA of B.subtilis 168……………………………

111 Tab. 13 Comparison of all screened signal sequences used for export of

heterologous cutinase in B. subtilis…………………………………………………...

113

Contents

V

Abbreviations

A Ampere aa Amino acid A. dest Distilled water Amp Ampicillin ATP Adenosine 5`-triphosphate bp Base pair BSA Bovine serum albumin °C Degree Celsius Cm Chloramphenicol CM Cell membrane Da Dalton DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid dATP Desoxyadenosine-5`-

triphosphate dCTP Desoxycytosin-5`-

triphosphate dGTP Desoxyguanosin-5`-

triphosphate dTTP Desoxythymidin-5`-triphosphate dNTP Desoxyribonucleoside

triphosphate DW-MTP Deepwell microtiter plate EDTA Ethylendiamine tetra acetic acid eP-PCR Error-prone PCR EtOH Ethanol Ffh Fifty four homologoue Fig Figure g Gram GRAS Generally recognized as safe h hour HTS High-throughput screening IPTG Isopropyl-β-D-thiogalactoside k Kilo kb Kilobases kDa Kilodalton Km Kanamycin L Litre M Molarity (mol/L) m Mili µ Micro MCS Multiple cloning site min Minutes n Nano NCBI National Center for

Biotechnology Information OD Optical density ON Over night orf Open reading frame ori Origin of replication p.A. Per Analyse

PAGE Polyacrylamid gel electrophoresis PCR Polymerase chain reaction PDB Protein database bank pNPC para-nitrophenyl-caproat pNPP para-nitrophenyl-palmitate pI Isolelectric points of proteins rbs Ribosome binding site

RNA Ribonucleic acid rpm Rounds per minute RT Room temperature scRNA Small cytoplasmic RNA sec Seconds SDS Sodium dodecyl sulfate SP(s) Signal peptide(s) SRP Signal recognition particle ss Signal sequence t time Tab. Table TBE Tris-Borat-EDTA TCA Trichlor acetic acid Tm Melting temperature TM Transmembrane segment Tris Tris(hydroxyl methyl) amino

methane U Units V Volt v/v Volume per volume w/v Weight per volume

Introduction

1

1. Introduction

1.1 The necessity of protein translocation in cells

The permanent adaptation is obligatory for the survival of living cells in a

continuously changing environment. Therefore the whole cell metabolism must be

strictly regulated to avoid a waste of energy resources. Cell membranes (CM) serve

as protective barriers to maintain composition and concentration of cytoplasmic

molecules, on the other hand they must allow and control the transfer of molecules

and proteins from and to the environment to enable essential events like energy

supply (e.g. chemical gradients or nutrition mobilization and uptake), cell division, cell

communication, enemy defence and DNA uptake. This dilemma is solved by a

membrane composition of amphiphilic lipids and transmembranel proteins. The lipids

form a flexible but nearly impermeable structure whereas proteins can build

membrane channels for regulated molecule and enzyme transport. While eukaryotic

cells contain a complex network of membranes separating different organelles,

bacteria belong to the prokaryotic organisms that show only a single cell

compartment surrounded by one or two membranes.

Nevertheless, protein transfer from the site of synthesis, the cytoplasm, to other cell

compartments or the exterior is a quite complex process even in bacteria (151, 178).

Due to the extended use of microorganisms in commercial production processes

many efforts have been undertaken to decipher their secretome. But despite the

increasing knowledge of bacterial protein export at molecular level continuously

documented by numerous publications over the last decades (33, 47, 62, 142, 144,

151, 167, 178, 188, 200, 220) there are still many secrets of the bacterial secretome

left behind building a platform for extensive future research studies.

Introduction

2

1.2 Protein sorting in cellular traffic- The crucial role of signal peptides

Proteins destined for the transport and secretion across the cytoplasmic membrane

in pro- and eukaryotes are synthesized as preproteins containing a similar N-terminal

amino acid extension, the so-called signal peptide (SP). The domain organization of

signal sequences shows a common pattern (Fig. 2). They consist of a positively

charged amino terminus (N-region) showing high affinity for phospolipids of CMs, a

central hydrophobic core (H-region) of certain length (10-12 residues) enough to

span a hydrophobic region of 35 Å in a β structure, and a more polar carboxyl-

terminal end (23, 58, 59, 173, 202) recognized by a signal peptidase. Each region

was shown to be essential for a functioning SP.

However, based on the primary structure of a SP its functionality is hardly predictable

except the change of essential parts or residues like the hydrophobic core or the

cleavage recognition site (59, 225). Several studies focussed on the conformation

and orientation of isolated SPs interacting with monophospolipids (22, 26, 34, 59,

123). In summary, a conformational change of a SP was found depending on the

environmental conditions: Most SPs form a random structure in aqueous solution,

adopt a β structure after interaction with a lipid phase without insertion, but prefer an

α-helical conformation after insertion into a lipid phase. The structural change of a SP

into an α-helix was proposed to result in the further insertion of 10 to 15 residues of

the mature exoprotein into the membrane as shown in Fig. 1.

Introduction

3

These findings demonstrate that the conformational flexibility is an important feature

of a SP to support the insertion of a protein into a CM. Furthermore, the SP and its

secondary structure influences the folding and therefore the targeting of a secretory

protein on the whole way to the CM (52, 145, 154).

During the targeting process along the general secretion pathway (Sec pathway) of

bacteria (151) or into the lumen of the endoplasmatic reticulum (ER) in most

eukaryotes (97) SPs interact with several components of the cell: (i) Binding to the

signal recognition particle (SRP) (127) or other cytoplasmic factors (see Fig. 3) acting

as intracellular chaperones. (ii) Binding to the membrane. (iii) Initiation and facilitation

of translocation. (iv) Recognition and cleavage by a signal peptidase (SPase) at the

trans-site of the membrane that are highly conserved processes as shown for

archaeal and eukaryotes SPs fully functional in bacteria (59, 149, 183). Due to the

Fig. 1: Simplified model for the interaction of a signal peptide with a cell membrane. A SP contains a positively charged N-region (N, highlighted in blue), a central hydrophobic region (H, marked in black) and the cleavage site recognized by a signal peptidase I (C, highlighted in red). The N-terminal SP is fused to the target protein (T) forming the precursor protein. (A) The SP is unstructured in the aqueous cytoplasm. As the initial step the positively charged N-region of a SP interacts with the negatively charged phospholipids of the membrane. The high tendency of the SP to insert into the membrane is indicated by black arrows. (B) While the N-region stays “fixed” at the cytoplasmic site of the membrane, the hydrophobic part of the SP continuously inserts into the membrane thereby forming an α-helical structure. (C) Due to this “threading” process the N-terminal part of the mature target protein is pulled through the membrane. The exposed C-region of the SP at the trans-site of the membrane is recognized and cleaved by a signal peptidase I (coloured in green). For clarity cytoplasmic targeting factors (e.g. SRP, SecA, SecB) or other components essential for protein secretion are not considered in this model.

A B

T

++N

CH

C

cytoplasm

extracellular

cell membrane

++N

T

T

++N

C

H

SPase I

H

C

Introduction

4

involvement in all stages of protein export from synthesis until membrane crossing,

SPs can be regarded as zip codes essential for the selective protein targeting.

Stimulated by the relevance of SPs for protein export in microorganisms, prediction

tools have been developed (58) with SignalP being the most popular and user-

friendly program, which is freely accessible via the world wide web

(http://www.cbs.dtu.dk/service/SignalP/) (9, 135). The SignalP 3.0 server is able to

predict the likelihood of a particular amino acid sequence to act as a signal peptide or

not by calculating a discrimination score termed D-score. Sequences leading to

values above 0.5 are classified as signal peptides. Sequences showing calculated D-

scores of > 0.7 are signal peptides with the highest probability.

1.3 Signal peptide classification in B. subtilis

B. subtilis, a Gram-positive endospore forming soil bacterium, is ubiquitous and

developed to a paradigm for Gram-positive bacteria due to intensive research over

the last decades. Even if B. subtilis shows a relatively simple cell organisation, non-

cytoplasmic proteins can be addressed to various destinations via different pathways

depending on the presence or absence of a SP and/or retention signals (Table 1).

Table 1: Various destinations of exoproteins and their export pathways in B. subtilis. Abbreviations are TM, transmembran segments; CWB, cell wall binding repeats.

destination of

exoprotein signal peptide retention signal export pathway

Sec-type - Sec pathway

Tat-dependent - Tat pathway (90) extracellular

medium leader peptide - ABC transporters (119)

pseudopilin-SP TM Com pathway (30, 31)

Sec-type TM trans-site of

membrane Sec-type lipobox

cell wall Sec-type CWB

Sec pathway

Introduction

5

In contrast to Gram-negative bacteria secretory proteins have to overcome only one

CM in B. subtilis before being released into the extracellular medium. By using the

SignalP prediction tool, Tjalsma et al. were able to predict essentially all secreted

proteins in the Gram-positive bacterium Bacillus subtilis (188). The classification of

Fig. 2: Classification of cleavable N-terminal signal peptides in B. subtilis modified according to Tjalsma et al. (187, 188). The predicted SPs were divided into five distinct classes on the basis of SPase cleavage site and the export pathways via which the preproteins are exported. Amino acids (aa) are shown in the one-letter code. “X” is defined as any aa. The N-, the H- and the C-regions (if known) of a SP are distinguished by blue, white and red coloured boxes, respectively. The N-terminal part of the mature protein is indicated as a grey coloured open box. SP cleavage sites are indicated by a black arrow. (A) Sec-type SPs, precursors are targeted across the CM by the general secretion pathway. The cleavage site is recognized by one of the five type I SPase. (B) Sec-type lipoprotein SPs contain a so-called lipobox with the consensus motif L-X-X-C. After transport across the CM via the Sec pathway, the Cys of the lipobox is modified by lipids, before the type II SPase cleaves the peptide bond in front of the Cys which is afterwards attached to an additional acyl group serving as a membrane anchor in the CM. (C) The proteins ComGC, ComGE, ComGD and ComGG are required for DNA binding and uptake during competence and contain prepilin-like SPs. These are targeted by the Com pathway completely bypassing the Sec pathway. Since the C-region is located next to the N-region, the H-region stays fused to the mature protein after cleavage by ComC, a Com specific SPase. (D) N-terminal SP of bacteriocins and pheromones are often called leader peptides and lack a H-region. So far three secretory proteins, the bacteriocins sublancin 168, subtilosin and the pheromone ComX were identified. The leader peptides prevent premature activity and provide secretion via ABC transporters. (E) Several exoproteins were identified containing a Sec-type SP with the consensus motif R-R-X-#-# indicating the potential to be secreted by the Tat pathway. So far, only two proteins, PhoD and YwbN, showed a Sec-independent export by the Tat machinery.

Sec-type signal peptidesA

N H

++ AXA A

SPase I

C +1

C +1

Sec-type lipoprotein signal peptidesB

++ LXX C

SPase II

HN

++ R-R-X-X- # - # AXA

C +1

SPase I

N H

Twin-arginine signal peptidesE

Prepilin-like signal peptidesC

EF

ComC

H N C +1

KG

D Bacteriocin and pheromone signal peptide

SunT, AlbE/F

N C +1

Sec pathway

Com pathway

ABC transporter

Tat pathway

Introduction

6

SPs into five distinct groups is based on the export pathway and the SPase cleavage

sites as shown in Fig. 2. Regarding the relevance of each SP class for the complete

secretome in the following this study will focus only on the Sec-type SPs recognized

and targeted by the general secretion pathway which is described in detail in the next

chapter.

1.4 The Sec pathway as the major export route

Next to secretion by ABC transporters or via the Tat pathway, the majority of the

predicted ~300 potential secretory proteins in B. subtilis use the Sec pathway,

therefore called the general secretion pathway (188, 195), even if a proteomic

analysis of B. subtilis culture supernatant showed a discrepancy between genome-

based prediction and the experimentally detected secretome: For several proteins no

secretion was predicted due to a lack of a SP or cell retention signals. Surprisingly,

these proteins were found in the growth medium instead of at the predicted sites in

the cytoplasm or bound to the CM. These results imply the presence of additional

mechanisms for protein release into the extracellular medium like protein shedding

(proteolysis or inefficient retention) for membrane proteins and lipoproteins (5). The

occurrence of cell wall-bound proteins in the extracellular medium might be a result

of cell wall turnover, cell lysis or even a so far unknown secretion pathway as

discussed for protein release lacking a SP (187).

However, this study focusses on the Sec pathway in B. subtilis, the main road for

export proteins, which is most likely co-translational, since no clear evidence of a

SecB homologon or analogon to E. coli was described (187, 220). SecA, the

preprotein translocase ATPase, is the central motor protein of the Sec pathway,

interacting with most of the contributing components like acidic phospolipids of the

CM, the SecYEG proteins which form the membrane pore, intracellular chaperones,

the signal peptide as well as the mature protein of a precursor (44, 126, 144, 205).

The translocation process is driven by the SecA protein through multiple rounds of

ATP binding and hydrolysis. SecA alternates between “the inserted” and “deinserted”

state, resulting in processive translocation along the precursor substrate and export

of the translocating protein across the membrane in steps of 20-30 amino acids (175,

192).

Introduction

7

Whether SecA functions as a monomer or a dimer during the translocation process

has been the subject of considerable controversy (40, 42, 76, 89). Depending on

--- ---

----

- -

scRNA

HBsu

Ffh

SP

ribosome

mRNA

cytoplasm

cell membrane

cell wall

extracellular

FtsY

Sec

A

FtsY

SecA

Sip

SecYEG

SecD

F

Sp

pA

TepA

PrsA

BdbB/C

Yq

jG

Sp

oIIIJ

proteases

mature exoprotein

--- ---

----

- - --- ---

----

- ---- ---

----

- -

--- ---

----

- ---- ---

----

- -WprACa2+ Ca2+

Fe3+ Fe3+

Fe3+

Ca2+

1 2

3

4

Fig 3: Schematic overview of the targeting of secretory proteins containing a Sec-type signal peptide in B. subtilis . The presented model summarizes findings reported in various publications (131, 136, 181, 187-189, 195, 220, 221, 227) and does not claims for completeness. (1) Ribosomally synthesized exoproteins are directly bound at the exposed N-terminal SP by the Ffh protein to guarantee a translocation-competent state in an (partially) unfolded conformation. Ffh is next to HBsu and scRNA part of the signal recognition particle (SRP)-complex providing co-translational translocation. (2) The SRP-precursor complex interacts with the SecA protein via binding to the SP. (3) The SRP-precursor-SecA complex is targeted to the membrane embedded translocase consisting of the pore forming proteins SECY, SecE, SecG and accessory proteins SecDF, SpoIIIJ and YqjG. After cleavage of the SP at the trans-site of the membrane by one of the SPases I (SipS-W), the precursor protein is pushed step by step through the Sec-pore by SecA energized mainly by ATP and supported by the proton motif force. The FtsY protein can bind to Ffh and is required for release of the SRP from the membrane to ensure the next targeting cycle. (4) Whereas the SP is degraded by one of the two SPPases SppA and TepA, in the late stage of secretion the processed exoprotein must pass through cell wall thereby undergoing quality control by proteases like the cell wall associated WprA and folding to its native conformation supported by the extracellular folding catalyst PrsA, a lipoprotein anchored into the CM. Extracytoplasmic thiol-disulfide oxidoreductases like BdbB and BdbC provide disulfide bond formation if required. The negatively charged cell wall environment harbours several proteases and huge amounts of Ca2+- and Fe3+-ions, which are necessary to stabilize some exoproteins.

Introduction

8

temperature and salt concentration, the occurrence of both, SecA monomers and -

dimers in solution, was reported (212).

Interestingly, even if the common assumption is that SecA is active as a dimer, a

recent study reported the full functionality of only monomeric SecA for the cell (138).

The complete process can be subdivided into the early stage of secretion comprising

the targeting to the membrane by several intracellular chaperones including SecA (1-

2), the actual translocation process across the membrane (3) and the late stage of

secretion including extracellular folding, degradation and release of the target protein

into the environment (4). A more detailed description is given in figure 3.

1.5 Industrial use of Bacillus species

Long before useful genetic techniques for strain improvement were developed,

B. subtilis and its relatives were used in traditional fermentation processes (63). An

important feature of this diverse group of organisms is a tremendous protein export

capacity up to 25 g/L for homologous proteins (81, 82) allowing a direct enzyme

transport into the cell-surrounding environment avoiding intracellular accumulation.

The phenomenon of protein accumulation occurs during protein overexpression

especially in Gram-negative bacteria in the cytoplasm as well as in the periplasm and

can lead to toxicity problems to the cells depending on the kind of product (8). Yet, as

another positive effect of the protein export, the genus Bacillus bypasses the

reducing conditions in the cytoplasm that might support formation of misfolded

proteins and inclusion bodies (124). The secretion is a natural separation step

simplifying further purification processes of the product. In contrast to Gram negative

bacteria, B. subtilis and other representatives like Bacillus licheniformis or Bacillus

amyloliquefaciens are classified as endotoxin free microorganisms by the Food and

Drug Administration and therefore generally recognized as save (GRAS) what makes

them preferred candidates for commercial applications especially in the food industry.

Additionally, Bacillus species show easy culturing conditions and high cell growth

during fermentation processes. In consequence since the early 60es several Bacillus

spp. were used and further improved for the fermentative production of primary

homologous hydrolytic enzymes. In 2004, the world market for industrial enzymes

was estimated to be 1,6 billion $US with increasing tendency. Between 50% and

Introduction

9

60% of the commercially available enzymes are produced by Bacillus species (172).

Two of the most important bulk enzymes are alkaline proteases (subtilisins) used in

household detergents and amylases used in industrial applications (143). Table 2

gives an overview of homologous and heterologous protein production in various

Bacillus strains with respect to industrial applications.

Table 2: Protein production of commercial interest in different Bacillus species. In the case of B. subtilis as the production host the corresponding column is shaded in grey.

1.6 Export bottlenecks for foreign recombinant proteins in B. subtilis

Despite this dominant position in industrial enzyme production it is remarkable that

E. coli is still preferred over Bacillus species for the commercial therapeutic

production (160). Although a variety of heterologous proteins could be successfully

produced in Bacillus species on laboratory scale (see also Table 2), there still exists

product application origin production

host reference

alkaline protease detergents B. subtilis/licheniformis B. subtilis/

licheniformis (155)

alkaline amylase detergents B. licheniformis B. licheniformis (172) α-amylase starch degradation B. licheniformis B. licheniformis (172) pullulanase starch degradation B. halodurans B. halodurans (129) glucose isomerase starch processing B. coagulans B. coagulans (172)

CGTase starch conversion to cyclodextrins

B. firmus B. firmus (57)

β-glucanase glucan modification B. subtilis B. subtilis (17) xylanase food processing B. subtilis B. subtilis (103) cellulase cellulose degradation B. subtilis B. subtilis (75) chitinase chitin degradation B. thuringiensis B. thuringiensis (185)

levansucrase Hydrolase/transferase activity for

B. circulans B. circulans (140)

esterase lipolytic degradation B. circulans B. circulans (91, 92) growth hormon medicin human B. subtilis (64) interleukin-1beta medicin human B. subtilis (172) proinsulin medicin human B. subtilis (172) penicillin G acylase medicin B. megaterium B. subtilis (222) cyclic oligopeptides antibiotic B. licheniformis B. licheniformis (95) basic peptides antibiotic Brevibacillus brevis Brevibacillus brevis (150) endotoxin biodegradable insecticide B. thuringiensis B. thuringiensis (172) purine nucleotides medicin, flavour enhancer B. subtilis B. subtilis (172) riboflavin vitamin ingredient B. subtilis B. subtilis (37)

D-ribose flavor enhancer B. subtilis, B. pumilus B. subtilis,

B. pumilus (39)

2-acetyl-1-pyrroline popcorn flavour B. cereus B. cereus (162) polyhydroxybutyrate biodegradable plastics bacterial B. megaterium (74) streptavidin biotin-binding protein Streptomyces species B. subtilis (214) antigen displaying spore oral vaccination tetanus toxin fragment C B. subtilis (45)

Introduction

10

a clear reservation to use Bacillus in their industrial applications. Even for the model

organism of Gram positive bacteria B. subtilis, whose complete genome is available

since 1997 (104) and diverse DNA recombination methods led to improved

production strains over the last years, several bottlenecks still limit the universal

application of B. subtilis for heterologous protein production. An overview is given

in Table 3.

(i) Since the phenomenon of plasmid instability for replicating vectors was

reported for Bacillus species, much more integration vectors have been

constructed which show stable integration in the chromosome (25). But the

clear disadvantage of these one-copy plasmids is the lower expression

rate in contrast to replicating plasmids (~50 copies per cell).

(ii) The problem of plasmid instability can partly explain the lack of versatile,

stable replicating expression vectors for B. subtilis (208) comparable to the

various available, user-friendly vector systems in E. coli (e.g. pUC-Vectors,

or the pET-vectors series commercialised by Novagen, Madison, USA).

(iii) The Signal peptides, labelling proteins to be secreted, accompany them all

the way to the CM thereby getting in contact to most of the Sec

components (see Fig. 3). Thus SPs play one of the most important roles in

the efficient translocation across the membrane. Numerous secretion

studies demonstrated a key role of SPs for efficient production of

homologous and heterologous proteins in B. subtilis (13, 32, 55, 58, 70,

111, 112, 130, 137, 164, 210, 225).

(iv) Interestingly, even for the secretion of foreign proteins the formation of

inclusion bodies can be a limiting factor as reported for the antidigoxin

single-chain antibody (SCA) production in B. subtilis (215, 217), although

the intracellular concentration should continuously be reduced by the Sec

machinery.

Introduction

11

(v) Another critical factor for protein export is the sufficient processing of

precursors at the outer site of the membrane by one of the five type I signal

peptidases (5, 187, 188).

(vi) At least 8 extracellular proteases are expressed by B. subtilis resulting in

proteolytic degradation of particularly heterologous products (208). These

foreign proteins are more accessible for extracellular proteases than

homologous enzymes due to their very slowly folding process into the

mature conformation.

(vii) A frequent problem is the misfolding of foreign exoproteins after the

translocation across the CM as a consequence of missing or insufficient

chaperone activity or their misfolding after translocation. (11, 20, 83, 101,

102). The misfolding of heterologous proteins, that contain structural

relevant disulfide bridges, represent a potential bottleneck as shown for the

secretion of a staphylokinase fusion with the fibrin-targeting Kringle domain

of human plasminogen, for the secretion of human pancreatic α-amylase

(HPA) as well as for secretion of human serum albumin (12, 171, 213).

(viii) Before processed proteins finally reach the culture medium, they must

pass through the 10-50 nm thick cell wall. It is composed of peptidoglycan

and anionic polymers forming a negatively charged environment thereby

expose affinity to especially positively charged secretory proteins. These

can interact with the cell wall leading to low yields in the culture

supernatant like shown for the human serum albumin, an α-amylase from

B. licheniformis and a recombinant B. anthracis protective antigen (171,

180, 186).

1.7 Strategies to bypass the limitations in protein production

For all listed reasons (1.6) the well studied E. coli expression systems are still mostly

used in large scale production of heterologous proteins. Meanwhile various projects

have been started to enhance the productivity of B. subtilis for foreign proteins:

Introduction

12

(i) One possibility to overcome the problem of plasmid instability might be the

deletion of the responsible BsuM restriction-modification system in

B. subtilis as done in a previous study (207). This system was found to be

responsible for the limitation of using plasmids for high level production

(60).

(ii) Over the last years, some novel plasmids were constructed with different

and improved properties and applications to extend the range of available

Bacillus expression systems (106, 108, 147).

(iii) The export inefficiency for foreign products could be improved

conspicuously by various modifications of the SPs: For instance a study of

Hemilä et al. showed that a modified junction between SP and a

heterologous pectinase from Erwinia carotovora yielded in a high level

production of 0,8 g/L protein (68). Furthermore, Saunders et al. changed

the C-region of a SP from a B. amyloliquefaciens neutral protease in a way

to increase export of a 34 aa fragment of human parathyroid hormone

(170). A structural modified SP α-amylase was the reason for a

significantly enhanced secretion of the β-lactamase (132).

(iv) However, another approach to enhance the secretion yield in B. subtilis

was the additional overexpression of intracellular chaperones GroE and

DnaK. It decreased the insoluble amount and increased the secreted

amount of the antidigoxin single-chain antibody (scFv) in B. subtilis

significantly (215).

(v) Limitations in the crucial processing step can be decreased by an

overexpressed SPase I like shown in a previous study (193). But enhanced

processing does not inevitably result in enhanced amount of secreted

protein like observed in a study by Vitikainen et al. (198).

(vi) Several B. subtilis strains were constructed showing low proteolytic activity

by elimination of extracellular proteases and could be used in successful

production of some heterologous proteins, which were sensitive to

Introduction

13

proteolytic degradation (96, 211, 214, 216, 217, 223). On the other hand,

different studies reported undesirable side effects of the inactivation of

proteases like increased cell lysis and growth reduction in protein-rich

industrial media what might lower the production yield (161, 179). A

change of growth temperature or pH variation of the medium can also help

to reduce proteolytic activity (208).

(vii) The occurrence of misfolded proteins often results in massive degradation.

Yet the single overexpression of extracellular chaperon PrsA led to an

increase of export yield of a protease and an α-amylase (102, 198). A

simple overexpression strategy applied for the thiol-disulfide

oxidoreductases BdbB and BdbC to reach a better disulfide bond formation

might be a successful strategy, but needs to be tested in the future.

(viii) To overcome the problem of protein retention in the cell wall due to a high

pI (> 9) protein variants can be engineered bearing equal enzymatic

properties but a lowered net charge resulting in a better release into the

culture medium as described in a previous study (180).

(ix) Since the whole genome sequence of B. subtilis is available, a totally

different approach to overcome production limits in B. subtilis is the

minimization of the chromosome as reported for E. coli (100). However, of

the total amount of ∼ 4100 protein encoding genes in B. subtilis only 270

genes were revealed to be indispensable for growth in rich medium at

37°C (99). The idea is the elimination of all unnecessary gene products to

avoid useless gene expression what might prevent a maximal production

yield for the protein of interest or lead to enhanced cost due to additional

contaminations of the product.

Table 3 summarizes the known secretion problems for B. subtilis and shows potential

solutions.

Introduction

14

Table 3 : Performed or planned strategies to overcome export bottlenecks in B. subtilis. Improved/not improved production yields of correctly folded protein are indicated by a “+” or a”-“. A”?” indicates a strategy that still needs to be tested. “+/-“ indicate both positive and negative effects of an applied strategy in different studies.

1.8 Scope of this thesis

Because several bottlenecks still limit the production for heterologous proteins in the

Gram-positive host B. subtilis, this work focusses on the optimization of secretion

capacity by different strategies:

(i) In the broad field of biotechnology the availability of useful Bacillus expression

plasmids is limited. Especially high throughput screening processes require

user-friendly cloning and expression strategies. Therefore a clear preference

exist for the Gram-negative bacterium E. coli as expression host. To overcome

this dilemma, several useful Bacillus plasmids should be constructed and

bottleneck strategy resulting

production yield stage of

secretion

(i) plasmid instability deletion of BsuM-rstriction-modification system ?

(ii) lack of expression vectors construction of new plasmids +

(iii) “wrong” signal peptide

change or modification of the signal peptide +/-

lowering the growth Tm (iv) inclusion bodies overexpression of intracellular

chaperones GroE and DnaK +

early

(v) inefficient processing

overexpression of Sip proteins +/-

construction of protease deficient strains +

(vi) extracellular proteases adaptation of Tm or pH to

reduce protease activity +/-

overexpression of extracellular chaperone PrsA +

(vii)

misfolding of exoproteins/ inefficient disulfide bond formation

overexpression of extracellular Bdb proteins ?

(viii) retention to cell wall engineering of protein variant with a lower pI +

late

(ix) unwanted site products

deletion of relevant genes- minimizing the genome ?

Introduction

15

tested with respect to (a) non inducible, optional intracellular expression, (b)

secretion, (c) easy purification and (d) application for HTS processes.

(ii) Since highest secretion efficiencies up to 20 g/L are obtained only with

homologous proteins from Bacillus species fused to the original signal peptide

(SP), one major limitation in secretion of foreign proteins is caused by using

the wrong SP resulting in inefficient and unsatisfying low yields.

Hence the main goal of this work was to solve this secretion handicap by a so

far new strategy. The idea was a systematic approach to identify the optimal

SP in a pool of all natural B. subtilis SPs with respect to its heterologous

secretion partner. The first step to realize this project was the generation of a

functional secretion reporter system in B. subtilis using a heterologous

enzyme of biotechnological relevance. Consequently, this system must allow

testing of different SPs fused to a heterologous marker enzyme for secretion

efficiency. Next, this system should be adapted to a high throughput screening

(HTS) format in B. subtilis enabling a general fast SP-optimization for export of

any target protein. Once established, this HTS system can form the basis to

use various mutagenesis techniques as an alternative way to improve the

functionality of a SP.

(iii) Another target for optimization was SecA, the central motor protein of the

general secretion pathway. Since translocation across the CM strongly

depends on the efficient formation of a complex of SecA and the precursor

proteins, variants of SecA should be constructed by Directed Evolution and

screened in the generated HTS system for improved export of a secretion

reporter.

Materials and Methods

16

2. Materials and Methods

2.1 Chemicals and enzymes

All used chemicals and enzymes were obtained in p.a. quality from the following

companies:

Antibiotics: Serva (Heidelberg), Sigma-Aldrich (Taufkirchen)

Chemicals: Fluka (Sternheim), Merck (Darmstadt), Roth (Karsruhe), Sigma-Aldrich

(Taufkirchen), DIFCO (Detroit, USA), Roche (Grenzach, Germany)

Enzymes: Lysozyme (Sigma-Aldrich), Pfu-DNA Polymerase (Stratagene,

Heidelberg), TurboPfu-polymerase (Stratagene, Heidelberg, Germany), Taq-DNA

polymerase (MGI Fermentas (St. Leon-Roth, Germany), Taq-Goldstar Polymerase

(Stratagene, Heidelberg, Germany), restriction enzymes (MGI Fermentas (St. Leon-

Roth), T4 DNA Ligase (MGI Fermentas, St. Leon-Roth), T7 DNA Polymerase (MGI

Fermentas, St. Leon-Roth),

2.2 Bacterial strains and plasmids

The strains and plasmids used in this study are listed in Table 4.

Table 4: Bacterial strains and plasmids used in this study.

Strains or plasmids Genotype/ Characteristics Source or reference

Strains

E. coli JM109

e14-(McrA-) recA1 endA1 gyrA96 thi-1 hsdR17(rK- mK+)

supE44 relA1 D(lac-proAB) [F’ traD36 proAB lacIqZDM15]

Stratagene, Heidelberg

B. subtilis 168 trpC2 (104)

B. subtilis DB430 his nprE aprE bpf ispI (41)

B. subtilis TEB1030 B. subtilis DB430 lipA lipB (48)

B. subtilis Marc0 B. subtilis TEB1030 amyE::pMCut This study

B. subtilis Marc1 B. subtilis TEB1030 amyE::pMCut1 This study

Plasmids

pMA5 ColE1 repB Kmr Ampr P HpaII (228)

pDG268 spoVG-lacZ Ampr Cmr (6)

pDG268-∆lacZ

pDG268 plasmid EcoRI / BpU1102I restricted and religated

after blunting; thereby, deleting the spoVG-lacZ fragment

This study


17

Plasmids Characteristics Source or reference

pMCut pDG268-XY containing a PHpaII-cutinase-fusion This study

pMCut1

pMCut containing a HindIII / EcoRI fragment coding the

B. subtilis lipase A signal sequence (sslipA), thereby

generating an in-frame fusion of sslipA and cutinase under

control of PHpaII

This study

pBSMuL1

B. subtilis – E. coli shuttle vector for overexpression

(ribosome binding site, PHpaII ) , secretion (sslipA) and

purification (C-terminal 6x-His-Tag); ColE1 repB Kmr Amp

r

This study

pBSMuL1-Cut pBSMuL1 containing a 662 bp EcoRI-XhoI fragment of

Fusarium solani pisi cutinase in frame with H6-Tag

This study

pBSMul2 pBSMuL1 vector with a second constitutive promoter P59 This study

pBSMuL2-Cut pBSMuL2 containing a 662 bp EcoRI-XhoI fragment of

Fusarium solani pisi cutinase in frame with H6-Tag

This study

pBSMuL3

pBSMuL1 derivative without ribosome binding site and

without sslipA; inverted MCS

This study

pBSMuL3-Cut

pBSMuL3 containing a 655 bp EcoRI-BamHI fragment of

Fusarium solani pisi cutinase without signal sequence

This study

pBSMuL3-Cut-ss

pBSMuL3-Cut containing an in-frame fusion of all Sec-

dependent signal sequences of B. subtilis ; ss is an

abbreviation for the paticular signal sequence; according to

the 174 different signal sequences of B. subtilis, 174

different pBSMul3-Cut-ss plasmids were constructed

This study

pBSMuL3-Cutintra pBSMuL3 containing a 655 bp HindIII-BamHI fragment of

Fusarium solani pisi cutinase without signal sequence This study

pBSMuL3-EstCL1

pBSMuL3 containing a 964 bp EcoRI-BamHI fragment of a

metagenome lipase (EstCL1) without signal sequence

This study

pBSMuL3-EstCL1-ss

pBSMuL3-Cut containing an in-frame fusion of all Sec-

dependent signal sequences of B. subtilis comparable to

pBSMuL3-Cut-ss

This study

pBSMuL3secA

pBSMuL3 containing a 2500 bp XhoI / BamHI fragment

encoding secA∆SacI of B. subtilis

This study

pBSMuL3secA-ep

pBSMuL3 containing 2500 bp XhoI / BamHI fragments

encoding ep-PCR-mutated secA of B. subtilis

This study

2.3 Oligonucleotides and PCR-primers

All listed oligonucleotides were ordered online from Thermo Electron Corporation at

website “http://www.thermohybaid.de/cgi-bin/oligos-new/order.py” and were obtained


18

as high purified salt free and lyophilized. The primers were dissolved in A. dest

resulting in a concentration of 50 pmol/µl and stored at –20°C.

Table 5: Oligonucleotides used in this study*. Restriction sites are highlighted in bold letters, start or stop codons are underlined. Wobbled bases (N = equimolar mix of all dNTPs; S = equimolar mix of dCTP and dGTP) are marked in italics.

Primer Sequence 5´ →→→→ 3´ Modifications

Cut1 ATATGAATTCGCGCCTACTAGTAACCCTGCT EcoRI

Cut2 ATATCTCGAGAGCAGAACCACGGACAGCCCG XhoI

Cut_low ATATGGATCCTCAAGCAGAACCACGGACAGC BamHI,

Cut_up ATATAAGCTTATTTGAATTCGCGCCTACTAGTAACCCTGCT HindIII, EcoRI

cutintra-up ATATAAGCTTAAGGAGGATATTATGGCGCCTACTAGTAACCCTGCT HindIII

Cut_down ATATGCATGCATATGGATCCTCAAGCAGAACCACGGACAGC PaeI, BamHI,

hpaII_up ATATGCATGCTTAAAGGTGGAGATTTTTTGA PaeI,

hpaII_down ATATAAGCTTCCCTGATTTCACTTTTTGCATTCT HindIII

EstCL1_up ATATGAATTCATGACCGATCCCTATGTGCGC EcoRI

EstCL1_low ATATGGATCCTCATGCTTCTGCCATAACCCC BamHI,

PPX_up ATTTCTGGACAAGCTGCAAAA -

PPX_down CAACGTCATGCTTTCGTTTTG -

secA_up ATATCTCGAGCTCTAAAATAGGCGTGTGATGATAGAGGAGCGTTATAA

ATG

XhoI,

secA_low ATATGGATCCATGGTACCTCAGTAAACTTGCCGGGGCGAACTA BamHI,

secA∆SacI-up GATGAAATGCTTGAACTCATTATGGATCGC

secA∆SacI-low GCGATCCATAATGAGTTCAAGCATTTCATC

silent base exchange to delete internal SacI restriction site in the secA gene

U1 TAATATATTAATAAGGAGGACATATGAAATTTGTAAAAAGAAGGATCAT

TGCACTTGTAACAATTTTGAT

VspI

U2 GCTGTCTGTTACATCGCTGTTTGCGTTGCAGCCGTCAGCAAAAGCCGC

CGAATTCGGTACCGTCGACCCC

-

U3 GGGAAGCTTGCGGCCGCGATATCTCTCGAGCACCACCACCACCACCA

CTGAATTAATATAT

VspI

L1 TGCAATGATCCTTCTTTTTACAAATTTCATATGTCCTCCTTATTAATATA

T VspI

L2 GGCTGCAACGCAAACAGCGATGTAACAGACAGCATCAAAATTGTTACA

AG -

L3 CCGCAAGCTTCCCGGGGTCGACGGTACCGAATTCGGCGGCTTTTGCT

GAC -

L4 TAATATATTAATTCAGTGGTGGTGGTGGTGGTGCTCGAGAGATATCGC

GG

VspI

AmyE-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCAAA HindIII

AmyE-2NNS-up ATATAAGCTTAAGGAGGATATTATGNNSGCAAAACGATTCAAA HindIII, Phe2 saturated

AmyE-3NNS-up ATATAAGCTTAAGGAGGATATTATGTTTNNSAAACGATTCAAAACC HindIII, Ala3 saturated

AmyE-4NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCANNSCGATTCAAAACCTCT HindIII, Lys4 saturated

AmyE-5NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAANNSTTCAAAACCTCT

TTA HindIII, Arg5 saturated

AmyE-6NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGANNSAAAACCTCT

TTACTG

HindIII, Phe6 saturated

AmyE-7NNS-up ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCNNSACCTCT

TTACTGCCG

HindIII, Lys7 saturated


19

Primer Sequence 5´ →→→→ 3´ Modifications

ssamyE-low ATATGAATTCAGCAGCACTCGCAGCCGCCGGTCC EcoRI

ssamyE

(+1NNS)– low ATATGAATTCSNNAGCACTCGCAGCCGCCGGTCC EcoRI, +1 position

saturated

P59-up ATGGCTTGACAGGGAGAGATAGGTTTGATAGAATATAATAGTTGTCGC

GGAAGCCATCCATGAAG BstXI

P59-low CTTCTACCGAACTGTCCCTCTCTATCCAAACTATCTTATATTATCAACA

GCGCCTTCGGTAGGTA BstXI

*All oligonucleotides used for PCR-amplification of all Sec-dependent signal sequences from genomic DNA of B. subtilis 168 are listed in the Appendix.

2.4 Culture media and growth conditions

LB-medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl

LB-agar: LB-medium,15 g/L agar

TB-medium: 5g/L glycerol, 12 g/L tryptone, 24 g/L yeast extract, 12,54 g/L K2HPO4,

2,31 g/L KH2PO4 (pH7)

If not stated elsewhere the following growth conditions were applied:

E. coli cells were grown at least for 16 hours in selective LB-medium at 37°C.

B. subtilis precultures were incubated overnight in selective LB-medium at 37°C and

diluted 1:1000 to inoculate expression cultures. The expression B. subtilis cultures

were grown for 20 hours in selective LB-medium at 37°C.

Tab. 6: Relevant antibiotics used for growth selection

substrate final concentration [µg/mL] organism

ampicillin 100 E. coli

kanamycin 50 B. subtilis

chloramphenicol 5 B. subtilis


20

2.5 Storage of bacteria

E.coli and B. subtilis strains grown on agar plates were stored at 4°C and RT,

respectively. For longer storage 1,3 mL of a liquid culture was mixed with DMSO

(8 % v/v end concentration) and stored at -80°C.

2.6 Isolation of nucleic acid

Isolation of plasmid DNA was prepared by using the plasmid purification kits from

QIAGEN (Hilden, Germany). Genomic DNA of bacteria was prepared by using the

Genomic-tip 20/G-Kit from QIAGEN (Hilden, Germany) under conditions

recommended by the manufacturer.

2.7 Agarose gel electrophoresis

Agarose gel electrophoresis for DNA analysis and DNA isolation was carried out in a

i-Mupid mini gel system (Eurogentec, Köln, Germany) as described by Sambrook et

al. (165). For DNA electrophoresis 0,5x TBE buffer was used (89 mM Tris, 89 mM

Boris acid, 2,5 mM Na2-EDTA). An Eagleeye II (Stratagene, Heidelberg, Germany)

was used for visualisation and documentation of DNA. For purification of DNA

fragments out of agarose gels, the QIAEX II kit (Qiagen, Hilden, Germany) was

applied according to the protocol provided. The “Gene Ruler 1 kb DNA Ladder” from

Fermentas (St. Leon-Rot, Germany) was used as a molecular weight standard.

2.8 In vitro recombination of DNA Restriction reactions, blunting ends and ligation reactions were performed using

enzymes from Fermentas (St. Leon-Rot, Germany) under conditions recommended

by the manufacturer.


21

2.9 Transformations of bacteria

2.9.1 Chemical transformation of E. coli

Preparation of transformation competent E. coli cells and transformation of plasmid

DNA was performed according to the RbCl2 method of Hanahan (61).

2.9.2 Electroporation of E.coli

E. coli strain JM109 was used for electroporation in a MicroPulser (BioRad,

München, Germany). The preparation of electrocompetent cells and the

electrotransformation was performed following the manufacturer`s protocol (BioRad,

München, Germany).

2.9.3 Transformation of B. subtilis

Transformation of B. subtilis cells was conducted by the following procedure (4, 224):

the B. subtilis culture was grown in Paris-Medium until it reached a cell density of

OD580 = 1. The culture was divided into 0.5 mL aliquots, plasmid DNA (0.1-1 µg) was

added and the samples were incubated for 1-3 hours at 37°C. Selection for plasmid

bearing Bacillus cells was provided on LB-agar plates containing the suitable

antibiotic. For efficient DNA uptake of pBSMuL vectors by B. subtilis, the plasmid

DNA was SacI digested and self-ligated to generate plasmid multimers before adding

to the competent cells.

Paris-medium: 60 mM K2HPO4, 40 mM KH2PO4, 3 mM Na3-citrate, 1% (w/v) glucose,

20 mM K-glutamate, 2,2 mg/L Fe(III)NH4-citrate, 0,1% (w/v) casamino acids, 20 mg/L

tryptophan, 3 mM MgSO4


22

2.10 Polymerase chain reaction (PCR)

2.10.1 Standard PCR

Amplification of DNA-fragments was performed under standard PCR-conditions in a

reaction mix containing genomic or plasmid DNA (~1 ng) as the template, primer

oligonucleotides (each 50 pmol), dNTP’s (0.2 mM each), Taq- (2.5 U), Pfu-(2.5 U) or

TurboPfu-Polymerase (2.5-5 U) Buffers containing MgCl2 or MgSO4 were used as

recommended by the manufacturers. Conditions for standard PCR were: 1 x (2 min

95°C); 35 x (30 sec 95°C; 30 sec 58°C; 30 sec up to 3 min 72°C, depending on the

length of target gene), 1 x (7 min 72°C). If (Turbo-)Pfu-Polymerase was used, 1%

DMSO was added to the reaction mix. The PCR was performed using a Mastercycler

Gradient (Eppendorf, Hamburg, Germany). The resulting PCR-products were purified

by gel extraction using QIAEX II kit (Qiagen, Hilden, Germany).

2.10.2 Site directed mutagenesis

If a defined base exchange should be inserted close to the 5`- or 3`-ends of a target

gene, a standard PCR reaction was performed using mutagenesis oligonucelotides.

Other defined base exchanges were insetted into a target gene by using a modified

protocol of the Quik Change Site-Directed Mutagenesis Kit (Stratagene, Heidelberg,

Germany): a plasmid containing the target gene was extracted from an E. coli strain

with a functioning DNA-methylisation system (e.g. E. coli JM109) and used as a

template in a following PCR reaction to amplify the hole plasmid DNA. The standard

conditions were changed by the following modifications: Since the mutagenesis DNA

primers are complementary to each other, more template DNA (~10-100 ng) was

used to ensure proper product synthesis. TurboPfu-Polymerase (5 U), and a reduced

amount of PCR cycles (≤ 20) were chosen to minimize random point mutations. The

elongation steps were perfomed at 68°C, the elongation time depends on length of

the plasmid. After PCR reaction the sample was incubated with 10 U of restriction

enzyme DpnI for one hour at 37°C resulting in a complete elimination of methylised


23

template DNA. The remaining PCR product was directly transformed into E. coli

JM109 cells.

2.10.3 Saturation mutagenesis

To saturate a defined position in a protein with all possible amino acids and to

minimize the chance of stop codons and amino acid redundancy at the same time, a

standard PCR was performed using mutagenesis primer with wobbled bases for the

defined base position. This primer consists of a nucleotide mix of all 4 bases (dATP,

dCTP, dGTP and dTTP) in equimolar concentration for the first two codon positions

and a reduced base composition (only dCTP and dGTP) at the degenerated codon

position 3 (2).

2.10.4 Random mutagenesis

Random point mutations were introduced into a target gene by error-prone

polymerase chain reaction (ep-PCR) as described before (27, 226). The reaction mix

contains ~1 ng as the template, 50 pmol of each primer, 0.2 mM of each dNTP and

2.5 U Taq-Goldstar-Polymerase (Stratagene, Heidelberg, Germany). As reaction-

buffer the (NH4)2SO4-containing Taq-reaction-buffer from Fermentas (St. Leon-Rot,

Germany) was used. Each approach contains 6 mM MgCl2 and in addition 0.15- 0.3

mM MnCl2 yielding an average error rate of 1 up to 10 base substitutions per 1 kb.

The cycler conditions for ep-PCR were: 1 x (2 min 95°C); 30 x (30 sec 95°C; 30 sec

58°C; 30 sec up to 3 min 72°C, depending on the length of target gene) and 1 x (7

min 72°C).

2.10.5 Cassette mutagenesis of the PPX-domain of secA

To insert random point mutations only in the preprotein-crosslink domain of secA, a

method was used based on the QuickChange II Site-Directed Mutagenesis Kit from

Stratagene (Heidelberg, Germany). In the first step an ep-PCR of the PPX-domain


24

using primer pair ppx-up/ ppx-down and methylated plasmid DNA pBSMuL3secA

from E. coli JM109 as the template lead to a 350 bp fragment containing the library of

the PPX-domain of SecA. The DNA of gel extracted PPX-fragments was taken as the

primer pair in a second PCR amplifying the whole plasmid pBSMuL3secA resulting in

a plasmid mix of methylised wildtype plasmid and and generated plasmid variants.

To eliminate methylised wildtype secA (template) the PCR sample was incubated

with endonuclease DpnI. An aliquote of this sample was directly used for

electrotransformation into E. coli JM109 for plasmid library construction. A

schematical overview is given in Fig. 34 (Appendix).

2.11 Construction of integration plasmid pMCut

The 642 bp cutinase gene was amplified from E. coli expression plasmid pMac5-8

(176) by standard PCR using the primer pairs Cut_up and Cut_down containing

unique restriction sites for HindIII/EcoRI and BamHI/PaeI for cloning. The amplified

cutinase lacks a Bacillus ribosome binding site and an ATG-start codon. In a second

standard PCR a 430 bp fragment containing the strong constitutive HpaII promoter

sequence was amplified using oligonucleotides hpaII_up and hpaII_down containing

PaeI and HindIII restriction sites, respectively (Table 5) and the B. subtilis plasmid

pBSMuL1 (24) as the template. The 682 bp cutinase PCR-fragment and the 430 bp

HpaII promoter PCR-product were digested with endonuclease HindIII, ligated and

subsequently used as a DNA-template in another standard PCR using the primer pair

hpaII_up and Cut_down to amplify the promoter-cutinase gene-fusion. The resulting

1102 bp PCR-product was cloned into the unique PaeI site of Bacillus integration

vector pDG268-∆lacZ (Table 4). The resulting plasmid was analyzed by DNA-

sequencing and named pMCut.

2.12 DNA sequencing DNA sequencing was performed by SequiServe (Vaterstetten, Germany).


25

2.13 Enzyme assays

2.13.1 Lipolytic plate assay

Esterase indicator plates were prepared by addition of 15 ml of an emulsion of 50%

(v/v) tributyrin and 5% (w/v) gum arabic to 500mL of molten LB-agar with appropriate

antibiotica. Tributyrin was emulsified by sonication for 3 min at 75 W (duty cycle

100%) in a Branson 250 sonifier. Esterase and lipase activity is indicated by the

formation of clear halos around the colonies.

2.13.2 α-amylase activity plate assay

Starch agar (pH 6.8) was prepared by addition of 15 g of agar, 10 g of starch, 5 g of

gelatine, 3 g of beef extract to 1 L A. dest. After steam sterilisation the suitable

antibiotic was admixed. By adding 5 % (w/v) iodine solution (Lugol`s solution) to the

plate amylase activity is indicated by yellow halos around the colonies.

2.13.3 Lipolytic activity assay

Detection of lipolytic activity of cutinase was performed as described by Winkler and

Stuckmann using a spectrophotometric assay (209). As the substrate 30 mg

p-nitrophenyl-palmitate (pNPP) was dissolved in 10 mL isopropanol and mixed with

90 mL of Sørensen phosphate buffer (pH 8), supplemented with sodium desoxycholic

acid (207 mg) and gum arabic (100 mg). The final concentration of the substrate was

0.8 mM. The culture supernatant was separated from the cells by 5 min

centrifugation at 10.000 g and micro-filtration (membrane filter, pore size 0,22 µm,

Millipore Corp, Billerica, MA, USA). Culture supernatant (5-20 µL) was mixed with 2.5

mL of the substrate emulsion. After incubation at 37°C (15-30 min) the absorbance at

410 nm was recorded. The enzymatic activity was calculated using a molar

absorbtion coefficient of 15000 M-1·cm-1.


26

2.13.4 Esterase activity assay

For the detection of esterase activity of EstCL1 23.7 mg of pNPC (p-nitrophenyl-

caproat) was dissolved in ethanol (5 ml) and added to 95 ml of 100 mM potassium

phosphate buffer (pH 7.2) containing 10 mM MgSO4 to yield a final concentration of 1

mM pNPC. Culture supernatant (5-20 µL) was added to the substrate solution to give

a final volume of 1 ml, and the ∆OD410 was recorded for 15-30 min at 37°C. The

enzymatic activity was calculated using a molar absorbtion coefficient of

12500 M- 1·cm-1.

2.13.5 High throughput screening (HTS) assay for cutinase/EstCL1 secretion using a pipetting robot

B. subtilis colonies expressing SPs fused to the cutinase or EstCL1 encoded by

pBSMuL3-Cut-ss/ pBSMuL3-EstCL1-ss were transferred from LB-agar plates into

96er deepwell microtiter plates (DW-MTP, 2mL, Greiner Bio-One, Frickenhausen,

Germany). The strains were cultured in 1 mL LB-medium per well at 37°C using a

microplate shaker (600 rpm, TiMix 5, Edmund Bühler GmbH, Hechingen, Germany).

After 16 hours of growth, culture supernatant was prepared by centrifugation (30 min,

4°C, 5.000 g). Culture supernatants (200 µL) of each clone were carefully transferred

into a new MTP (0.5 mL, Greiner Bio-one, Frickenhausen, Germany).

The lipolytic activity of the supernatants against the substrate p-NPP for cutinase or

p-NPC for EstCL1 was determined by an automated lipolytic activity assay which was

integrated in a HTS process using a TECAN workstation Genesis 200 Freedom

(Tecan, Crailsheim, Germany):

In the first part of the Gemini V4.2 pipetting script “Ulf pNPP1_10 tiptouch_bis_10

Pl.gem (Fig. 35.A, Appendix)” 20 µL of each culture supernatant of one “source” plate

is diluted 10-fold in selective LB-medium provided in a 200 mL trough. The second

part of the Gemini script (Fig. 35.B, Appendix) defines the pipetting of the assay

plate: supernatant (20 µL of 10-fold diluted) are mixed with 180 µL of substrate

emulsion provided in a second 200 mL trough. After completing the Gemini script the

roboting arm (ROMA) is continuing the Process Manager program

Ulf_PnPP1_10V_Trog200 (Fig. 12) transporting the assay plate directly to the

preheated (30°C or 37°C) Genios spectrophotometer (Tecan, Crailsheim, Germany).


27

The changing of absorbance at 410 nm was detected by recording a 15 min kinetic at

30°C (cutinase) or 37°C (EstCL1).

2.14 Determination of protein concentration

The protein concentration was measured spectrophotometrically at 595 nm according

to the method of Bradford (19) using bovine serum albumin (BSA) as the standard.

2.15 TCA precipitation of proteins

1/10 volume of 1% (w/v) sodium dodecyl sulfate was added to the protein sample

and incubated for 5 min at RT. Proteins were precipitated with 1/10 volume of 70%

(v/v) trichloracetic acid (TCA) and incubated for 5 min at RT. After a centrifugation for

30 min (RT, 14000 rpm), samples were washed twice with ice-cold 80% (v/v) acetone

and dried at RT. Finally, proteins were resuspended in a suitable volume of SDS

loading buffer (10% Glycerol, 0,2% (v/v) SDS, 0,125 M Tris-HCL pH 6.8, 0,1% (w/v)

Bromphenol blue, 2% (v/v) β-Mercaptoethanol).

2.16 SDS-polyacryamide gel electrophoresis (SDS-PAGE)

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Mini-

PROTEAN 3 Cell (BioRad, München, Germany) under reducing conditions using a

5 % stacking gel and a 12-15% separating gel as described before (105). Protein

samples in SDS loading buffer were incubated 2-5 min at 95°C before separated by

SDS-PAGE. Afterwards, proteins were stained with Coomassie Brilliant Blue R250.

As the molecular weight standard the “Prestained Protein Marker” from Fermentas

(St. Leon-Roth, Germany) was used.


28

2.17 Cell culture and production of cutinase-specific antibodies by hybridoma

cells

Polyclonal anti-cutinase antibodies were produced by hybridoma cells (1) resulting

from the fusion of B-lymphocytes from immunized Balb/c mice with SP2/0 myeloma

cells (163) and secreted into the culture media. Cells were routinely cultured in

serum-free hybridoma media (Ex-Cell 610 – HSF, JRH biosciences, Lenexa, Kansas,

USA). Stock holding was performed in T-flask cultures (Greiner, Frickenhausen,

Germany) and under selection pressure with 400 µg mL-1 geniticin (G418, Gibco

BRL, Eggenstein, Germany) in the cultivation media. Batch cultivations for production

of the antibodies were all carried out in spinner flasks (100 – 300 mL working volume,

Techne, Cambridge, UK) with headspace aeration at agitation speed of 40 rpm.

Cultures were incubated at 37°C in an atmosphere of 95% air saturation, 5% CO2

and 99% humidity (Heraeus Instruments, Hanau, Germany). Viable cells were

counted whether manually by hemacytometer using the Trypan Blue exclusion

method (Roche Diagnostics, Mannheim, Germany) or by using an automated cell

viability analyzer (Vi-Cell XR, BeckmanCoulter, Inc., Miami, FL, USA). Initial cell

densities were 0.2 × 106 mL-1 for all cultivations and cell stock holding was

appropriately splitted to keep cell densities below 1.5 × 106 mL-1. Supernatants

containing anti-cutinase antibodies were recovered by centrifugation (200 × g, 10

minutes, 4°C) and kept refrigerated (4°C) for short-term usage or frozen (-80°C) for

long-term storage, respectively.

2.18 Immunodetection of proteins

For immunodetection proteins were concentrated by trichloroacetic acid precipitation,

separated by SDS-PAGE and transfered under semi dry conditions onto a

polyvinylidene difluoride (PVDF-) membrane in a Mini-PROTEAN 3 Cell (Biorad,

Munich, Germany). Blotting was carried out for 15 min at 15 mA followed by 20 min

at 300 mA using Dunn Carbonat buffer (10mM NaHCO3, 3 mM Na2CO3, 20% (v/v)

Methanol) as described before (46). After the transfer, blocking of the membrane was

achieved by incubation in TBST (50 mM Tris/HCL pH 6.8, 150 mM NaCl, 1 mM


29

MgCl2, 0.2% (v/v) Tween 20) containing 5% (w/v) milk powder. The following steps

were then carried out in TBST. For detection of cutinase protein a polyclonal

antiserum was used (1:1000 diluted) produced in hybridoma cells (2.17). For

detection of B. subtilis SecA a polyclonal antiserum was used (1:1000) raised against

SecA in a rabbit. A sample was kindly provided by M. Caspers (Prof. Dr. R. Freudl,

Bacterial Protein Secretion Group, IBT 1, Research Centre Jülich, Germany). For

protein detection horseradish peroxidase-labelled goat-anti-mouse antibody (BioRad,

München, Germany) was used as the second antibody. Detection was performed

with the ECL system (Amersham Pharmacia Biotech, England) using a luminograph

(LB 980, EG &G Berthold, Bad Wildbad, Germany) as recommended by the

manufacturer.

2.19 Purification of overexpressed cutinase in E.coli

The overexpression of cutinase from the fungus Fusarium solani pisi and the

following purification procedure was performed as follows: A preculture (50 mL) of

E. coli cells WK6 harbouring cutinase expression plasmid pMac5-8 (176) were grown

overnight in TB-medium (17 mM KH2PO4, 72 mM K2HPO4, 1,2 % (w/v) tryptone,

2,4 % (w/v) yeast extract, 0,4% (v/v) glycerine, pH 7,0) at 37°C. The main culture

was inoculated 1:1000 from the preculture and grown in TB-medium at 25°C until a

cell density of OD580= 1. After induction with a final IPTG concentration of 0.1 mM the

culture was incubated for further 16 hours at 25°C. After a centrifugation step (30

min, 4°C, ~14.000g) the accumulated cutinase in the periplasm was released by cold

osmotic shock: The cell pellet was resuspended in icecold digestion buffer (100 mM

Tris-HCl, 500 mM Sucrose, 1 mM EDTA). After centrifugation (30 min, 4°C,

~14.000g) the pH of the periplasmic fraction (supernatant) was lowered to 4.7 by

adding 50% (w/w) acetic acid. After overnight incubation at 4°C the precipitated

material was removed by centrifugation (30 min, 4°C, ~14.000g). The supernatant

was filtered through a cellulose-acetat filter (0.2 µm, Firma) and loaded on a

preequilibrated G25 Sephadex column (20 mM Tris-HCl, pH 7,6). The eluat was

loaded on a preequilibrated (20 mM Tris-HCl, pH 7,6) DEAE Sepharose Fast Flow-

column (Pfizer, Wien, Austria). After a desorbing step with a gradient (0-10%) of

elution buffer (20 mM Tris-HCl, 1 M NaCl, pH 7,6) the eluat was loaded on a


30

preequilibrated Q-Sepharose Fast Flow column (20 mM Tris-HCl, pH 7,6). After a

desorbing step with a step gradient (25%) of elution buffer (20 mM Tris-HCl, 1 M

NaCl, pH 7,6) the eluat was concentrated using a PL10 Centricon membrane (Pall,

Dreieich, Germany). In a final gel filtration step the sample was loaded on a

preequilibrated G200 Sephadex column (20 mM Tris-HCl, 150 mM NaCl, pH 7.6).

The eluat was concentrated using a PL10 Centricon membrane (Pall) followed by

buffer exchange to PBS buffer (4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl, pH

7,2) using a PD 10 column (Pfizer, Wien, Austria). Finally the sample was sterile

filtrated through a Pall Acrodisc Syring Filter (0,2 µm, Pall, Dreieich, Germany). The

purification of overexpressed cutinase was performed by E. Knieps-Grünhagen (Dr.

J. Hubbuch, Bioseparation group, IBT 2, Research Centre Jülich, Germany).

2.20 Pulse-chase protein labelling

Exponential phase cells of B. subtilis TEB1030 grown in S7 minimal medium (197) at

37°C were washed twice with methionine free S7 medium, normalized to an OD600 of

0.8 and incubated in methionine free S7 for 60 minutes at 33°C. Subsequently, the

cells (2.5ml) were labelled with 150µCi [35S]- methionine for 1 minute, followed by

chasing with a vast excess of non- radioactive methionine (2mg/ml). Samples (600µl)

for each strain were withdrawn at certain time points after the chase (10 sec, 30 sec

and 1 min) and precipitated overnight with ice- cold TCA (final concentration 8%).

Precipitates were washed three times in 80% acetone, resuspended in 100 µl lysis

buffer (10 mM Tris/HCl pH 8,0, 25 mM MgCl2, 200 mM NaCl, 5 mg/ml lysozyme) and

incubated at 37°C for 45 min. Lysis was completed by addition of 15µl of 10% (w/v)

SDS and heating for 10 min at 95°C. Immunoprecipitation and SDS-PAGE were

carried out as described by van Dijl et al. (194). For the immunoprecipitation, a

polyclonal antibody raised against cutinase in a rabbit was used. Radioactively

labelled Protein bands were visualized using a “Fuji BAS 1800 Bio Image Analyser.”

The pulse-chase experiments were performed by M. Caspers (Prof. Dr. R. Freudl,

Bacterial Protein Secretion Group, IBT 1, Research Centre Jülich, Germany).


31

2.21 Cell fractioning of B. subtilis

After a centrifugation step (30 min, 7.500 UpM, 4°C ) the cells were separated from

the culture supernatant. The cell pellet was resuspended in phosphat buffer (100 mM

K2HPO4, 100 mM KH2PO4) containing protease inhibitor “Complete” (1x, Roche,

Grenzach, Germany). Ultrasonic treatment (20 min, 75 W, “Branson Sonifer 250”,

Branson, Danbury, USA) and the incubation of the cells with lysozyme (10 mg/mL,

37°C, 30 min) was performed to ensure complete cell lysis. The fractionation in cell

wall, cell membrane and cytoplasm was performed to the protocol of Harwood et al.

(66).

2.22 Computational methods

Analyses of DNA, amino acid sequences and construction of plasmid maps were

carried out using the programs Edit Sequencer, Clone Manager for Windows 7.0,

Plasmid MapEnhancer 3.0 (SCIENTIFIC AND EDUCATIONAL SOFTWARE,

Durham, USA) and CS ChemDraw Pro 7.0 (CAMBRIDGESOFT, Cambridge, USA).

Sequence alignments, database searches and protein structure analysis were

performed using the standard programs of the NATIONAL CENTER FOR

BIOTECHNOLOGY INFORMATION (http://www.ncbi.nlm.nih.gov). DNA sequence

informations about B. subtilis 168 were obtained from the SubtiList-database

(http://genolist.pasteur.fr/SubtiList/). Signal peptide prediction of given amino acid

sequences were carried out using SignalP 3.0

(http://www.cbs.dtu.dk/services/SignalP/).

Results

32

3. Results

3.1 New expression and secretion vectors for Bacillus subtilis

3.1.1 Choice of expression host: B. subtilis versus E. coli

Today a large pool of cloning and expression plasmids is available for the gram-

negative bacterium Escherichia coli (e.g. pUC-Vectors, or the pET-vectors series

commercialized by Novagen, Madison, USA). Therefore, most protein expression

strategies in microbiological research focus on this organism. However, beside the

obvious advantages of the E. coli-systems, serious problems can occur during the

process of heterologous gene expression and purification: (i) low expression rates,

(ii) formation of inclusion bodies, (iii) improperly protein folding and/or (iv) toxicity

problems might oblige to change the expression host (8).

An alternative expression host also for large scale production of foreign proteins is

the Gram-positive bacterium Bacillus subtilis. One major advantage of the strain is its

classification as a GRAS – generally recognized as safe – organism free of any

endotoxin. Furthermore, in contrast to E. coli the bacterium B. subtilis offers an

efficient secretion apparatus guiding the expressed protein directly into the culture

supernatant (195); thereby bypassing the time-consuming cell disruption which

makes a subsequent protein purification much easier. In addition, in case of efficient

secretion the formation of inclusion bodies in the cytoplasm is reduced, leading to

higher amounts of properly folded and active enzymes. For all these reasons

B. subtilis has developed into an important expression strain frequently used in

industrial fermentations during the past decades (172). Various kinds of secretion

vectors have been published already for B. subtilis (106, 128); however, until now the

choice of available expression plasmids combining similar properties like E. coli-

systems is still limited.

The intended plasmids shall combine several advantages in comparison to available

Bacillus expression systems: an appropiate multiple cloning site consisting of 13

unique restriction sites, one or two strong constitutive promoters, a highly efficient

Results

33

signal sequence for protein secretion and the possibility to express proteins as His-

tagged fusions for easy detection and purification.

3.1.2 Construction of vectors pBSMuL1 and pBSMuL2

The multi-copy vectors pBSMuL1 and pBSMuL2 (Fig. 4) were constructed by

excision of the original cloning site (EcoRV, KpnI and HindIII) of pMA5 (36) by EcoRI

and HindIII double-digestion, blunting using the Klenow fragment and subsequent

religation resulting into an intermediate plasmid named pMA5∆MCS. The DNA-

fragment containing the new MCS of both vectors was constructed in vitro using

seven oligonucleotides (U1-U3, L1-L4; Table 5). A pool of all these oligonucleotides

(5 pmol each, dissolved in A. dest) with a final volume of 14 µL was incubated at 95-

100°C for 10 min and cooled down to room temperature afterwards. This sample was

directly used as a template in a standard PCR reaction using additional 50 pmol of

oligonucleotide U1 and L4 (Table 5) as flanking primers. The expected 201 bp DNA

fragment was amplified, flanked by VspI restriction sites (ATTAAT). After

gelextraction this product was digested using VspI and ligated into the NdeI-

linearized pMA5∆MCS plasmid; thereby eliminating the original NdeI site of the pMA5

vector. The resulting plasmid was sequenced and named pBSMuL1. In contrast to

the original pMA5 vector the cloning site is directly downstream of the strong HpaII

promoter; therefore, no additional cloning steps are necessary to get the target gene

under control of the promoter (36). To achieve an even higher expression and

secretion level a second constitutive promoter P59 (191) was inserted upstream of the

HpaII promoter. The P59 DNA-fragment was synthesized in vitro as described before

by using the oligonucleotides P59-up and P59-low (Table 5). The final 65 bp long

promoter DNA was cloned directly into the unique BstXI site of pBSMuL1. The

resulting plasmid was sequenced and named pBSMuL2 (Fig. 4).

Results

34

3.1.3 Overexpression, secretion and purification of His-tagged cutinase in

pBSMuL vectors

To confirm the functionality of these vectors, the cutinase from the fungus F. solani

pisi (51) was chosen as a heterologous enzyme for expression in B. subtilis. The

cutinase gene was amplified by a standard PCR from E. coli expression plasmid

pMac5-8 (176) using the primer pair cut1/cut2 (Table 5) containing restriction sites for

EcoRI and XhoI without including the cutinase stop codon at the 3`-end. The 0,7 kb

PCR product was cloned into the MCS of pBSMuL1 and pBSMuL2 to obtain an in

frame fusion of the cutinase gene and the 6x-His-sequence. The resulting plasmid

pBSMuL1-Cut was used to transform the lipase deficient B. subtilis strain TEB1030

(48) which lacks both genes for the extracellular lipolytic enzymes LipA and LipB as

Fig. 4: Plasmid map of the pBSMuL series: pBSMuL1 (7494 bp) and pBSMuL2 (7559 bp). (A) Overview of relevant elements: PHpaII, constitutive Gram-positive promoter; P59, second constitutive gram-positive promoter only present in pBSMuL2; MCS, multiple cloning site; ori E. coli, gene required for replication in E. coli; amp

r, β-lactamase

gene conferring ampicillin resistance in E. coli; repB, gene required for replication in B. subtilis; kan

r, kanamycin resistance gene; (B) Properties of the artificial MCS: rbs, ribosome binding site recognized in B. subtilis; sslipA, lipA signal sequence of B. subtilis for efficient protein secretion into the culture medium; the signal peptidase cleavage site (Ala-Lys-Ala) is marked with an arrow; His-Tag, C-terminal in-frame fusion to six histidine residues for protein purification.

PHpaII

MCS

EcoRI

Acc65I

KpnI

AccI

SalI

SmaI

HindIII

NotI

EagI

EcorV

XhoI

NdeI

pBSMuL

ori E. coli

repB

kanr

ampr

BamHI

rbs NdeI

.....TAATAAGGAGGACATATGAAATTTGTAAAAAGAAGGATCATTGCACTTGTAACAATTTTGATG

MetLysPheValLysArgArgIleIleAlaLeuValThrIleLeuMet

Acc65I

EcoRI KpnI

CTGTCTGTTACATCGCTGTTTGCGTTGCAGCCGTCAGCAAAAGCCGCCGAATTCGGTACC

LeuSerValThrSerLeuPheAlaLeuGlnProSerAlaLysAlaAlaGluPheGlyThr

XmaI EagI

SalI SmaI HindIII NotI EcoRV XhoI His-Tag

GTCGACCCCGGGAAGCTTGCGGCCGCGATATCTCTCGAGCACCACCACCACCACCACTGAAT.....

ValAspProGlyLysLeuAlaAlaAlalleSerLeuGluHisHisHisHisHisHisEnd

pBSMuL multiple cloning site (MCS)

signal peptidase

sslipA

yy

A

B

P59

Results

35

well as the extracellular proteases NprE and AprE. Consequently the strain TEB1030

shows no disturbing lipolytic background activity and less proteolytic degradation in

the supernatant. The resulting Bacillus strain expressing the cutinase was cultured in

selective LB-medium 20 hours at 37°C. About 25 µg of total protein of culture

supernatant was separated using SDS-polyacrylamide gelelectrophoresis (SDS-

PAGE) showing the successful overexpression and efficient secretion of 20 mg

cutinase per litre (Fig. 5, lane 1). Using the Ni-NTA Spin Column (Qiagen, Hilden,

Germany) as recommended by the manufacturer’s standard protocol, the

recombinant cutinase was purified to electrophoretic homogeneity as detected by

SDS-PAGE (Fig. 5, lane 4). The enzyme has been expressed and secreted in

catalytically active conformation as determined spectrophotometically using p-

nitrophenyl-palmitate (pNPP) as the substrate (209). The specific activity of the His-

tagged cutinase towards pNPP was 200 U/mg and thus similar to the native cutinase

(225 U/mg) expressed and purified from E. coli.

Fig. 5: Purification of His-Tag cutinase from the B. subtilis culture supernatant. The expression culture was grown 20 hours in LB-medium supplemented with kanamycin (50 µg/mL) at 37°C. 600 µL of culture supernatant (pH 8.2) was loaded on a Ni-NTA Spin Column (Qiagen, Hilden, Germany) and the His-tagged cutinase was purified as recommended by the manu-facturers standard protocol. 25 µg protein of the supernatant (lane 1) and comparable volumes of flow-through (lane 2), wash fraction (lane 3) and elution (lane 4) were concentrated using 70% trichloride acid precipitation and separated on a protein gel (12% SDS-PAGE) stained with coomassie brilliant blue. The protein band of the His-Tag cutinase is marked by an arrow. As a control 2 µg of purified native cutinase expressed in E. coli (22.2 kDa) was loaded as a positive control (lane +).

elu

tio

n

flo

w-t

hro

ug

h

mark

er

supern

ata

nt

wash f

racti

on

po

sit

ive

co

ntr

ol

55 kDa

45 kDa

35 kDa

25 kDa

15 kDa

M 1 2 3 4 +

Ni-NTA purification

Results

36

For higher production yields the second plasmid pBSMuL2 was tested for cutinase

overexpression and secretion. Because of the additional constitutive promoter P59 it

was possible to increase the secreted amount of mature cutinase three-fold.

Recombinant cutinase (60 mg/L) was detected in the culture supernatant by activity

determination using pNPP as the substrate (Fig. 6).

3.1.4 Stability of pBSMuL vectors in B. subtilis under non selective conditions

Both pBSMuL-plasmids multiply by the so-called rolling-circle type of replication

(repB from Staphylococcus aureus) which might lead to the disadvantage of

segregational instability (25) in non selective media. But especially industrial

applications require economical processes avoiding additional costs for antibiotics.

Therefore, we tested the stability of both plasmids under selective and non-selective

conditions as shown in Fig. 7:

Fig. 6: Expression and secretion efficiency of cutinase in Bacillus subtilis using vectors pBSMuL1 and pBSMuL2. The lipase-deficient strain Bacillus subtilis TEB1030 was transformed with cutinase overexpression plasmids pBSMuL1-Cut and pBSMuL2-Cut. As negative controls B. subtilis TEB1030 was transformed with vectors pBSMuL1 and pBSMuL2, respectively. All strains were incubated in selective LB-medium for 20 hours at 37°C and showed no significant growth differences. The culture supernatants were harvested by centrifugation. All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay. The lipolytic activity was adapted to an OD580= 7. Standard deviations are indicated by error bars. The results represent data from five independent experiments.

0

2

4

6

8

10

12

14

pBSMuL1 pBSMuL2 pBSMuL1-Cut pBSMuL2-Cut

expression strains

U/mL

Results

37

Overnight cultures of both strains grown under selective conditions were used to

inoculate 25 mL of fresh LB-medium and cultured up to 24 hours at 37°C with and

without antibiotic selection: No reduction in cutinase secretion was detected under

non-selective growth conditions compared to the control Bacillus strain cultured

under antibiotic selective pressure. Even after inoculation of fresh LB-medium and

another 24 hours of growth at 37°C no differences were observed. However, after

another round of inoculation and cultivation in fresh medium the expression strain

started to loose cutinase activity due to plasmid instability as described for repB-

containing vectors (25). Therefore, even batch cultures grown under non-selective

conditions inoculated with selective starter cultures will lead to sufficient protein

production and secretion as demonstrated.

time (h)

25 µL 25 µL 25 µL

0- 24 25- 48 49- 72

A

time (h)

B

0

20

40

60

80

100

120

24 48 72

pBSMuL1+pBSMuL1-

pBSMuL2+pBSMuL2-

relative lipolyticactivity

(%)

time (h)

Fig. 7: Stability of pBSMuL vectors in B. subtilis. (A) After inoculation with 25µL from a preculture grown under selective pressure the Bacillus subtilis TEB1030 strains bearing cutinase overexpression plasmids pBSMuL1-Cut and pBSMuL2-Cut were grown three times in 25 mL LB-medium for 24 hours each with and without antibiotic selection. (B) The culture supernatants were harvested by centrifugation after 24 h, 48 h and 72 h of growth with (+) or without (-) kanamycin (50µg/ mL). At these time points the cultures showed no significant growth differences. All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay. The lipolytic activity was adapted to an OD580= 7. The lipolytic activity of strains under selection pressure were defined as 100 %.The results represent means of two independent experiments.

Results

38

3.2 In search of the most functional signal peptide for heterologous protein

secretion in B. subtilis

In addition to the components of the secretion apparatus the signal peptides (SPs) of

the secreted proteins play a crucial role in the efficient translocation across the

cytoplasmic membrane (201). Tjalsma et al. used the computer tool SignalP to

predict essentially all secreted proteins in the Gram-positive bacterium B. subtilis

(188). Nevertheless, the prediction of secretion efficiency becomes even more

complicated when heterologous proteins are fused to the SP instead of the natural

secretion partner: former secretion studies in B. subtilis compared SP variants to the

wildtype when fused to a mature protein and revealed defects or differences for

export efficiency of the hybrid protein (13, 29, 130). But they could not detect this

effect for the same mature protein when fused to another SP. Therefore a strong

influence of the mature sequence was postulated for the secretion effect of a signal

peptide like described by Lehnhardt et al. (110).

Consequently one might suggest that a signal peptide and a secreted protein

constitute a unique unit, where the N-terminus of the mature protein, the so-called

signal-mature junction, plays an important role in the secretion efficiency making the

prediction of “good” SPs for heterologous protein secretion impossible. Therefore, the

high yield of industrially interesting proteins in heterologous fermentation hosts may

still be limited by the secretion efficiency due to the use of a “wrong” SP.

For the first time a strategy was chosen to bypass this limitation by generating a

library of all 173 native Sec-type B. subtilis SPs allowing to test each for export

efficiency in combination with a heterologous target protein.

3.2.1 Establishment of a high-throughput screening (HTS-) system to evaluate

secretion efficiency for heterologous proteins in B. subtilis

Next to the immense potential of automated screening processes for finding and

analysis of enzymes with improved or new properties for biotechnological

applications, HTS systems bear numerous factors leading to fluctuations that can be

critical to a successful screening especially for comparative studies. The most

relevant factors are (i) pipetting errors, (ii) different growth/expression rates if

Results

39

microorganisms (often E. coli) are used as cell factories mostly in combination with

an inducible expression system and (iii) instability of substrate and/or enzyme in the

performed assay.

Yet a screening in the Gram-positive bacterium B. subtilis provides even more

challenges in relation to E. coli: Most of the available vector systems are integrated

plasmids due to a higher transformation efficiency compared to replicating plasmids.

But if those are used for the screening of gene libraries, they cannot easily be

recovered for the purpose of necessary sequence analysis. Anyway, the general

transformation process is more extravagant, the common transformation rate is

significantly lower than in E. coli making the library construction a more time-

consuming procedure. In summary, the discovery and analysis of all factors that

reduce the screening capacity are important preconditions for an efficient HTS

system and are presented in this chapter.

3.2.1.1 Construction of a Bacillus vector system using cutinase as a secretion

reporter

In this study the heterologous lipoloytic enzyme cutinase from the fungus Fusarium

solani pisi was chosen as a secretion reporter for heterologous protein secretion in

B. subtilis, because of its eukaryotic origin and the availability of easy

spectrophotometrical activity assays (Fig. 8). Furthermore, cutinase itself and more

important the class of enzymes it belongs to – the so-called α/β-hydrolases – are of

high biotechnological importance (84, 121). For example the protease (subtilisin)

NO2

O (CH2)14

CH3

O

H2O

NO2

OH

+

HO (CH2 )14

CH3

O

p-nitrophenyl-palmitate (pNPP) p-nitrophenol palmitic acid

cutinase

Fig. 8: Schematic overview about the hydrolysis-reaction which the assay is based on. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C. The formation of p-nitrophenol leads to a yellow colour of the assay solution whose intensity is a measure for activity increase.

Results

40

present in washing powder also belongs to the family of α/β-hydrolases showing high

structural similarities to cutinase used in this study (84, 117, 120, 121).

The first step in the optimization was to choose a suitable expression system for the

cutinase under the control of a constitutive promoter. Therefore the E. coli-B. subtilis

shuttle plasmid pBSMuL3 was constructed (Fig. 9): The artificial MCS was introduced

in the opposite direction to pBSMuL1

(compare Fig. 4) downstream from the

constitutive promoter PHpaII resulting in

ten unique restriction sites without a

Bacillus ribosome binding site or a signal

sequence. The two SacI restriction sites

flanking the Bacillus part of the vector

enable the formation of vector

multimeres with internal repeats (65)

which can be transformed efficiently

(~ 5X103 transformants/µg DNA) into

B. subtilis thereby suitable for gene

library construction. The cutinase gene

encoding the marker enzyme was

amplified by a standard PCR from E. coli

expression plasmid pMAC5-8 (176)

using the primer pair cut1/cut_low and

EcoRI/BamHI cloned into pBSMuL3 resulting in plasmid pBSMuL3-Cut. Once

transformed into B. subtilis no expression of cutinase occurred (data not shown)

since neither a rbs nor a start codon exists upstream from it. The cloning of different

SPs into the MCS of vector pBSMuL3-Cut in front of the cutinase gene results in

expression of a possibly transportable precursor form, thus allowing to study the

effects of any defined SP for cutinase export.

Fig. 9: Plasmid map of shuttle vector pBSMuL3 (7494 bp). PHpaII, constitutive Gram-positive promoter; MCS, multiple cloning site introduced in opposite direction as in pBSMuL1 (Fig. 4); ori E. coli, gene required for replication in E. coli; amp

r, β-lactamase gene conferring

ampicillin resistance in E. coli; repB, gene required for replication in B. subtilis; kan

r, kanamycin resistance gene. two SacI restriction sites for multimeres forming, required for high transformation rate in B. subtilis.

BamHI

ori E.coli

pBSMuL7494 bps

repB

kan r

amp r

pBSMuL37494 bps

repB

PHpaIIPHpaIIPHpaII

kan r

amp r

ori E.coli

MCSMCS

XhoI

EcoRV

NotI

HindIII

SmaI

SalI

KpnI

EcoRI

NdeI

SacI

SacI

Results

41

3.2.1.2 Analysis of growth differences in microtiter plates

A reliable screening system

claims an equal or at least a

similar growth of all cultures

compared. Since the

cultivation of B. subtilis was

intended to be performed in

DeepWell microtiter plates

(DW-MTP, 96 wells, 2 mL

total volume each), it was

imperative to analyse the

growth of B. subtilis with

respect to the minimization of

growth differences between

each well. Therefore

B. subtilis TEB1030 was

transformed with the cutinase

overexpression plasmid

pBSMuL3-Cut-ssLipA. The

resulting Bacillus strain was

able to secrete cutinase into the culture supernatant (see Fig. 17). About 660

colonies of this strain, grown not longer than 24 hours on selective LB-agar plates,

were transferred to DW-MTPs and incubated in 1 mL selective LB-medium each

(37°C, 600 rpm). At defined time points the cell density of one DW-MTP (= 95

cultures plus one blank without cells) was measured spectrophotometrically at a

wavelength of λ= 580 nm. The lowest growth variation was found in the time period

between 9 and 20 hours of growth (Fig. 10.A). With respect to practical lab work the

time point after 16 hours of growth was supposed to be suitable and chosen for a

more extended growth study: The cell density of 780 clones grown for 16 hours

under the same growth conditions distributed over eight DeepWell plates was

determined and revealed a standard deviation lower than 10 % (Fig. 10.B). In

addition no significant growth differences occurred between wells of the inner part

compared to wells of the outer regions of the plate (data not shown). This similar

0

1

2

3

4

5

6

0 100 200 300 400 500 600 700 800

0

1

2

3

4

5

6

0 10 20 30 40 500

20

40

60

80

100

120

A

Btime [h]

number of Bacillus clones

standarddeviation

[%]

cell growth [OD580]

cell growth [OD580]

Fig.10: Growth of B. subtilis in DeepWell microtiter plates. B. subtilis TEB1030 bearing plasmid pBSMuL3-Cut-ssLipA was incubated in 1 mL selective LB-medium per well at 37°C continiously shaking at 600 rpm. (A) The recorded cell density [OD580] is indicated by a blue line, the respective standard deviation (%) is indicated by a red line. Each value represents the average of 95 individual cultures of a DW-MTP. (B) The cell density [OD580] of 780 clones was recorded after 16 hours of growth. The mean value of OD580= 3,4 is indicated as a red line. The standard deviation was 9,6 %.

Results

42

growth behaviour of B. subtilis cultures under the chosen conditions in DeepWell

plates was considered to be sufficient for a reproducible high throughput screening.

3.2.1.3 Development of a HTS-process for detection of export efficiency using a

pipetting robot

Since the cutinase was chosen as the secretion reporter for HTS, the lipolytic activity

towards the standard substrate p-nitrophenyl-palmitate (p-NPP) had to be adapted to

an automated pipetting process in MTPs using a TECAN workstation Genesis 200

Freedom (Tecan, Crailsheim, Germany) as shown in Fig. 11. For this purpose a

pipetting program had to be designed and several parameter of the instruments had

to be coordinated in order to automate all required steps of the assay. (Fig. 12, 35). A

more detailed description is given under 2.13.5. The generated process was adapted

for the efficient MTP transport from the hotel (Fig. 11.B) to the pipetting station (Fig.

11.D), to the spectrophotometer (Fig. 11.E) and back to the hotel using the ROMA

(Fig. 11.C). Furthermore, a minimized pipetting programm containing permanent

B

D E A C

Fig. 11: The pipetting robot “TECAN workstation Genesis 200 Freedom”. Relevant features are (A) A commercially available computer system using Windows 2000 containing softwares of Facts, Gemini and Magellan. (B) A rotatable “hotel” to store and to release MTPs. (C) A roboting arm (ROMA) to grip, move and release MTPs. (D) A pipetting station containing eight needles (green coloured) for simultaneous uptake and release of liquids. (E) A heatable spectrophotometer “GENios” able to measure absorption, fluorescence or luminescence.

Results

43

washing steps with desalted water between the necessary pipetting and dilution

steps ensures a fast screening progress and avoids contaminations among the

different wells (Fig.12.B). Additionally the pipetted volumes were chosen to be no

smaller than 20 µL to minimize pipetting standard deviation to less then 3 % (data not

shown). However, the theoretically screenable number of 60 MTPs per day in a

maintenance-free process is clearly limited by the instability of the substrate pNPP in

the assay solution which leads to autohydrolysis of the substrate after five hours at

A

Fig. 12: Two different software scripts providing the complete pipetting process for one source plate. (A) the higher Process Manager programm “Ulf_PnPP1_10V_Trog200” and (B) the secondary Gemini Script “Ulf pNPP1_10 tiptouch bis 10 Pl.gem” controlling the pipetting station. For more details see text and chapter 2.13.5.

Results

44

RT (data not shown). This results in a screening process of 13 MTPs maximum

according to the time schedule calculated by the Facts software.

3.2.1.4 Validation of the automated HTS process in B. subtilis

The generated process was simulated for ten MTPs. The Facts software scheduled a

total run time of four hours (Fig. 13.A). Therefore the stability of cutinase stored at RT

up to four hours had to be defined. The supernatant of one batch culture B. subtilis

TEB1030 pBSMuL3-cut-sslipA was transferred into a MTP and used as the same

source plate for five measurements (Fig. 13.B) using the created process as shown

in Fig. 12.

B C

020406080

100120

0 1 2 3 4

incubation time at RT (h)

lipol

ytic

act

ivity

(%

)

A

Fig.13: Reproducibility and stability study of cutinase activity of B. subtilis culture supernatant over four hours performed with the pipetting robot. B. subtilis TEB1030 expressing cutinase on plasmid pBSMuL3-cut-sslipA was grown in 25 mL selective LB-medium for 20 hours and the culture supernatant was transferred into a MTP and used in the created HTS process. (A) Screenshot of the time schedule of a HTS process calculated from FACTS software for ten “source” plates. The time periodes of the ROMA in action is indicated by a red bar, those of the pipetting station and the GENios spectrophotometer in action are indicated by green and blue bars, respectively. The hole process was automatically interlocked optimizing the procedure time to 4 hours. (B) The HTS process for one source plate was started after incubating the supernatant for a defined time at RT. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C in the GENios spectrophotometer. The cutinase activity of supernatant used for the HTS without incubation time was defined as 100 %. The standard deviation calculated of 94 wells each was always lower than 8 % and is indicated in error bars. (C) Magellan software transformes the raw data into a graph recorded by a GENios spectrophotometer. Exemplarily the recorded results for one MTP after 2 hours incubated at RT are shown as a screenshot. In each well the blue line indicates the increase of absorbance during 15 min of measurements. Wells A1 and A2 are negative controlls showing no lipolytic activity. The recored data are automatically transferred to Excel and are available for further calculations.

Results

45

The standard deviation of the assay was continiously less than 8 % (Fig. 13. B,C)

and even after four hours at RT the cutinase showed only minimal reduction of

lipolytic activity of about 5 % (Fig. 13. B).

A further approach was followed to define possible variations after retransformation

experiments. Thus the same process was performed with five “source” plates

containing supernatant that was collected from five different DW-MTPs inoculated

from different B. subtilis TEB1030 clones transformed with the same plasmid

pBSMuL3-cut-sslipA. This study confirmed the stability of the cutinase over the time

but revealed a significant increase in fluctuations of cutinase activity up to 25 % (Fig.

14).

3.2.2 Library construction and high-throughput screening of all Sec-type signal

sequences fused to cutinase.

In a so far unique approach the DNA sequences encoding for all presently identified

173 Sec-signal peptides (SPs) (188) ranging from 57 bp up to 138 bp were amplified

0

20

40

60

80

100

120

140

0 1 2 3 4

Fig. 14: Fluctuations in cutinase activity of different B. subtilis cells expressing and secreting wildtype cutinase after transformation with the same plasmid. B. subtilis TEB1030 was transformed with plasmid pBSMuL3-Cut-ssLipA. Single colonies grown on selective LB-agar plates were transferred each into one well of five DW-MTPs altogether and incubated in 1 mL selective LB-medium at 37°C continiously shaking at 600 rpm. After 16 hours of growth the supernatant was transferred into a new MTP. The HTS process for one “source” plate was started after incubation of the supernatant for a defined time at RT. The absorbance at 410 nm was detected by recording a 15 min kinetic at 30°C in the GENios spectrophotometer. The cutinase activity of supernatant without incubation time was defined as 100 %. The standard deviation calculated from 94 wells each (Two wells are blanks) was not higher than 25 % and is indicated by error bars. It represents growth differences, assay fluctuations and variations by plasmid copy numbers after retransformation.

incubation time at RT [h]

lipolytic

activity

[%]

Results

46

by standard PCR from genomic DNA of B. subtilis 168 using the specific primer pairs

(Table 12, Appendix) and extracted from agarose gel after electrophoresis (Fig.

15.A). All upstream primers contain a Bacillus consensus ribosom binding site (rbs)

and the ATG-start codon essential for efficient expression of cutinase in B. subtilis.

The plasmid pBSMuL3-Cut was used for in-frame cloning of all SPs in front of the

marker enzyme cutinase resulting in plasmid library pBSMuL3Cut-ss (Fig. 15.B).

Since the hpaII promotor and the rbs were also recognized in E. coli, the

prescreening was performed in E. coli JM109. Here, only the fusion of an amplified

SP, containing the rbs and the ATG-start codon and cutinase leads to expression of

cutinase protein and to the formation of halos around the E. coli colonies on tributyrin

A B C

D

cutinase BamH I EcoR I Hind III

pBSMuL3 ori E.coli

repB

kanr

ampr

MCS

7987 bp

P

ss

E F

genomic DNA B. subtilis ss

174 PCR reactions

Fig. 15: Schematic overview of amplification, cloning and screening of all Sec-type signal peptides (SPs) of B. subtilis. (A) PCR amplification of all SPs using specific primer pairs and genomic DNA from B. subtilis 168 as the template. (B) Cloning step into the E. coli–B. subtilis shuttle vector pBSMuL3Cut upstream of the heterologous secretion target cutinase. (C) Prescreening in E. coli JM109 on lipolytic indicator plates and plasmid isolation of halo-forming colonies. (D) Transformation of all plasmid encoded SP-cutinase fusions into B. subtilis TEB1030 and growth in MTPs. (E) Isolation of culture supernatant by centrifugation. (F) Automated spectrophotometric HTS-assay for cutinase quantification in isolated culture supernatants.

Results

47

indicator agar (Fig. 15.C). The plasmid DNA from halo-producing cells was isolated,

analyzed by restriction analysis or sequencing. The produced plasmid library

containing all B. subtilis Sec-signal sequences was transformed into B. subtilis

TEB1030, a strain lacking any disturbing extracellular lipolytic activity (48). After

growth on selective agar the colonies were transferred into Deepwell microtiter plates

and cultured for 16 hours in 1 mL LB-medium per well at 37°C using a microplate

shaker (Fig. 15.D). After a centrifugation step the culture supernatant of each clone

was carefully transferred into a new microplate for quantification of lipase activity

(Fig. 15.E). The lipolytic activity towards the standard substrate p-nitrophenyl-

palmitate (p-NPP) was quantified by the automated lipase activity assay using the

TECAN workstation Genesis 200 Freedom (Fig. 15.F) as described under 2.13.5.

3.2.3 The highest D-score does not necessarily predict the optimal signal

peptide for secretion of the heterologous cutinase.

The D-score calculated by the SignalP3.0 (9, 135) indicates the probability of an

amino acid sequence to function as a signal peptide. Therefore, high D-scores (0.8-

1.0) usually indicate high chances of secretion. Commonly, these sequences are

believed to work efficiently in protein translocation across the membrane. In contrast,

D-scores below 0.7 (0.5 - 0.7) indicate lower secretion efficiencies and values below

0.5 are commonly regarded as not recognized by the secretion machinery. However,

by evaluating the complete set of native signal peptides from B. subtilis we were able

to prove this generally accepted strategy to classify SPs.

Therefore SignalP 3.0 was used to determine the D-score for the first 70 amino acids

of all hybrid proteins (SP-cutinase) as shown in Table 13 (Appendix). In most cases

the SignalP-data confirms a signal peptide and the predicted cleavage site position

(C-site) in front of the +1 alanine to be recognized by a SPase I (Fig. 16).

Results

48

Among the plasmid library pBSMuL3Cut-ss 148 different SP-cutinase fusions were

successfully transferred into B. subtilis TEB1030. The screening of this signal peptide

library resulted in a clear ranking of all SPs as summarized in Figure 17 and Table 7.

The detailed results are given in Table 13 (Appendix).

AAGGAGGATATT ATG........................ GCTGAATTC GCG...

Met... .................... AlaGluPhe Ala...

Rbs Spacer

+1-1-3 -2

junction mature cutinaseSignal Sequence

N ++ H C

AAGCTT

A

B

1

0,8

0,6

0,4

0,2

0

10 20 30 40 50 60

score

Fig. 16: (A) Overall composition of signal peptides (SPs) fused to the mature junction of cutinase and (B) its computer analysis exercising SignalP 3.0. (A) The Bacillus ribosome binding site (rbs) and the spacer in front of the ATG-start codon ensure efficient mRNA-translation of the signal sequence gene-fusion. The signal peptides are devided into three domains: the positively charged N-region, the hydrophobic H-region and the C-region coding the recognition site for SPaseI. All SP-cutinase fusions contain the junction built of the amino acids alanine, glutamate and phenylalanine. The HindIII (AAGCTT) and EcoRI (GAACTT) restriction sites for cloning are underlined. (B) The graphical output of a SignalP 3.0 computer analysis is shown exemplarily for the first 70 amino acids of the hybrid protein SP-LipA cutinase calculated with the hidden Markov model (SignalP3-HMM ) for gram positive bacteria which was performed for all 173 hybrid proteins of SP fused to cutinase. The score represents the probability (1.0 = 100 %) for n-region (green line), h-region (dark blue line), c-region (turquoise blue line) and cleavage site (red line). The critical score of 0.5 for of an amino acid indicating significance to be part of a SP region is marked as a punctured line.

amino acid position of the hybrid protein

Results

49

This method to determine cutinase export based on an activity assay was verified by

immunoblotting experiments using cutinase-specific antibodies. Exemplarily, 7

different SP-cutinase fusions were analyzed by Western-blotting with the SP-fusions

of protease Epr (4.67 U/mL), unknown protein YncM (4.12 U/mL), lipase LipA (2.79

U/mL) and phosphodiesterase GlpQ (1.96 U/mL) chosen because of their relatively

high cutinase activity in the culture supernatant. Furthermore SP-fusions of α-

amylase AmyE (0.67 U/mL) leading to moderate cutinase activity and nuclease NucB

(0.34 U/mL) as well as protease WprA (0.12 U/mL) showing low cutinase activity in

the culture supernatant. This direct cutinase quantification on protein level (Fig. 18)

was in accordance of our activity based quantification of cutinase in the culture

medium (Table 7).

Fig. 17: Comparison of all 148 screened signal peptides (SPs) used for export of heterologous cutinase in B. subtilis. The SPs of Epr, YncM, LipA, GlpQ, AmyE, NucB and WprA which have been analyzed further by Western-blotting and pulse-chase experiments are highlighted in red. The lipolytic activity assay of the culture supernatants were performed at 30°C. The enzymatic activity was calculated using a molar absorbtion coefficient of 15000 M-1·cm-1. The results represent data of 12 independent experiments. The standard deviation was not higher than 25 %.

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145

Sec-type SPs fused to cutinase

lip

oly

tic a

cti

vit

y [

U/m

L]

AmyE, 0.67

Epr, 4.67

YncM, 4.12

LipA, 2.79

GlpQ, 1.96

WprA, 0.12NucB, 0,34

Results

50

The size of cutinase identified in the supernatants corresponds to the purified mature

cutinase (22 kDa) isolated from recombinant E. coli. The exclusive occurence of

processed protein in B. subtilis culture supernatants indicates complete cleavage of

the SP by a SPaseI at the trans-site of the membrane. This processing results in

release of only mature cutinase into the medium and strongly indicates successful

secretion via the Sec-apparatus and cancels out cell lysis as an alternative

explanation. Interestingly, intracellularly expressed cutinase lacking a SP could not

be detected in the cytoplasm of B. subtilis or in the supernatant showing rapid

intracellular degradation of mature cutinase (Fig. 18, lane 2 and 3). Additionally,

when the cutinase was expressed with a SP, in all cases no precursor protein was

detectable in the cytoplasmic fraction (immunoblot not shown). However, a general

observation for the 25 best SPs for cutinase transport was the divergence of the

amino acid at position -3 in the C-region of the B. subtilis. consensus motif A-X-A in 8

cases to V-X-A. In contrast to that, only alanine was found at position -1. The C-

region is responsible for recognition and cleavage of the SP by one of the SPases I.

Thus at least for the fusion with the cutinase it is possible to define the most efficient

cleavage site as A/V-X-A.

Surprisingly, these results suggest that high secretion efficiencies can not simply be

correlated with known D-scores. Most of the best performing SPs identified in the

4 5 6 7 8 9 101 32

cutin

ase

C S

intracellularexpression

SP-Ep

r

SP-Y

ncM

SP-L

ipA

SP-G

lpQ

SP-Am

yE

SP-N

ucB

SP-W

prA

Figure 18: Immunodetection of cutinase in culture supernatants of B. subtilis expression strains. Western blot analysis of B. subtilis strains expressing different SP-cutinase fusions. Of each sample 20 µg protein were subjected to immunodetection by cutinase-specific antibodies produced in hybridoma cells. Lane 1: As positive control 1,5 µg purified cutinase was loaded. Lanes 2-3: intracellular expression of cutinase using vector pBSMuL3Cutintra; cellular fractions (C) and supernatants (S) were isolated by centrifugation. Lanes 4-10: Culture supernatants isolated from B. subtilis strains expressing various SP-cutinase fusions (SP-Epr, SP-YncM, SP-LipA, SP-GlpQ, SP-AmyE, SP-NucB and SP-WprA).

Results

51

screening show D-scores around 0.93 (e.g. Bpr, 0.936; YjfA, 0.924; Epr, 0.919) as

expected; nevertheless, SPs showing D-scores around 0.5 – 0.6 were also identified

(e.g. YncM, 0.507; Csn, 0.684) that work highly efficient in cutinase secretion (Table

7).

Furthermore, the screening revealed many SPs with high D-scores showing

moderate (e.g. GlpQ, AmyE), low (e.g. DacF) or no secretion at all (e.g. YodV) as

summarized in Table 7. In addition, further SP-characteristics like the number of

charged amino acids in the N-region as well as the overall hydrophobicity do not

correlate with secretion efficiencies, indicating no clear patterns or rules to predict the

No.

Name

Signal sequence

Secreted cutinase [U/mL]

a

Charge

N-region b

Hydro-

phobicity [% ]

c

D-

Score d

1 Epr MKNMSCKLVVSVTLFFSFLTIGPLAHA 4.67 2 62.96 0.919 2 YncM MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADA 4.12 4 76.19 0.507 3 YjfA MKRLFMKASLVLFAVVFVFAVKGAPAKA 3.84 3 78.57 0.924 4 YfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA 3.67 3 68.97 0.906 5 Csn MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA 3.35 3 62.86 0.689 6 LytD MKKRLIAPMLLSAASLAFFAMSGSAQA 3.33 3 70.37 0.87 7 Bpr MRKKTKNRLISSVLSTVVISSLLFPGAAGA 2.97 5 56.67 0.936 8 WapA MKKRKRRNFK RFIAAFLVLA LMISLVPADVLA 2.88 8 65.63 0.918 9 BglC MKRSISIFITCLLITLLTMGGMIASPASA 2.87 2 65.52 0.839

10 LytB MKSCKQLIVCSLAAILLLIPSVSFA 2.83 2 64.00 0.916 11 LipA MKFVKRRIIALVTILMLSVTSLFALQPSAKA 2.79 4 64.52 0.874

15 PhrK MKKLVLCVSILAVILSGVA 2,53 2 73,68 0,669 … 26 GlpQ MRKNRILALFVLSLGLLSFMVTPVSA 1.96 3 69.23 0.921 … 65 AmyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA 0.67 3 78.79 0.904 … 75 NucB MKKWMAGLFLAAAVLLCLMVPQQIQGASS 0.34 2 72.41 0.746 … 88 DacF MKRLLSTLLIGIMLLTFAPSAFA 0.14 2 73.91 0.909 89 TyrA MNQMKDTILLAGLGLIGGSIALA 0.13 0 73.91 0.466 90 LytF MKKKLAAGLTASAIVGTTLVVTPAEA 0.13 3 65.38 0.744 91 WprA MKRRKFSSVVAAVLIFALIFSLFSPGTKAAA 0.12 4 67.74 0.941 …

114 YnzA MELSFTKILVILFVGFLVFGPDKLPALG 0 0 78.57 0.468 115 YobV MKLERLLAMVVLLISKKQVQA 0 1 61.9 0.623 116 YocH MKKTIMSFVAVAALSTTAFGAHA 0 2 65.22 0.88 117 YodV MKVPKTMLLSTAAGLLLSLTATSVSA 0 2 61.54 0.927

a The lipolytic activity assay was performed at 30°C; the enzymatic activity was calculated using a molar extinction coefficient of 15000 M-1·cm-1. b The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine and lysine defined as +1 and any other amino acid defined as 0. c The percentage of hydrophobic amino acids of each signal sequence was calculated with amino acids G, A, V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. d D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) for the first 70 amino acids of the hybrid proteins

Table 7: Comparison of chosen screened signal sequences used for export of heterologous cutinase in B. subtilis. The results represent data of 12 independent experiments. The standard deviation was below 25 %. The complete data is listed in Table 13 (Appendix). The SPs which have been analyzed further by Western-blotting and pulse-chase experiments are highlighted in red.

Results

52

a The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine and lysine defined as +1 and any other amino acid defined as 0. b The percentage of hydrophobic amino acids of the each signal sequence was calculated with amino acids G, A, V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. c D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) for the first 70 amino acids of the hybrid proteins

optimal SP fusion-partner: One of the best SPs for cutinase secretion was SP-WapA

with 2,88 U/mL containing a huge number of 8 positive charges in the N-region. On

the other hand the SP-BglC has almost the same sequence length, shows an equal

cutinase export (2,87 U/mL) but possesses only 2 positively charged aa in the N-

region. It is remarkable that there is no direct dependency not even between the

length of the SPs and export efficiency. For instance the shortest screened SP-PhrK

(19 aa) shows a good export capacity of 2,5 U/mL, but the SP-YncM which

represents one of the longest sequences (42 aa) provides also an efficient cutinase

export of more than 4 U/mL. It is noteworthy that among the 15 best SPs many

sequence lengths occur around the average length for B. subtilis of about 28 aa.

Table 8: Signal peptides (SPs) not tolerated in B. subtilis when constitutively expressed in front of heterologous cutinase. C-regions which diverge drastically from the B. subtilis consensus cleavage site are highlighted in red.

No.

Name

SP: N- and H-region

SP: C-region

Charge N-region

a

Hydro-phobicity

[% ]b

D-Score

c

149 CwlD MRKKLKWLSFLLGFIILLFLFKYQ FSN 5 62,86 0.708 150 DltD MKKRFFGPIILAFILFAG AIA 3 85,71 0.653 151 FliL MKKKLMIILLIILIVIGALG AAA 3 86,96 0.777 152 LytR MRNERRKKKKTLLLTILTIIGLLVLGTGGYAYYLWH KAA 7 53,85 0.554 153 MreC MPNKRLMLLLLCIIILVAMI GFS 2 78,26 0.718 154 PbpB MIQMPKKNKFMNRGAAILSICFALFFFVILG RMA 5 70,59 0.704 155 PbpX MTSPTRRRTAKRRRRKLNKRGKLLFGLLAVMVCITI WNA 12 48,72 0.667 156 PhoA KKMSLFQNMKSKLLPIAAVSVLTAGIF AGA 4 66,67 0.745 157 PhrG MKRFLIGAGVAAVILSGW FIA 2 85,71 0.513 158 SpoIIP MRNKRRNRQIVVAVNGGKAVKAIFLFIVSLIVIFVL SGV 7 66,67 0.436 159 SpoIIR MKKTVIICIYIFLLLSG ALV 2 70 0.649 160 YdjN MKKRIILLLAVIIAAAAA GVA 3 85,71 0.557 161 YjcN MKKKTKIILSLLAALIVILIVLPVLSPVVFT ASS 4 70,59 0.801 162 YocA MKKKRKGCFAAAGFMMIFVF VIA 5 73,91 0.711 163 YolI MKKWIVLFLVLIAAAISIFVYVST GSE 1 70,37 0.638 164 YopL MKKLIMALVILGALGTSY ISA 2 71,43 0.605 165 YpmB MRKKALIFTVIFGIIFLAVLLVSASIYKS AMA 4 71,88 0.774 166 YqfZ MKRLTLVCSIVFILFILFYDLKIGTIPIQDLPVYE ASA 0 63,16 0.397 167 YrrL MYINQQKKSFFNKKRIILSSIVVLFLIIGG AFL 5 60,61 0.610 168 YrrR MKISKRMKLAVIAFLIVFFLLLLRL AEI 4 75 0.706 169 YrrS MSNNQSRYENRDKRRKANLVLNILIAIVSILIVV VAA 4 51,35 0.586 170 YunA MITDIFKPGCRKLCVFNMKGDYFVKVLLSALLLL LFA 3 64,86 0.338 171 YunB MPRYRGPFRKRGPLPFRYVMLLSVVFFILSTT VSL 6 62,86 0.690 172 YveB MNYIKAGKWLTVFLTFLGILLF IDL 1 72 0.568 173 YyaB MVYQTKRDVPVTLMIVFLILLIQ ADA 0 65,38 0.565

Results

53

25 SP-cutinase fusions were not expressed in B. subtilis (Table 8). Here, the

plasmids constructed in E. coli were not successfully transferred into the expression

and secretion strain B. subtilis TEB1030; by this, indicating a lethal effect. About 23

of these SPs do have calculated D-scores below 0.7 suggesting inefficient cleavage

of the signal peptide (Table 8). It can be speculated that these SP-cutinase fusion

proteins accumulate in the cytoplasmatic membrane, which finally prevents cell

growth.

3.2.4 Translocation efficiency is not solely responsible for the varying export

amounts of cutinase

The varying amounts of overexpressed cutinase in the supernatants when fused to

various signal peptides could be explained by different translocation efficiencies

across the cytoplasmic membrane via the Sec- translocase. Therefore, the

processing kinetics of selected hybrid proteins were analysed by pulse-chase

experiments. Since all newly synthesized protein precursors were labelled for a short

time with radioactive 35S-methionine, a defined pool of labelled proteins was

generated. In the next step, the processing of only the target precursor could be

detected by protein precipitation with specific antibodies raised against the cutinase.

If the C-region of a SP is recognized efficiently by a SPase I, the precursor form

should vanish rapidly and simultaneously the amount of mature cutinase protein is

expected to increase.

For these experiments, five SP-cutinase-fusion proteins (SP-AmyE, SP-GlpQ, SP-

LipA, SP-YncM and SP-Epr) with significant differences in cutinase export rate into

the supernatant were selected: two of which evoked the highest cutinase protein

amounts and lipolytic activities in the supernatants (SP- Epr and SP-YncM). Two

hybrid proteins resulted in significantly lower protein amounts and lipolytic activities

but were still counted among the members of the group of “efficient” signal peptides

with respect to the lipolytic activity in the supernatant (SP-LipA, SP-GlpQ). The fifth

hybrid protein yielded in a comparably low amount of cutinase protein and enzymatic

activity in the supernatant (SP-AmyE). In Fig. 19, the processing of cutinase

precursor proteins into the mature form by removal of the signal peptide is shown. A

strain carrying only the vector pBSMuL3 as a control shows no protein bands at all,

Results

54

demonstrating that the proteins observed in the other samples are indeed specific for

the cutinase precursor and the mature form.

Processing kinetics vary dramatically between the different signal peptides, with the

Epr and AmyE signal peptides mediating the fastest processing. For SP-Epr-

cutinase, no precursor is visible even after the shortest time point (10 seconds) of the

chase, while very low amounts of the precursor of the SP-AmyE-cutinase can be

detected after 10 seconds and 30 seconds of the chase. After one minute, no

precursor protein is detectable for the SP-AmyE- cutinase. In case of the SP-GlpQ

and SP-LipA fusion proteins, the amounts of the precursor after 10 seconds of the

chase are significantly higher than those observed for the SP-AmyE- cutinase and

decline rapidly over time. For the SP-LipA- fusion, processing into the mature form is

complete after one minute of the chase, whereas for the SP-GlpQ- fusion only very

low amounts of precursor are present at that time point. The YncM- fusion shows the

slowest export kinetic. After one minute of chase there is still more precursor than

mature form of the cutinase present, and even after 3 minutes of chase, large

amounts of the precursor form of this fusion were visible (data not shown).

10´´ 30´´ 1´ 10´´ 30´ ́ 1´

SP

-A

myE

SP

- YncM

SP

Epr

SP

-G

lpQ

10´´ 30´´ 1´ 10´ ́ 30´ ́ 1´

SP

- Lip

A

10´ ́ 30´ ́ 1´ 10´´ 30´ ́ 1´

contr

ol

10´´ 30´´ 1´ 10´´ 30´ ́ 1´

- - --10´´ 30´´ 1´ 10´ ́ 30´ ́ 1´

-

10´ ́ 30´ ́ 1´ 10´´ 30´ ́ 1´

1 2 3 4 5 6pm

Fig. 19: Analysis of the processing kinetics of cutinase precursor proteins with different SPs in B. subtilis TEB1030 via pulse-chase experiment. Kinetics were measured for strains with either the vector pBSMuL3 as control (lane 1) or cutinase fused to SP-AmyE, SP-GlpQ, SP-LipA, SP-YncM and SP-Epr on plasmid pBSMuL3Cut-ss (lane 2-6, respectively). The Bacilllus strains were pregrown in S7 minimal medium containing methionine at 37°C to an OD600nm of 0.8, washed twice and starved in S7 minimal medium without methionine for 60 min at 33 °C. Subsequently cells (2,5 mL) were labelled with 150µCi [35S]- methionine for 1 min, followed by chasing with vast excess of non-radioactive methionine. Samples (600µL) for each strain were withdrawn at certain time points (10 sec, 30 sec and 1min), precipitated with ice-cold TCA and further processed as described under 2.20. Sample volumes were equalized to 7.500 cpm. Positions of precursor form (p) and mature protein (m) are indicated.

Results

55

If the varying amounts of cutinase in the supernatant for the different signal peptide

cutinase fusion proteins would only be due to differences in the efficiency of protein

translocation across the cytoplasmic membrane, those fusions with the highest

amounts of protein in the supernatant should show the fastest processing kinetics.

However, this is clearly not the case. While the processing of the SP-Epr- fusion

protein, which shows the highest lipolytic acitivity and large amounts of cutinase

protein in the supernatant, indeed is very fast, the second “best” signal peptide with

respect to the amounts of cutinase protein and activity in the supernatant, YncM,

shows the slowest processing of all the proteins investigated in the pulse chase

experiment. Furthermore, the SP-AmyE fusion, which shows the lowest protein

amounts and lipolytic activity in the supernatant of the five proteins analysed in the

pulse- chase, is processed very rapidly. Thus translocation efficiency is not solely

responsible for varying amounts of secreted cutinase, present in the culture

supernatant.

3.2.5 High level production of cutinase in B. subtilis using the best identified

signal peptide

To analyse cutinase yield under batch culture conditions, B. subtilis TEB1030 strains

with cutinase fused to SP-Epr, SP-LipA and SP-AmyE on plasmid pBSMuL3-Cut-ss

were grown in Erlenmeyer flasks. Culture supernatants were analysed by SDS-

PAGE and a spectrophotometric lipase activity test. As shown on the SDS-gel (Fig.

20.A), there is a distinct cutinase band in all supernatants for cutinase fusions to a

SP compared to the negative control that corresponds to the calculated molecular

weight of about 22 kDa for the cutinase. The different amounts of cutinase were

confirmed by the lipolytic activities in the culture supernatant (Fig. 20.B). Additionally,

they show the same SP ranking as listed Fig. 17. According to the specific activity of

200 U/mg for cutinase against the substrate pNPP, about 35 mg cutinase can be

secreted into the medium by B. subtilis using the SP of Epr.

Results

56

B

A

1 2 3 4

cont

rol

SP-Epr

SP-LipA

SP-Am

yE

kDa

50

30

20

0

2

4

6

8

10

lipolyticactivity[U/mL]

1 2 3 4

Fig. 20: Cutinase production by B. subtilis via secretion into the medium. B. subtilis TEB1030 strains with either the vector pBSMuL3-Cut as control (lane 1) or cutinase fused to SP-Epr, SP-LipA, SP-AmyE on plasmid pBSMuL3Cut-ss (lane 2-4, respectively) were incubated in 250 mL flasks with 25 mL selective LB-medium for 20 hours at 37°C under continuous shaking (150 rpm) and showed no significant growth differences. The culture supernatants were isolated by centrifugation. (A) 20 µg of total proteins of each supernatant were concentrated using 70% trichloride acid precipitation and separated on a protein gel (15 % SDS-PAGE) stained with coomassie brilliant blue. The position of the cutinase band is marked with an arrow. The positions of molecular mass reference markers are indicated (kDa). (B) All culture supernatants were tested for lipolytic activity using the spectrophotometric pNPP-assay at 37°C. The lipolytic activity was adapted to an OD580= 7. Standard deviations are indicated by error bars. The results represent data from 3 independent experiments.

O cutinase palmitic acid nitrophenol - p ) NPP p ( palmitate - nitrophenyl - p O 3 H C 4

Results

57

3.3 One-step optimization of the heterologous esterase EstCL1 for secretion

using the SP-library

In the first approach using cutinase as the heterologous secretion reporter, every SP-

cutinase fusion was ligated and cloned separately to ensure the completeness of the

signal peptide fusion library (Fig. 15). Nevertheless, this procedure is tedious and

cost extensive. Therefore, in another approach we tested the possibility to use the

SP-library to find the optimal fusion partner easily in a standardized procedure of (i)

cloning, (ii) transformation and (iii) screening.

As positive control an equimolar mixture of all PCR-amplified SPs was cloned in front

of the cutinase gene. The ligation – theoretically containing all SP-cutinase fusions –

were transferred into B. subtilis TEB1030 and subsequently 930 randomly chosen

clones were screened for cutinase activity in the culture supernatant (data not

shown). The 12 best performing clones leading to secretion efficiencies of 4 – 5 U/mL

cutinase were isolated and sequenced. As the result, the 8 best performing SPs

according to Table 7 were identified again, with SP-YncM identified 3-times as well

as SP-LytD and SP-WapA both identified twice. Thereby it was demonstrated that

the fast random approach identifies the same SPs being highly efficient in cutinase

secretion.

In order to demonstrate the general applicability of this system, a second

heterologous protein was chosen for secretion optimization in B. subtilis. The

esterase EstCL1 of metagenomic origin was amplified from plasmid p11EstCL1 (109)

by a standard PCR using the primer pair EstCL1_up / EstCL1_low and cloned into

vector pBSMuL3 resulting in vector pBSMuL3-EstCL1. An equimolar mix of all

amplified SPs was cloned into plasmid pBSMuL3-EstCL1 resulting in the plasmid

library pBSMuL3-EstCL1-ssMix which was finally transformed into B. subtilis

TEB1030. The culture supernatants of more than 950 clones were tested in a

modified HTS assay using the TECAN workstation towards the standard esterase

substrate p-nitrophenyl-caproate (p-NPC). Plasmid DNA of ten clones showing

highest secretion efficiencies of EstCL1 was isolated and sequenced. Signal peptides

NprE and YfhK were identified twice; therefore, 8 SPs were characterized in more

detail (Fig. 21), with SP-YwmC being the most efficient one with respect to EstCL1-

secretion transporting up to 1400 U/L across the membrane.

Results

58

The measured activity corresponds to 17,5 mg protein/L calculated with a specific

activity of 80 U/mg against substrate pNPC determined at same conditions. Again,

like in cutinase secretion, the D-scores do not correlate in every case with the

secretion efficiencies of the target proteins. Furthermore, the best performing SPs in

EstCL1-secretion do not correlate with the SP-ranking of cutinase secretion (Fig. 21)

indicating the uniqueness of each hybrid protein.

Figure 21: Identification of the most efficient signal peptide in secretion of the heterologous esterase EstCL1 in B. subtilis. (A) The plasmid library pBSMuL3EstCL1-ssMix containing a mix of all Sec-type signal peptides fused to esterase EstCL1 was transferred into B. subtilis TEB1030. Culture supernatants of about 950 transformants were screened towards esterase activity using the HTS system described before with p-NPC as an esterase substrate. Plasmid DNA of ten clones showing highest activity was sequenced and tested again towards esterase activity. The increasing absorbance at 410 nm was detected by recording a 15 min kinetic at 37°C. The results represent data from three independent experiments. The standard deviation is indicated with error bars and was below 20 %. The D-score was calculated for the first 70 amino acids of each hybrid protein SP-EstCL1 using Computer program Signal P 3.0. (B) The ranking/secretion efficiency of the identified SPs are listed for SP-cutinase hybrid proteins according to Table 13 in the Appendix. The secretion efficiency of the best SP-Epr was defined as 100 % and is according to cutinase activity of 4,67 U/mL.

013,3078,6003,225,1

ranking 1126614541421188653

D-score

secretionefficiency

[%]

0,5880,7990,6950,8550,7250,7420,8170,875

SP-name

este

rase

Est

CL1

cutin

ase

YwmC YpjP YojL YwsB YfhK YxaK NprE YlxW

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

0,5

0,55

0,6

0,65

0,7

0,75

0,8

0,85

0,9

es

tera

se

ac

tiv

ity

[U/m

L]

D-s

co

re

A

B

Results

59

3.4. Directed Evolution of SP-AmyE to achieve high yield of cutinase export

As described before the secretion efficiency of the heterologous model enzyme

cutinase from the fungus Fusarium solani pisi in B. subtilis was improved by

identifying the best natural B. subtilis SP. Another possibility to increase the

functionality of a SP might be the usage of mutagenesis techniques. Directed

Evolution is a powerfull method for enzyme optimization based on random

mutagenesis of the target gene and does not stringently require detailed knowledge

of 3D-structure or reaction mechanism (54, 87, 125, 146, 159). So far Directed

Evolution was rarely used in combination with a screening in B. subtilis mainly due to

the lack of a suitable cloning and screening system resulting mostly in the choice of

E. coli as expression host.

But the establishment of an automated screening process for secretion (chaper 3.2)

offers the opportunity to screen large gene libraries in B. subtilis which is essentiell

for successful Directed Evolution approaches.

Here, two different non-recombinative methods were used to increase the export

capacity of a SP for the cutinase: i) The random insertion of mutations into a SP

using error-prone PCR and ii) saturation mutagenesis at defined regions of a SP

using wobbled oligonucleotides (2, 18, 27, 226).

After the construction of the corresponding signal sequence mutant library the

following cloning procedure using plasmid pBSMuL3-Cut and the automated

screening for lipolytic activity was performed as described in Chapter 3.2.

Based on the screening results summarized in Table 7 the SP of AmyE was chosen

for optimization by Directed Evolution showing a moderate secretion of cutinase

(0.67 U/mL). An overview of the detailed composition of the hybrid protein SP-AmyE-

cutinase and its computer analysis using SignalP 3.0 is given in Fig. 22.

m

Results

60

3.4.1 Random mutagenesis of SP-AmyE did not lead to increased amount of

exported cutinase

Random point mutations were generated performing an ep-PCR by variation of the

MnCl2-concentration in the reaction buffer. The ep-PCR was performed using primer

pair ss-AmyE-up/ -low and plasmid pBSMuL3-Cut-ssAmyE as the template. The

mutation rate was analysed for different concentrations of MnCl2. A compromise

between sufficient PCR product and a reasonably high error rate 0.35 mM MnCl2 was

chosen resulting in an error frequency of 10 base exchanges per kb DNA. This

results in about one mutation per SP. According to the size of 33 amino acids of SP-

AmyE the theoretical library size of about 6000 variants was calculated by using a

formula described elsewhere (7, 87) considering a 10 fold oversampling.

Nevertheless, the screening of more than 3200 clones revealed no significant

improvement of cutinase export compared to wild type SP-AmyE (data not shown).

Fig. 22: Composition of hybrid protein SP-AmyE-cutinase and its computer analysis exercising SignalP 3.0. (A) The signal peptid of AmyE consists of 33 amino acids and is devided into three domains: the N-region (light blue) with three positively charged amino acids, the hydrophobic H-region (black) and the C-region coding (green) the recognition site for SPaseI. The junction (J) between SP and mature protein is indicated in gray. (B) Computer analysis of first 70 amino acids of SP-AmyE fused to cutinase using Signal P 3.0. The graphical output from SignalP was calculating with the hidden Markov model (SignalP3-HMM ) for gram positive bacteria. The score represents the probability (1.0 = 100 %) for a cleavage site (red line), n- and c-region (light blue line) and the h-region (dark blue line).

+1

CN (7) H (23)

TSLLPLFAGFLLLFHLVLAGPAA A E F CutinaseA S AMFAKRFK

J

SPase I A

B

-1 +1 -2 -3

Results

61

3.4.2 Saturation mutagenesis of SP-AmyE revealed improved variants

Since the N- as well as the C-domain of a SP are known to have high impact on the

secretion level of the fusion partner (13, 28, 164, 170), both regions of SP-AmyE

(Fig. 22.A) were subjected to saturation mutagenesis. Additionally the Ala at the +1

position in the junction region was mutated because the most frequent amino acid

Ala at this position for all Bacillus Sec-type exoproteins does not necessarily

represent the most effective one for foreign protein export. Except for the C-region,

the saturation libraries were generated in a standard PCR reaction according to the

oligonucleotides listed in Table 9.

Table 9: Positions of SP-AmyE chosen for saturation mutagenesis. Randomization of bases was performed using an equimolar mix of all dNTPs (N) for the first two bases and an equimolar mix of only dCTP and dGTP (S) for the third base of a codon. Plasmid pBSMuL3-Cut-ssAmyE was used as the template.

Upstream Primer1 Downstream Primer1 Saturated position2 Saturated region of SP-AmyE

AmyE-2NNS-up AmyE-low Phe2

AmyE-3NNS-up AmyE-low Ala3

AmyE-4NNS-up AmyE-low Lys4

AmyE-5NNS-up AmyE-low Arg5

AmyE-6NNS-up AmyE-low Phe6

AmyE-7NNS-up AmyE-low Lys7

N-region

AmyE-up AmyE-low+1NNS Ala+13 +1 region

1 complete sequences listed in Table 5

2 positions as indicated in Fig. 25

3 Ala+1 is the first position of the mature protein as shown in Fig. 23

In order to create a saturation library covering the whole C-region, a mix of wobbled

oligonucleotides synthesized in cooperation with the biotechnological company

BioSpring AG (Frankfurt, Germany) was used. This single stranded (ss-) DNA mix

contains the whole sequence for SP-AmyE with partially wobbled bases encoding for

the three amino acids of the C-domain as listed in Table 10. The base compositions

were chosen to prefer the consensus motif of the C-region Ala-X-Ala (188) (ratio 9:1)

in the resulting SP-AmyE library.

Results

62

Table 10: Base composition of oligonucleotide mix to saturate the C-region of SP-AmyE. Randomization of bases was performed using an equimolar mix of all dNTPs (N) for the first two bases and an equimolar mix of only dCTP and dGTP (S) for the third base of a codon. “X” is defined as any aa.

1 positions as indicated in Fig. 23

2 ratio was chosen with preference for the consensus motif Ala-X-Ala in the C-region of a B. subtilis Sec-type SP

Every synthesized ss-DNA forms a hairpin at the 5ènd that was used as a primer in

a Polymerase-reaction. This was perfomed by using the enzyme T7-Polymerase

(Fermentas, St. Leon-Rot, Germany) as described by the manufacturers protocoll

resulting in double stranded DNA ready for subsequent cloning.

The screening of saturation libraries of the C-domain (∼150 variants) as well as the

+1 position (∼260 variants) revealed no further enhancement of the secretion level

(Fig. 23.B,C). This result was confirmed by a pulse chase experiment performed with

the hybrid protein formed by wildtype SP-AmyE and cutinase: very low amounts of

the precursor of the SP-AmyE-cutinase protein could be detected after 10 seconds

and 30 seconds of the chase indicating no bottleneck in the signal peptidase

catalyzed liberation of the mature protein (Fig. 24).

However, of the C-domain three clones and of the +1 position six clones were

sequenced which were screened with the highest activity but still ranging in the

fluctuation range (25 %) of wild type activity. All sequenced clones showed the AXA-

motif of the C-domain of the SP-AmyE with the amino acids Cys, Val and Gly at

position –2 compared to Ser of wild type SP-AmyE. Thus this consensus motif for the

C-region in Bacillus subtilis might hardly be improvable at least in case of this hybrid

protein. Five of the six sequenced clones found in the +1 saturation library possessed

an Ala, one clone bore a Val at position +1 indicating a clear preference for the amino

acid Ala directly after the SP which can be regarded as the optimal amino acid.

position of SP-AmyE1 -3 (Ala) -2 (Ser) -1 (Ala)

base composition

GCC NNS NNS GCC NNS

ratio [%]2 90 10 100 90 10

resulting aa Ala X X Ala X

oligo-nucleotide mix

prefered aa motif

Ala X Ala

Results

63

Screening of the saturation libraries mutated in the N-region of SP-AmyE revealed

four interesting variants leading to highly efficient cutinase secretion (Fig. 25.B,C). In

order to cover the complete saturation library about 200 SP-variants were screened

per position (ten-fold oversampling), except for position R5. Here the library size was

60 SP-variants leading to a three-fold oversampling.

Fig. 23: Screening results after saturation mutagenesis generated in the C-region and +1 position of SP-AmyE fused to cutinase. The screening was performed using the automated high-throughput assay for lipolytic activity in B. subtilis culture supernatants using p-nitrophenyl-palmitate as the substrate. Amino acids are indicated by the one-letter code. (A) Amino acid sequence of SP-AmyE fused to cutinase. N- and H-regions are indicated by open boxes. The C-region and the +1 position are highlighted as red boxes. (B, C). The lipolytic activity of every single clone is shown as relative activity referring to the B. subtilis strain using wild-type SP-AmyE which is defined as 100 % and indicated as a black line. Amino acid substitutions of sequenced SP-variants are shown close to its data-spot, the respective amino acids of wildtype SP-AmyE are shown close to the black line indicating wildtype activity. The standard deviation of the screening system was 25 % and is indicated as a grey shaded field.

rela

tive

lipol

ytic

activ

ity[%

]

screened variants

0

20

40

60

80

100

120

140

0 50 100 150 200 250 300

VA

A

AA

A

+1

CN (7) H (23)

TSLLPLFAGFLLLFHLVLAGPAA A CutinaseA S AMFAKRFK

SPase I

E F

+1

C+1 position

BC-region

rela

tive

lipol

ytic

activ

ity[%

]

screened variants

0

20

40

60

80

100

120

140

0 50 100 150 200

AGAAVA

ACA

ASA A

A

Results

64

Changes at positions Phe2,

Lys4 and Phe6 were identified to

improve secretion of cutinase up

to three-fold compared to the

wild-type SP-AmyE. The best

four SP-variants were

sequenced, thereby identifying

two beneficial substitutions.

Amino acid exchanges from Phe

to Asp and Phe to Glu at

position Phe2 of the N-domain

both increased the secreted

amount of cutinase more than

three-fold. Therefore, the

exchange of the non polar

aromatic phenylalanine residue

to the negatively charged amino

acids aspartate or glutamate at position 2 leads to a drastically increased cutinase

export. Interestingly, by this exchange the net charge of the SP was reduced from +3

to +2. In contrast, a significant reduction in secretion occurred when the signal

peptide showed uncharged amino acids like alanine or threonine at this position (Fig.

25. B,C).

10´´ 30´´ 1´ 10´´ 30´´ 1´

SP

-A

myE

contr

ol

10´´ 30´´ 1´ 10´´ 30´´ 1´

-

1 2

p

m

Fig. 24: Processing kinetics of cutinase precursor protein with SP-AmyE in B. subtilis TEB1030 analysed by pulse-chase experiment. Kinetics were measured for strains with either the vector pBSMuL3 as control (lane 1) and cutinase fused to SP-AmyE on plasmid pBSMuL3Cut-SP-AmyE (lane 2). Both strains were treated as described in 2.20. Sample volumes were equalized to 7.500 cpm. Positions of precursor form (p) and mature protein (m) are indicated.

Results

65

100 2000 100 2000 100 2000

100 2000 100 200030 600

100

200

300

400

100

200

300

400

100

200

300

400

100

200

300

400

100

200

300

400

100

200

300

400

F2 A3 K4

R5 F6 K7

B

C

ASAMFAKRFK

N (7) H (21)

TSLLPLFAGFLLLFHLVLAGPAA A Cutinase

+1A

F2D F2E

F2A F2T

K4L

K4G

F6W

F6S

0

100

200

300

400

w t F2D F2E F2T F2A K4L K4G F6W F6S

Figure 25: Screening results of saturation mutagenesis libraries generated in the N-region of SP-AmyE fused to cutinase. (A) Amino acid sequence of SP-AmyE given in one-letter code. N-, H- and C-regions are indicated by open boxes. The saturated positions of the N-region are highlighted in red letters. (B) Detailed screening results of the saturation libraries at position 2-7 of SP-AmyE. The lipolytic activity of every single clone is given as relative activity refered to the B. subtilis strain using wild-type SP-AmyE which is defined as 100 %. Amino acid substitutions of sequenced SP-variants are given in the one-letter code close to its data-spot. (C) Reproduction of the secretion efficiency results for eight SP-variants in comparison to the wild-type SP-AmyE as reference. The results represent data from 7 independent experiments. The standard deviation is indicated by error bars. The amount of secreted cutinase fused to the wild-type SP-AmyE (wt) is defined as 100%. The results were confirmed by Western Blot analysis using cutinase specific antibodies (data not shown).

1. A +1

CN (7) H (23)

TSLLPLFAGFLLLFHLVLAGPAA CutinaseA S AMFAKRFK A E F

+1

C

lipolytic activity

[%]

Results

66

Another important position of the SP-AmyE N-region was identified to be Lys4. Most

of the SP-variants showed less than 50% of wild-type activity. However, the

substitution of Lys4 by a leucin residue leads to a three-fold increase in cutinase

secretion. The third important residue has been identified at position Phe6. The

substitution of Phe6 by tryptophan resulted in a two-fold improved export, whereas

the exchange to a serin residue had the opposite effect reducing the secreted

cutinase amount to less than 20%. At the positions Ala3, Arg5 and Lys7 no SP-

variants were identified showing significantly improved secretion amounts but seem

to be no critical position neither. All SP-variants showing improved properties were

confirmed by retransformation and repeated independent growth experiments (Fig.

25.C). Regarding the correlation between the change in export efficiency and the

change in the D-score caused by one defined amino acid exchange results in a

surprising finding for the sequenced variants (Fig. 26): for any amino acid exchanges

leading to higher cutinase export rates the D-score was at least minimally reduced up

to a strong decrease for variants whose amino acid Phe2 was changed to the polar

residues Asp or Glu. In summary, by using saturation mutagenesis in the N-region of

the SP-AmyE cutinase export was increased more than three-fold thus changing the

Fig. 26: Comparative analysis of export efficiency and calculated D-score for chosen variants of SP-AmyE with amino acid exchanges in the N-region. The SPs were found in the saturation library of the N-region of SP-AmyE (data taken from Fig. 25.C). The amount of secreted cutinase fused to the wild-type SP-AmyE is defined as 100%. Amino acid substitutions of sequenced SP-variants are given in the one-letter code. The D-score was calculated for the first 70 amino acids of each hybrid protein using Computer program Signal P 3.0. The D-score of hybrid protein cutinase and wild-type SP-AmyE was 0,904 and is indicated with a red bar.

0

100

200

300

400

0,92

0,86

0,88

0,9

0,94

0,96

D-s

core

Lipo

lytic

activ

ity(%

)

F6WF2A F2T F2D F2E K4G K4L F6S

different variants of SP-AmyE fused to cutinase

Results

67

SP of AmyE from a “lower class” SP (ranking No. 65, Table 7) to a “moderate“ SP”

(corresponding to ranking No. 23, Table 7) similar to SP-GlpQ (ranking No. 26, Table

7).

3.5 Coexpression and mutagenesis of B. subtilis secA to increase cutinase

export

The overexpression and secretion of heterologous proteins show major drawbacks

due to different bottlenecks in the secretory pathway (11, 20). The optimization of

SPs as described before comprises the efficient transport to the cell membrane as

welll the acceptable processing for release of the mature exoprotein outside the cell.

At least of the same importance is the essential protein SecA which is the central

component of the Sec pathway mediating the active translocation of proteins based

on interaction with the SP and the channel forming proteins. For some exoproteins

the overexpression of SecA had a positive effect on the secretion efficiency (111). To

study the influence of co-overexpressed SecA protein for cutinase export in a HTS

system, a suitable B. subtilis marker strain had to be constructed using the cutinase

of the fungus Fusarium solani pisi stably integrated into the chromosome as a fusion

with a Bacillus SP. In addition, variants of SecA were constructed by applying

random mutagenesis and coexpressed in the marker strain. Using the HTS assay

described under 3.2 the secA libraries were screened for enhanced cutinase export.

3.5.1 Construction and characterization of marker strain B. subtilis Marc1

The B. subtilis secretion marker-strain Marc1 was created by transforming B. subtilis

TEB1030 with the integration plasmid pMCut1 (2.11) which contains a DNA fragment

encoding for the hybrid protein SP-LipA-cutinase (Fig. 27.A). The correct double-

crossover integration of pMCut1 into the chromosomal locus amyE was confirmed by

an indicator plate assay using a starch-containing growth-medium (177) (data not

shown). A double crossover led to a phenotype expressing no Amylase E protein and

Results

68

thus no starch degredation on the indicator plate. In contrast to strain B. subtilis

TEB1030 the constructed marker strain B. subtilis Marc1 showed lipolytic activity in

the supernatant (Fig. 27.B). A further western blot analysis confirmed the presence of

cutinase in the supernatant of B. subtilis Marc1 (Fig. 27.C).

This marker strain shows a lipolytic activity of 18 U/L against p-NPP as the substrate

leading to a cutinase export amount of 90 µg protein/ L calculated with the specific

activity of 200 U/mg cutinase. The stability of the integrated cutinase was tested by

growth of B. subtilis Marc1 in 25 mL LB-medium under non selective conditions over

96 hours. After each 24 hours an aliquot was tested by a lipolytic activity assay as

well inoculated into fresh medium. The cutinase activity did not decrease over this

time period indicating a stable integration of the cutinase into the Bacillus

chromosome even under non selective growth conditions (data not shown).

Fig. 27: Construction and characterization of B. subtilis secretion marker strain Marc1. (A) E. coli-B. subtilis shuttle vector pMCut1 was integrated into the chromosomal amyE gene of B. subtilis TEB1030 via double crossover of two 500 bp flanking amyE fragments resulting in cutinase expressing strain B. subtilis Marc1. ss: signal sequence of LipA; The constitutive promoter PHpaII is symbolized as a rightangled black arrow; Cm: gene providing chloramphenicol resistance; the deletion of two genes of extracellular lipase A and B are indicated by crossed out yellow arrows. The expressed cutinase is recognized by its SP-LipA from the Sec machinery and translocated across the cell membrane. (B) B. subtilis TEB1030 and Marc1 were grown in 25 mL LB-medium for 20 hours and the culture supernatant was tested for lipolytic activity in a spectrophotometric assay using p-NPP as the substrate. Both cultures showed no significant growth differences. The lipolytic activity was adjusted to an OD580= 7. The data represent the average value of three independent experiments. The standard deviations were lower than 12 % and indicated by error bars. (C) Immunodetection of cutinase in culture supernatant of B. subtilis TEB1030 and Marc1 using cutinase-specific antibodies produced in hybridoma cells. From each supernatant a total protein of 20 µg was used.

pMCut1

chromosome

amyE gene

cutinase Cmss

lipA lipB

A

B. subtilis TEB1030 cell

B

0

5

10

15

20

TEB1030 Marc1

lipolytic activity[U/L]

C

cutinase

B. subtilis Marc1 cell

chromosome

lipA lipB

cutinase Cmss àmyEamyE`

Sec pore

Results

69

3.5.2 Co-overexpression of SecA improves cutinase secretion

Marker strain B. subtilis Marc1 was used to test the effect of overexpression of SecA

in addition to the chromosomal encoded secA controlled by its native promoter.

Therefore the secA gene containing its natural ribosome binding site was cloned into

plasmid pBSMuL3 resulting in pBSMuL3secA. To compare the export rate of

cutinase fused to SP-LipA with and without overexpressed SecA, B. subtilis Marc1

was transformed with pBSMuL3 and pBSMuL3secA (Fig. 28.A) and the culture

supernatants of both strains were tested for lipolytic activity. The plasmid encoded

expression of SecA increased the cutinase activity five-fold up to 100 U/L indicating a

protein export of 500 µg cutinase (Fig. 28.B). A Western blot analysis of the

Fig. 28: The effect of overexpressed SecA for cutinase export in marker strain Marc1. (A) Plasmid pBSMuL3secA overexpressing secA was transformed into B. subtilis Marc1. Vector features are described in Fig. 9. (B) B. subtilis Marc1 bearing empty plasmid pBSMuL3 and secA-overexpression plasmid pBSMuL3-secA were grown in 25 mL selective LB-medium for 20 hours and the culture supernatant was tested for lipolytic activity in the standard spectrophotometric assay. Both cultures showed no significant growth differences. The lipolytic activities were adjusted to an OD580= 7. The data represents means of four independent experiments. The standard deviations were lower than 10% and indicated by error bars. (C) Immunodetection of cutinase in culture supernatant (S) and cellular (C) SecA of B. subtilis TEB1030 (lane 2) and Marc1 (lane 3) after 20 hours. As a positive control 1,5 µg purified cutinase was used (lane 1). Cutinase-specific antibodies were produced in hybridoma cells. Polyclonal antibodies against B. subtilis SecA were produced in a rabbit. From each cell lysat/ supernatant a total protein amount of 20 µg was used.

amppBSMuL3

secA

repB

kan

MCS ori E.coli

secA2,5 kb

Transformation

A

control + secA

0

50

100

150

lipolyticactivity[U/L]

B C

1 2 3

cutinase

secA

S

C

cutinase

Marc1

B. subtilis Marc1 cell

lipA lipB

cutinase Cmss àmyEamyE`

secA

pBSMuL3secA

secA

cutin

ase

TEB1

030

Mar

c1

Results

70

supernatant confirmed the enhanced cutinase amount (Fig. 28C), a second smaller

band below the cutinase shows proteolytic degradation caused by one of the

extracellular proteases of B. subtilis. The immunodetection of cellular SecA revealed

an increased amount of SecA for the SecA-overexpression strain compared to the

control strain (Fig. 28C) confirming an overexpression of SecA in addition to the

chromosomal encoded SecA.

3.5.3 The precursor form of cutinase fused to SP-LipA is processed rapidly

under condition of SecA-overexpression.

The kinetic of precursor form processed into mature exoprotein is a quality control for

efficient translocation across the cell membrane. Therefore the cutinase export

kinetic of B. subtilis Marc1 with or without plasmid encoded secA was studied by a

pulse chase experiment (Fig. x): Control strain TEB1030 showed no signals

indicating high specificity of immunoprecipitation by using polyclonal cutinase

antibodies (lane 1). For TEB1030 containing plasmid encoded hybrid protein

cutinase-SP-LipA a precursor form is clearly visible up to 30 sec after moment of

chasing (lane 4). Nevertheless after 1 min all cutinase precursor is processed to

mature form reporting a rapid translocation process. Anyway Marc1 showed only the

1´10´´ 30´´

TE

B10

30

1

1´10´´ 30´´ 10´´1´10´´ 30´´

Mar

c1 +

secA

3

contr

ol4

Mar

c1 +

pB

SM

uL3

2

p

m

Fig. 29: Processing of precursor protein cutinase fused to SP-LipA in different B. subtilis strains. Kinetics were measured for the B. subtilis strains TEB1030 (lane 1), Marc1 bearing empty vector pBSMuL3 (lane 2) and Marc1 bearing secA-overexpression plasmid pBSMuL3-secA (lane 3). As a control B. subtilis TEB1030 bearing plasmid encoded hybrid protein cutinase-SP-LipA (pBSMuL3Cut-ss-LipA) was used (lane 4). The Bacilllus strains were pregrown in S7 minimal medium containing methionine at 37°C to an OD600nm of 0.8, washed twice and starved in S7 minimal medium without methionine for 60 min at 33 °C. Subsequently cells (2,5 mL) were labelled with 150µCi [35S]- methionine for 1 min, followed by chasing with vast excess of non-radioactive methionine. Samples (600µL) for each strain were withdrawn at certain time points (10 sec, 30 sec and 1min), precipitated with ice-cold TCA and further processed as described under 2.20. Sample volumes were equalized to 7.500 cpm. The different positions of unprocessed cutinase-SP-LipA precursor form (p) and the mature cutinase (m) are indicated.

Results

71

a pulse-chase experiment (Fig. 29): B. subtilis strain TEB1030 showed no signals

indicating high specificity of immunoprecipitation by using polyclonal cutinase

antibodies (lane 1). For control strain B. subtilis TEB1030 containing plasmid

encoded hybrid protein cutinase-SP-LipA the position of the precursor form is clearly

visible 10 sec after moment of chasing (lane 4). Anyway B. subtilis Marc1 with or

without overexpressed SecA showed at all measured time points only the mature

cutinase reporting an efficient processing (lane 2,3). This data suggested no export

bottleneck due to improper cleavage of SP-LipA at the trans-site of the membrane for

this B. subtilis secretion reporter strain.

3.5.4 A SecA variant leading to eight-fold enhanced protein export.

To achieve a further enhancement

of cutinase export in B. subtilis

Marc1, the strategy was to optimize

the recognition and binding affinity

of overexpressed SecA to the

hybrid protein SP-LipA-cutinase by

mutating the whole secA gene.

Therefore an ep-PCR was

performed to insert random point

mutations into the whole secA gene

by using primer pair secA_up/

secA_low and pBSMuL3secA as

the template. The generated secA

library pBSMuL3secA-ep was

finally transformed into B. subtilis

Marc1 and about 4300 B. subtilis

clones were screened for cutinase

activity. One SecA variant showed

Fig. 30:. The effect of two B. subtilis SecA variants for the export level of cutinase. B. subtilis Marc1 harbouring plasmid encoded secA and two secA mutants were grown in 1 mL selective LB-medium each in a DeepWell plate for 16 hours and the culture supernatant was tested for lipolytic activity in the automated spectrophotometric assay at 30°C described before. The data represents means of three independent experiments. The standard deviations were lower than 16 % and indicated by error bars. The amino acid exchanges are given in one letter code.

0

200

400

600

800

1000

SecA type variant + variant -wildtype

lipol

ytic

act

ivity

[U/L

]

amino acid exchange - A23T

K809D

A23TK809D

H289Y

Results

72

an eight-fold higher cutinase export than overexpressed wildtype SecA (Fig. 30). The

following sequencing revealed two amino acid exchanges at position 23 from alanine

to threonine and at position 809 from lysine to aspartate.

3.5.5 The improved export effect of SecA variant A23T, K809D was neutralised

by a further amino acid exchange H289Y in the PPX-domain

In a second approach only the preprotein-crosslink-(PPX-) domain was target of

mutagenesis. This domain is most likely responsible for the binding of SecA to the

signal peptide of a precursor protein as shown in former experiments (76, 144). To

mutate specifically the PPX-domain of secA by ep-PCR, a method was established

called “Quick Cassette Mutagenesis” based on the QuickChange II Site-Directed

Mutagenesis Kit from Stratagene (Heidelberg, Germany) as described under 2.10.5.

More than 2000 SecA variants were screened in Marc1 but showed no higher

cutinase export rate. Additionally the improved SecA variant A23T, K809D (Fig. 30)

was used for this “Quick Cassette Mutagenesis”: Under 350 screened PPX-variants

no further improvement could be observed. On the contrary, cutinase export of one

clone was decreased to wildtype level of overexpressed SecA. Interestingly, its

sequence showed a third amino acid substitution at position 289 from histidine to

tyrosine embedded in the SP binding domain (Fig. 31). In consequence this position

might be critical for the efficient interaction of SecA and the SP-LipA of the precursor.

Discussion

73

4. Discussion

Since many years Bacillus subtilis and related Bacilli like B. licheniformis are used as

expression and secretion host. The ability to express and secrete enzymes in high

amounts of up to 20 g/L into the culture medium as well as the classification as

GRAS, i.e. generally recognized as safe, organism free of any endotoxin, are

reasons for the wide use in industrial fermentation processes (172). In contrast to

high yield production of homologous proteins, heterologous enzyme secretion often

lead to disappointing low yields in Bacillus species due to numerous bottlenecks that

severely reduce the production capacity. In an already started process, identified

bottlenecks (e. g. inefficient targeting to the membrane, inefficient processing,

proteolytic degradation, misfolding, retention in the cell wall, inconvenient codon

usage, lack of suitable expression plasmids etc.) are targets of intensive studies to

eliminate or minimize them (11, 94, 114, 122, 180, 186, 208, 215).

This work focussed on the optimization of the secretion host B. subtilis for foreign

proteins with respect to the major role of the signal peptides for a successful

targeting to and across the cell membrane. Within the scope of this thesis a secretion

reporter system was established based on a biotechnological relevant lipolytic

enzyme of heterologous origin and (ii) adapted to an automated high throughput

screening (HTS) system in B. subtilis to (iii) analyse a generated library of all natural

B. subtilis signal peptides for optimal secretion efficiency of our target protein as well

as for other industrial applications. The established HTS system allowed (iv) the use

of Directed Evolution for the construction of mutant libraries of SPs or components of

the Sec pathway as a strategy for secretion enhancement. Another aspect of this

work deals with (v) the construction of useful Bacillus expression and secretion

vectors to broaden the range of biotechnological applications for B. subtilis.

Discussion

74

4.1 A new Bacillus vector series

pBSMuL vectors show a broad application spectrum

Although several new Bacillus plasmids were published over the last years (e.g.

(106, 108, 134, 147), there is a general need for stable and user-friendly expression

and secretion plasmids for B. subtilis (147, 208). Here, three multi-copy B. subtilis

expression vectors have been constructed, namely pBSMuL1,2,3.

For the first time, the pBSMuL series combines several convenient vector features for

B. subtilis comparable to available E. coli expression systems as shown in Table 11.

(i) The functionality for all vectors was demonstrated in this study using the

heterologous target genes cutinase and estCL1 as well as the homologous gene

secA. Additionally, the SP of pBSMuL1,2 can easily be cleaved off for intracellular

protein expression as shown in another study: plasmid pBSMuL2 was used for

intracellular overexpression of the homologous protein YtvA in B. subtilis resulting in

a production amount of more than 20 mg protein/L. The final yield after purification

was about 6 mg protein/L (Krauss, Gärtner, Jaeger, Eggert, unpublished results).

Since the pBSMuL vectors are based on an E. coli–B. subtilis shuttle plasmid (36,

228), the cloning procedure can be performed in E. coli. The E. coli part can be

cleaved off and the self-ligation of the remaining Bacillus vector fragment leads to

plasmid multimers, which are more efficiently uptaken by B. subtilis during

transformation (65). Consequently, this vector feature allows the generation and

screening of large gene libraries with one of the easiest transformation procedures

for B. subtilis (2.9.3) as done with vector pBSMuL3 (3.2, 3.4, 3.5) showing only a

slightly slower effectiveness than electroporation techniques (data not shown). Time-

consuming transformation methods like the “protoplasts” transformation can be

avoided (65).

Table 11: Properties and applications of the pBSMuL vector series

* C-terminal His-tag is optional

features constitutive promoter(s)

Bacillus

rbs MCS

signal peptide

His-tag multimeric

DNA formation

applications high level

expression high level

expression convenient

cloning secretion

intracellular expression

one-step purification

HTS-application

pBSMuL1 1 + + + + + * + pBSMuL2 2 + + + + + * + pBSMuL3 1 - + - + - +

Discussion

75

Especially pBSMuL3 is a promising tool for future projects using the SP-library for a

“one step optimization” of foreign secretory proteins in B. subtilis. This new strategy

was successfully applied for two heterologous lipolytic enzymes cutinase and EstCL1

(Fig. 20, 21) leading to secreted protein yields of 35 mg/L and 17,5 mg/L,

respectively. In comparison to results of other expression systems in B. subtilis used

for proteins of eukaryotic origin (21, 79, 133, 178, 206), the produced protein

amounts for the pBSMuL3 vector are quite satisfying. Moreover, since the second

promoter in the pBSMuL2 vector could increase three-fold the secretion yield for

cutinase up to 60 mg/L, the same insertion of this P59 promoter into the plasmid

pBSMuL3 could likely lead to an even higher production and secretion efficiency

which should be tested in the future.

Do pBSMuL vectors have the potential for industrial applications?

The construction of the pBSMuL series also addressed to industrial applications by

focussing on protein overexpression using constitutive promoters without the

necessity of induction, since common inducers like IPTG, xylose or heat represent

additional costs and can decrease dramatically the profit of a production system. This

money can be saved by the use of antibiotic-free growth medium, since a vector

stability was reported for pBSMuL1,2 overexpressing and secreting cutinase for at

least 48 hours in non selective medium without significant reduction (Fig. 7).

Nevertheless, a faster loss of the vector cannot be excluded for other target proteins

under non-selective growth conditions and/or under high cell density fermentation

due to enhanced selection for a plasmid-free cell culture. Moreover, in previous

studies it was reported that high secretion stress stimulates regulatory systems in

B. subtilis, which might have a negative influence in production yield (77, 78, 152).

In summary three multi-copy B. subtilis expression vectors for cloning of target genes

under control of one or two strong constitutive promoter(s) have been constructed.

The novel plasmid series enabled (i) convenient cloning into an artificial multiple

cloning site, (ii) high level overexpression without the necessity of induction, (iii)

generation of huge libraries for HTS processes, (iv) efficient secretion of

heterologous proteins, (v) application for the “one step optimization” by finding the

best natural SP for protein secretion and (vi) the easy one-step purification in the

Discussion

76

Gram-positive host B. subtilis. This system might be useful as an alternative in case

of serious problems (inclusion bodies, misfolding, low expression, toxicity)

concerning the well-established E. coli expression systems (8). However, an

industrial use for large-scale production needs to be chosen under fermentative

conditions in future projects.

4.2 A new secretion marker system for HTS processes in B. subtilis

As an alternative strategy to rational protein design (16) Directed Evolution enabled

enzyme design without much knowledge of structure or reaction mechanism. But the

analysis of large gene libraries requires a suitable High throughput screening (HTS)

assay, which allows a rapid and reliable determination of enzyme activity, a

significant reproducibility, a cheap and easily prepared reaction assay (176).

Lipolytic enzymes belong to the class of hydrolases. Some members of this group

show chemo- regio- and stereoselectivity, are very stable and active even in organic

solvent. Consequently they are of high commercial interest and are already used in

industrial production of detergents, food, pharmaceutics and agrochemicals (14, 15,

86, 88, 115). Since several reported HTS system were created successfully to detect

lipolytic enzymes with improved properties (49, 54, 85, 158), one of these

biocatalysts, the cutinase from Fusarium solani pisi (56), was chosen as a reporter

enzyme to analyse heterologous protein secretion in B. subtilis.

Since the secreted cutinase yield correlated with the lipolytic activity in the

supernatant in all measured cases, this lipolytic activity assay is a reliable method to

detect and compare easily the export capacity for cutinase in B. subtilis.

Chances and limits of the HTS system

The most important aspect of this automated HTS system, the reproducibility, was

good enough for a successful application using the lipase substrate pNPP due to an

overall standard deviation of about 10% including growth differences of B. subtilis,

stability of the assay and stability of secreted cutinase in Bacillus supernatant over

four hours (Fig. 13). The screening of mutant libraries in B. subtilis revealed an

Discussion

77

increased fluctuation in the cutinase activity up to a standard deviation of about 25%

(Fig. 14) most likely by a variation in the individual plasmid copy number (~50) per

cell after the transformation event. As a consequence of the performed mutant library

screening in B. subtilis, only clones with significantly improved or decreased cutinase

export (50 %) compared to wildtype activity were chosen for further analysis.

It is noteworthy that the incubation time of the Bacillus colonies on transformation

agar plates revealed to be a critical factor for reproducibility of cell growth in MTPs: if

Bacillus colonies were grown longer than 30 hours on agar plates before transferred

into the DW-MTP, the growth differences (compare Fig. 10) dramatically increased to

a standard deviation of more than 25 % (data not shown). Therefore it is crucial to

perform every step of the whole screening process stringently the same way to

guarantee the reproducibility and comparability of screened libraries.

In another study (38) the secreted cutinase showed almost the same activity even

over a time period of six hours incubated at -20°C, 4°C or RT confirming the enzyme

stability in the culture supernatant of B. subtilis TEB1030 that still harbours several

extracellular proteases (208). Nevertheless, processes for a duration of more than

five hours are not recommended due to substrate instability (chapter 3.2) resulting in

a maintenance free HTS process for maximal 1300 samples.

4.3 Improvement of heterologous protein secretion in B. subtilis by signal

sequence screening

Expression and survival of cutinase depends on the presence of a functional signal peptide

The generated Bacillus plasmid pBSMUL3-Cut, bearing only the cutinase gene

without its start codon, showed no cutinase expression since the vector lacks a

ribosome binding site and a start codon downstream from the promoter. This

property made plasmid pBSMuL3-Cut a versatile cloning and expression tool for the

comparison of cutinase export with different Bacillus Sec-type SPs. All 173 signal

sequences amplified by PCR (Table 12, Appendix) from the genome of B. subtilis

168 share a consensus sequence at the 5`-end encoding for a Bacillus rbs, a suitable

spacer region and the start codon ATG what is obligatory for a ribosomal translation

process (Fig. 16). Due to the phenomenon of SP-independent protein export (5, 187),

Discussion

78

the cutinase was overexpressed in B. subtilis on plasmid pBSMuL3-Cutintra lacking a

SP to test the occurrence of cutinase in the extracellular medium. But no cutinase

could be detected in the supernatant neither by immunodetection (Fig 18) nor by a

lipolytic activity assay (data not shown). Astonishingly, the cutinase was not even

detectable in the cytoplasmic fraction. This observation strongly suggests a massive

intracellular degradation as a consequence of a high sensitivity of the heterologous

cutinase to intracellular proteases in B. subtilis (67, 107). Contrary findings were

reported in previous studies for the production of foreign Bordetella pertussis toxin

subunits in B. subtilis: the secretion yield for most toxin subunits was low, whereas

the intracellular production without a SP expressed in the same vector revealed high

level production (73, 168, 169). One can speculate that these toxin subunits form a

more stable and protease resistant structure after intracellular expression compared

to cutinase, which might need a slower folding process to reach the final

conformation. This characteristic of the cutinase could explain the requirement for

secretion to prevent degradation (188). Furthermore, no precursor form of cutinase

was detected in the cytoplasmic when expressed as hybrid protein with different SPs.

This strongly supports the idea of a fast proteolytic degradation of precursor cutinase.

In conclusion, the intracellular transport for secretory cutinase seems to be a “race”

to the membran- the more efficient the binding of its fused SP to the targeting factors

(SRP, SecA) and the membrane, the higher is the amount of “surviving” precursor

cutinase that can be translocated across the cell membrane. This early secretion

step seems to be a major bottleneck for cutinase export.

The processing of cutinase precursor is not the limiting step

The observation that most of the functional SPs for cutinase export bear a suitable C-

terminal cleavage site suggested efficient processing (Table 13, Appendix). But the

simple analysis of a SP for a suitable C-region using prediction tools like SignalP

cannot guarantee an efficient processing rate of a precursor. For instance, the

precursor interacts with all involved Sec-components during the translocation

process across the membrane in an individual manner depending on its SP (131,

136, 178, 181, 189, 221, 227). Next, both SP and its mature protein might interact in

a unique way by its secondary structure (52, 145), thereby influencing the

Discussion

79

accessibility of the cleavage site (A-X-A) of the SP to a signal peptidase at the trans-

site of the membrane. But the C-region must be in an extended (β-) conformation for

an efficient cleavage event (141). Furthermore, the H-region must be of a suitable

length to guarantee the positioning of the C-region in the range of a SPase close to

the lipid headgroups at the trans-site of the membrane (72, 199). All these

possibilities, too complex yet to be considered in available prediction tools, could

disturb or prevent the suitable access of the C-region to the SPase leading to

insufficient processing and proteolytic degradation of the cutinase by extracellular

proteases (11).

Consequently, the processing kinetics of five selected SPs, showing different

cutinase export rates, were analysed by a pulse-chase experiment (Fig. 19). Even if

the processing rate of the precursors differed from each other significantly, in all

cases mature cutinase was visible indicating a successful processing event.

Remarkable is the observation that the secretion level of cutinase does not

corresponds to the processing rate. The SP-AmyE shows a similar fast export kinetic

than SP-Epr, although SP-AmyE results in a low, SP-Epr in a high secretion level of

cutinase. Moreover, SP-YncM leads to high yield of cutinase export but shows clearly

the slowest processing. In conclusion, the processing of all tested hybrid proteins is

efficient enough to provide cutinase secretion even if strong differences in the export

kinetics occurred. Hence, the processing of precursor to mature cutinase indicates no

export bottleneck for the secretion yield of cutinase in B. subtilis under the given

expression and culturing conditions.

Variations of the precursor synthesis rate might influence the secretion level

However, even if the chosen secretion marker system provides the same distance of

the gene to the promoter for all SP-cutinase fusion constructs, we cannot totally

exclude variations in cutinase export due to different precursor synthesis rate. Every

signal sequence fused to the cutinase gene leads to an individual mRNA, its

structure might influence the transcript stability or disturb the translation process

(112, 153). This effect could result in different amounts of precursor protein in the

cytoplasm and later result to different secretion yields of mature protein. A possible

feedback mechanism of a preprotein on translational level was postulated in earlier

Discussion

80

studies (174, 178, 190). Anyway, an additional analysis of transcriptional level was

not necessary during this study since the used secretion reporter system includes

this factor for export efficiency in the applied screening method.

In silico prediction of the most functional signal peptide for foreign proteins is

not yet possible

Our systematic approach using two different lipolytic enzymes as secretion targets

clearly demonstrate the inability to predict in silico an optimal SP for protein secretion

using the computer tools available so far. As one commonly used computer tool to

characterize a signal peptide, the SignalP-server generating the so-called D-score

was chosen (9, 135). There was no clear correlation between D-score and secretion

efficiency of the target proteins (Table 7, Fig. 21).

Since it was observed that defined alteration of net charge, hydrophobicity or length

of the SP could change secretion efficiency (13, 164), these criteria were analyzed in

the generated SP-library without clear correlation to the secretion amounts. Only the

overall composition of SPs – positively charged N-region, hydrophobic core region

and suitable cleavage site (A/VXA) – was obvious, in addition to this the SP-library

showed a strong heterogeneity in length, charge and hydrophobicity without

correlation to export efficiency (Table 13, Appendix).

Previous studies in B. subtilis compared SP-variants to the wildtype when fused to a

mature protein and revealed differences in export efficiency of the hybrid protein (13,

29, 130, 164). However, the effects could not be reproduced when other SPs were

fused to the same mature protein (29). Furthermore, secretion experiments in E. coli

reported an influence of the N-terminal part of the mature protein in secretion

efficiency, indicating the importance of an optimal interaction between signal peptide

and secretion target (110, 113, 118, 156, 210). Moreover, the junction between SP

and mature protein is a further critical variable that influences the secretion level of a

target protein (69, 93).

It looks like every fusion of a SP-mature protein forms a unique precursor interacting

with the components of the secretion apparatus suggesting that protein transport is

restricted severely by the efficiency of SP-recognition by all participating components

the translocation machinery.

Discussion

81

The new “one-step optimization” method could enable the secretion of any

target protein as demonstrated for EstCL1 in B. subtilis

The unpredictability of the secretion efficiency of a defined SP for an unknown

secretion target was discussed before. But the established HTS for B. subtilis offered

the screening of the complete SP library size in one step covering all possible

variations built of the generated pool of 173 Bacillus natural Sec-type SPs and the

target protein: and indeed, the best functional SPs for cutinase export, which were

found by a separately performed cloning procedure for each of the possible hybrid

protein, were found again in the combination of a one-cloning step and HTS. By this

new method for export optimization one can easily bypass the risk of low secretion

yield as a consequence of the “wrong” SP. This was successfully applied to the

secretion of a non-secretory protein EstCL1, an esterase of metagenomic origin.

Even if EstCL1 shows a pI of 4.5 suggesting no disturbing interaction in the cell wall

environment (171, 180, 186), its secretion into the growth medium up to 18 mg

protein/L is remarkable for two reasons. First, the encoding gene has a GC-content

of 60% which is significantly higher than the general GC-content of B. subtilis

(~ 50%) which might lead to expression problems by the wrong codon usage.

Second, the protein was defined as a non secretory protein due to the lack of any N-

terminal SP (109). Considering the minimal time expenditure of 1-3 weeks for the

complete cloning and screening process of EstCL1 to generate and test all natural

SPs, a general application of this “one-step optimization” method can be predicted for

B. subtilis for theoretically any heterologous protein production.

4.4 Saturation mutagenesis as a useful tool for the characterization and

optimization of a signal peptide

The HTS system enables Directed Evolution for secretion studies in B. subtilis

Besides the necessity for the overall criteria of a SP, no general statement could be

formulated so far how to make SPs more functional for a particular export target (29,

130, 182, 218, 219). Even if some studies performed a more systematic approach in

various Gram-positive bacteria (B. subtilis, Streptomyces lividans, Lactococcus lactis)

Discussion

82

by analysing a series of SP variants for export functionality (13, 29, 53, 130, 157,

225), the variants were small in number (< 30) and generated by site directed

mutagenesis.

In consequence, this lack of a generalized prediction for SP functionality claims for a

more random mutagenesis approach. So far, a systematic screening of mutant

libraries in B. subtilis with respect to secretion capacity was not found in literature.

Here, for the first time, Directed Evolution could be used in combination with a

systematic screening for secretion efficiency or differences in B. subtilis mainly due to

the establishment of a versatile Bacillus expression system with a sufficient

transformation rate for library construction (chapter 3.2).

The size of the signal peptide reduces the mutagenesis techniques

Among the already studied SPs of B. subtilis the SP of AmyE was a frequently used

object also for site directed mutagenesis. The screening of all SPs for cutinase

secretion revealed a medium level export for SP-AmyE (Fig. 17, Table 7) enabling

the detection of improvement and decrease in protein export. Therefore the SP-

AmyE was used as the target for random mutagenesis. Unfortunately, the classical

ep-PCR method (226), which was applied for enzyme improvement in many cases

(e.g. (49, 54, 85, 158) was not successful for the improvement of SP-AmyE after the

screening of more than 3000 B. subtilis clones. Regarding the shortness of the

encoding DNA of about 100 bp encoding for a SP, it is more likely that the highest

possible error rate of 1 base exchange/100 bp is not sufficient to generate enough

SP variants for a successful screening process. Especially since it was reported that

some amino acids are rarely possible to introduce by ep-PCR (50). However, for

future projects a more useful approach might be the so-called “hypermutagenic” PCR

that represents a modified ep-PCR. This method combines the use of varied

concentrations of the four different dNTPs in addition to MnCl2 what results in higher

mutation rates (196).

Discussion

83

The consensus motif A-X-A-A in the C-terminal part of a Bacillus SP is the most

functional sequence in the SP-AmyE for cutinase export

Tjalsma et al. (188) used the SignalP algorithm (135) to predict N-terminal signal

sequences. Furthermore, they analysed all found Sec-type SPs for amino acid

frequency around the SPase I cleavage site. A consensus motif “A-X-A-A” was

found, whereas the preference to alanine at positions –1 and -3 corresponds well to

the postulated interaction with the active site of a SPase I: according to the crystal

structure of an E. coli SPase I (141) the side chains of residues at the –1 and –3

positions are presumably bound in two hydrophobic substrate pockets (S1 and S3).

For this reason efficient binding occurs most likely only with uncharged and small

residues like alanine. The position -2 was predicted to allow any aa since its residue

points outwards from the SPase I. Accordingly, Tjalsma et al. found every aa except

cysteine and proline at position –2 for all Sec-type SPs. alanine was also the

preferred amino acid at the +1 position of the mature protein.

The question rised, if the most abundant amino acid sequence A-X-A-A is also the

most functional one for protein secretion. Using saturation mutagenesis and

subsequent HTS neither the C-region nor the +1 position of the SP-AmyE (A-S-A-A)

could be significantly improved for cutinase export (Fig. 23). On the contrary, the

screening results confirmed clearly the consensus motif since the most efficient

clones of the C-region as well as the +1 position showed wildtype activity and

contained the consensus motif A-X-A-A except for one case, which showed only a

slight deviation to sequence A-X-A-V. The presence of only alanine at the –1 position

corresponds to the findings in previous studies (164, 200) and to the postulated

interaction with a SPase I (141). More surprisingly was the finding of the amino acid

exchange to a cysteine at the position –2 in one case due to the total absence of

cysteine at this position in all Bacillus Sec-type SPs (188). But one has to be careful

with final conclusions for two reasons: first, the presence of alanine at the +1 position

might lead to a second cleavage site (Fig. 22) since a shift to another C-region was

observed for SP-AmyE by change of its amino acid sequence (164). Furthermore,

even if unlikely, amino acid exchanges at the N-terminus of the mature cutinase

might have an influence on enzyme activity. This can be critical since the cutinase

export was determined only indirectly by a lipolytic activity assay.

Discussion

84

Saturation mutagenesis revealed unpredictable amino acid exchanges in the N-

region for a higher functionality of SP-AmyE

A powerful method to generate all possible variants of a protein at a defined amino

acid position is called “Site-specific saturation mutagenesis”. This method introduces

all possible base triplets at a given codon position, thereby resulting in the formation

of all 20 amino acids at this position of the protein (2). Since the N-region of a SP is

important for functional interaction with SecA (3, 29) and the phospholipids of the cell

membrane (23, 203), each position of the N-region of SP-AmyE was completely

saturated and screened for cutinase export functionality (Fig. 25). The screening

revealed that the positions Ala3, Arg5 and Lys7 showed neither significantly

improved secretion but seem to be no critical position nor. On the contrary, the most

critical position for efficient cutinase export is position 4 since most of the

corresponding SP variants showed strong reduced export functionality. Interestingly,

among three of four sequenced improved SP-variants (F2D, F2E, K4L, F6W) a

reduction of the net charge of the SP-AmyE from +3 to +2 was observed. On the one

hand, this reduced positively charged N-terminus might suggest a less effective

interaction of the SP to its binding partners (3, 23) resulting in less secretion as

reported in a previous study (29). On the other hand this phenomenon of less

positive residues and improved secretion was observed before: in a secretion study

of the Gram-positive bacterium Streptomyces lividans a charge reduction from +3 to

+2 in the SP resulted in doubling of secreted α-amylase inhibitor tendamistat.

Furthermore, introduction of more positive charges significantly decreased secretion.

The authors (53) could exclude transcriptional effects and suggested a modulation of

precursor synthesis similar to the postulated feedback mechanism described before

(178). Furthermore, an additional lysine in the N-region of a SP drastically reduced

the export rate of a nuclease in Lactococcus lactis (157). An influence of the SP

charge variation on the synthesis of precursor protein was also observed in E. coli

(10, 80, 184) and might be the reason for the improved cutinase export by the SP-

AmyE variants with reduced net charge in B. subtilis.

However, possible effects on transcriptional level cannot be excluded for the cutinase

precursor. In another study the analysis of SP-AmyE variants for secretion of β-

Discussion

85

lactamase in B. subtilis revealed an increased secretion level that was caused by a

higher mRNA synthesis (130, 132).

Another aspect was the prediction of a distinct effect by one of the amino acid

exchanges for cutinase export. Thus the export efficiency of the variants was tested

for correlation to the D-score calculated by SignalP (Fig. 26). The D-score represents

the probability of a particular amino acid to function as a SP or not. Astonishingly,

there was no correlation between protein export and the D-score for any of the four

improved SPs. On the contrary, the D-scores were decreased in all cases indicating

a lower probability of a functional SP. Next, The prediction for decreased secretion

appeared to be only slightly better since there was a correlation in two of the four

examined variants.

Anyway, these result indicated again a clear lack for a suitable prediction of SP

optimization by in silico analysis using computer tools like SignalP and show a useful

application of Directed Evolution for significant improvement of protein export by

saturation mutagenesis in relevant parts of a SP.

4.5 Improvement of heterologous protein secretion in B. subtilis by co-

overexpression of SecA

Chaperone-like function of SecA might increase cutinase secretion in B. subtilis The translocation motor protein SecA is an essential component of the general

secretion pathway in bacteria and therefore of special interest. Here, the influence of

overexpressed SecA to secretion could be observed since a functional B. subtilis

reporter strain for export efficiency was constructed. This marker strain B. subtilis

Marc1 contains a chromosomal integrated cutinase fused to SP-LipA and showed a

stable cutinase secretion of about 90 µg protein/L (Fig. 27). This comparatively low

export level due to the integrated one-copy plasmid was useful since it suggested a

non-saturated Sec machinery without induction of secretion stress. And indeed, in a

pulse-chase experiment no cutinase precursor was detectable indicating efficient and

complete translocation of cutinase (Fig. 29). Although the Sec machinery was

obviously not overloaded, the constitutive overexpression of SecA in B. subtilis

Discussion

86

Marc1 led to a five-fold higher cutinase export (Fig. 28). In a previous study the

overexpression of SecA had a similar positive effect on the secretion of the

homologous levansucrase (111). The authors explained this by a low binding affinity

of the SP to SecA but argued against a requirement of an export-specific intracellular

chaperon since the absence of a SecB homologue in B. subtilis and the existence of

a self-stabilizing intermediate folding state of levansucrase under cytosolic

conditions. However, in contrast to the homologous levansucrase showing low affinity

of its SP to SecA (111), the heterologous cutinase was fused to the efficient SP-LipA

(Table 7, Fig. 17) what claims for another explanation. Interestingly, however, the

expression of SecA is strictly regulated in B. subtilis and reaches the maximum at the

end of the exponential growth phase and not, as one might expect, later in the actual

secretion phase during stationary growth (71) indicating a possible function of SecA

in the early stage of secretion. This idea is supported by the observation that SecA

exists next to a membrane-associated form also in a soluble form in the cell (35,

116). Additionally, no homologous protein to the export-specific intracellular

chaperone SecB of E. coli (43) could be found in B. subtilis (188). In consequence,

SecA might not only serve as the translocation motor but function itself as an

intracellular chaperone for secretory proteins. According to this study, SecA could

prevent the strong intracellular proteolytic degradation of the cutinase that was

shown before (Fig. 18).

Histidine 289 in the PPX-domain of SecA plays a key role for secretion of

hybrid protein SP-LipA-cutinase

The random mutagenesis of SecA and the subsequent screening in B. subtilis Marc1

yielded the SecA variant A23T, K809D showing an eight-fold export improvement of

cutinase compared to overexpressed wildtype SecA (Fig. 30). Alanine at position 23

is located in the nucleotide binding domain 1 (NBF1), which forms together with the

NBF2 the essential nucleotide binding site for ATP hydrolysis (Fig. 31). Lysine at

position 809 is located in the helical scaffold domain (HSD). The exact mechanism of

how the SecA variant A23T, K809D led to the improved export of precursor SP-LipA-

cutinase is hard to define and highly speculative. So far one might suggest that the

SecA protein becomes more active by the amino acid exchange at the nucleotide

Discussion

87

binding site resulting in a generally increased protein export. But first observations

did not detect an increase in the total amount of secretory proteins in this B. subtilis

Marc1 strain (data not shown). Another possible explanation is a higher binding

affinity of this SecA variant A23T, K809D for the N-terminal part of the precursor

protein SP-LipA-cutinase since an increased affinity of SecA to a preprotein was

reported before to have strong influence on the export efficiency (111) which has to

be investigated further.

However a further amino acid exchange

in the SecA variant A23T, K809D,

H289Y reduced the positive effect of

SecA A23T, K809D for the cutinase

export back to wildtype level indicating

an inhibitory effect for the binding event

of SecA to the hybrid protein (Fig. 30).

Interestingly, this non-conservative

amino acid exchange from the basic

histidine to an uncharged, but polar

tyrosine at position 289 is located in the

PPX-domain, that provides binding of the

precursor to SecA (144). It is noteworthy

that a hyperactive SecA variant H309Y

was found in E. coli, that could suppress

signal peptide defects. This amino acid

exchange corresponds to position 288 in

the PPX-domain of B. subtilis SecA

(139) which is in direct contact to amino acid H289. However, this result confirmed

the central role of the PPX-domain of SecA for precursor binding and suggested a

key role for histidine 289 for efficient binding and translocation of hybrid protein SP-

LipA-cutinase.

Homologous recombination disturbs the screening for SecA variants During the HTS for plasmid encoded SecA variants to improve cutinase secretion in

B. subtilis Marc1 the occurrence of false positive clones was frequently observed:

several B. subtilis clones showed an increased cutinase activity in the automated

N B F I-

d o m a in

P P X -d o m a in

L y s 8 0 9

H is 2 8 9

Fig. 31: Localisation of amino acid substitutions in the protein structure of B.

subtilis SecA that effected cutinase secretion. Structure of monomeric B. subtilis SecA modified after Hunt et al. (76). Highlighted in defined colours are the PPX-domain (blue), the NBFI-(pink), the NBFII-domain (brown) and the HSD-domain (green). Relevant positions of amino acid substitutions are indicated in red. The C- and N-terminus of SecA are indicated in white.

Discussion

88

lipolytic activity assay up to 40-fold, immunoblotting confirmed the strong increase of

secreted cutinase (data not shown). A subsequent sequence analysis of the

corresponding plasmids revealed an open reading frame encoding for cutinase fused

to SP-LipA instead of SecA downstream from the promoter. Since the B. subtilis

Marc1 strain was tested for a stable genome-integrated cutinase, the only possible

explanation was a homologous recombination between the plasmid pBSMuL3-secA

and the integrated cutinase cassette pMCut1. In a previous study the minimal

requirement for detectable homologous recombination was determined as 70 bp of

homology (98). The DNA analysis in B. subtilis Marc1 harbouring pBSMuL3-secA

revealed a homology of 400 bp due to the use of the same DNA fragment containing

the HpaII promoter. This could at least explain the high expression and secretion of

cutinase in the corresponding B. subtilis clones. The permanent chance of

recombination using the pBSMuL vectors in B. subtilis Marc1 strongly suggests the

use of either another integration vector for construction of a secretion reporter strain

or the use of other overexpression vectors in B. subtilis Marc1 with the total lack of

homologous DNA regions between plasmid and chromosome. Furthermore, another

possible source of homologous recombination is the secA gene itself, since plasmid

encoded SecA coexists to the chromosomally encoded SecA in B. subtilis Marc1

(Fig. 28) and shows 2500 bp of homology for possible crossing over events. For

intended future projects this additional critical factor must be considered since it can

decrease the efficiency of the HTS system.

4.6 High level secretion for any foreign protein in B. subtilis- prospects for a

general strategy

In this work, it turned out, that the best SP for the secretion of one target protein was

not automatically also the best for the secretion of another target protein. The

complex interaction of SP, mature protein and all participating Sec components might

explain the lack of correlation between the D-score calculated by SignalP to the

measured secretion efficiency. Based on the results for cutinase and EstCL1

secretion, novel computer tools need to be generated to identify the complex

interaction patterns between SP and mature part of the desired secretion target

protein.

Discussion

89

0

1000

2000

3000

4000

5000

6000

7000

8000

LB-medium TB-medium

SP-AmyE SP-YncMSP-Epr

Alip

olyt

icac

tivity

[U/L

]

B

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 5 10 15 20

growth time [h]

TB-medium

LB-medium

Sca

ttere

d lig

ht in

tens

ity I-

I 0[r

el. U

nits

]

However, our strategy to screen a complete natural SP-library fused to the secretion

target using the “one-step optimization” represents a powerful tool for a fast and easy

export optimization of virtually any target protein. It bypasses the single cloning and

screening procedure method saving up to 6-12 months of lab work. Furthermore, the

used B. subtilis strains were grown in simple LB-medium during all experiments

Fig. 32: Comparison of cutinase expressing B. subtilis strains in LB- and TB-medium using the on-line light scattering measurement technique for shaken MTPs. B. subtilis TEB1030 strains bearing plasmid pBSMuL3 encoding for hybrid proteins of cutinase and different SPs (SP-AmyE, SP-Epr, SP-YncM) were grown in a MTP (37°C, 995 rpm, 200 µL total volume each, inoculated with 10 µL of overnight culture) in LB- or TB-medium. The time point for the lipolytic activity assay after 20 hours is indicated by a red arrow. The results represent data from two independent experiments. (A) The growth behaviour was recorded over 20 hours using an excitation filter of 620 nm for continuous detection of the scattered light intensity difference from initial value (I-I0). For more details concerning the continuous on-line measuring technique see Samorski et al. (166). (B) The lipolytic activity assay of the culture supernatant was performed at 37°C in a MTP as described before.

Discussion

90

concerning secretion capacity. It could be possible to enhance the secretion yield by

improving the growth conditions for B. subtilis. This was tested for cutinase secretion

in B. subtilis using a new on-line measuring technique for shaken MTPs. The

experiments were performed by Dipl. Ing. Frank Kensy of the Department of

Biochemical Engineering of the RWTH Aachen University (Prof. J. Büchs, Aachen,

Germany). B. subtilis strains secreting cutinase with SPs of Epr, YncM and AmyE

were grown in LB-medium and TB-medium (Fig. 32). The growth as well as the

cutinase export was three-fold increased using TB-medium. However, these results

also suggest a further possible enhancement of protein yield for other targets.

Various industrially used Bacillus media could be tested for even higher cell growth

(148, 204).

Considering all aspects of SP-screening, Directed Evolution and growth

improvement, a general strategy could be formulated to optimize SPs for

heterologous proteins (Fig. 33). This strategy is speculative since it is based on the

assumption, that the improved export effects observed in single experiments might

be accumulative leading to an 8-fold total export improvement:

in a first step of a general optimization strategy it might be possible to enhance the

secretion yield up to three-fold compared to commonly used B. subtilis SPs (SP-

AmyE, SP-AprE or SP-SacB) as shown for the cutinase using SP-Epr resulting in 35

mg secreted protein/L.

The second step could be a Directed Evolution approach to function as a “fine-

tuning” of the best natural SP. Based on the saturation mutagenesis results of SP-

AmyE, the production yield of cutinase could be additionally increased two-fold up to

70 mg protein/L.

Finally, before an intended commercial application, the growth conditions could be

adapted to achieve an even higher secretion yield as shown for cutinase production

leading to a further 1.5-fold enhanced secretion by using a different growth medium

up to 100 mg protein/L.

Discussion

91

For the future, this general strategy will be tested for cutinase and other heterologous

targets in B. subtilis. But the established system might not only be limited to

B. subtilis- an application for other Gram-positive bacteria is intended and comprises

an interesting and very promising future perspective.

1 2 3

secr

etio

nyi

eld

[%]

different optimisation steps

frequently used SP

one-step optimization

Directed Evolution

growth conditions

0

100

300

500

700

900

1100

3-fold

2-fold

1,5-fold

1-2

1-2

estim

ated

time

sche

dule

[wee

ks]

4-6

Fig. 33: Idealized model of a general strategy to optimize secretion yields of heterologous proteins. The model is speculative since independent results of the export optimization of the cutinase are combined to an addititve model. The secretion yield is based on secretion level defined as 100% calculated from Table 13 as a mean of three commonly used natural SPs of B. subtilis for heterologous protein production (178), namely SP-AmyE, SP-AprE and SP-SacB. (1) In the first step the best natural SP of B. subtilis for the target protein is found in the SP-library (3.3). (2) A further enhancement of this SP can be obtained by using Directed Evolution strategies (3.4). (3) In another optimization phase the growth can be optimized to enhance the production yield (e.g. growth media). The presented time schedule for the complete optimization procedure with a total time of eight weeks is based on average rates observed for these experiments.

Summary

92

5. Summary Bacillus species are commonly used for industrial production of many biocatalysts.

Despite its powerful export machinery guiding up to 25 g/L of homologous

exoproteins directly into the growth medium, the Gram-positive host B. subtilis often

shows disappointingly low secretion yields of foreign proteins. This thesis deals with

new strategies to overcome some of the frequently described bottlenecks for

heterologous protein production in B. subtilis with respect to industrial applications.

(1) Due to a lack of stable and user-friendly expression plasmids for B. subtilis

comparable to available E. coli systems, a new B. subtilis vector series

pBSMuL1-3, for cloning of target genes under control of one or two strong

constitutive promoter(s), has been constructed. The novel plasmid series

provides a (i) convenient multiple cloning site, (ii) high level overexpression

without the necessity of induction, (iii) generation of libraries for HTS

processes, (iv) efficient secretion of heterologous proteins, and (v) an easy

one-step purification in the Gram-positive host B. subtilis. The functionality of

the plasmids was successfully tested for cutinase and EstCL1 export during

this project.

(2) One major limitation for secretion of foreign proteins is the use of unfavorable

signal peptides (SPs) resulting in inefficient secretion and unsatisfying low

production yields. The main goal of this work was to bypass this secretion

handicap by a so far new strategy called SP-screening. In a first step a

functional secretion reporter system for B. subtilis was generated using the

biotechnological relevant heterologous enzyme cutinase. For the first time, a

pool of all natural B. subtilis SPs was constructed, fused to the cutinase and

screened for export efficiency of cutinase. The screening results revealed a

ranking among the different SPs showing the SP of Epr being the best one

leading to a secretion yield of 35 mg protein/L. Pulse-chase analysis of the

processing kinetics of chosen SPs revealed the targeting of the precursor to

the translocase as the critical step for secretion of the cutinase. Comparative

analysis of all screened SPs with respect to computer prediction tool SignalP

and different relevant criteria of a SP indicated that the in silico prediction of

the best functional SP for a distinct protein is not possible so far due to the

Summary

93

complex interaction of a hybrid protein with all participating components of the

general secretion pathway.

(3) The convenient adaptation of the secretion reporter system to a high

throughput screening (HTS) process in B. subtilis using a pipetting robot

enabled the application of a new “one step optimisation” method to find the

best natural SP for export of any heterologous target in only one cloning and

screening event. This was demonstrated successfully for the export of the

cutinase and a second heterologous enzyme, the non secretory protein

EstCL1 leading to a production yield of 17,5 mg secreted protein/L.

(4) Once established, this HTS system using the cutinase as a secretion marker

was successfully applied to screen for improved signal peptides variants of

SP-AmyE generated by Directed Evolution. Several SP variants were detected

showing a three-fold improved export functionality for the cutinase.

(5) Most of the Sec components play a crucial role for a functioning Sec

machinery in B. subtilis. The most important one is the central motorprotein

SecA which mediates the translocation of precursor proteins through the Sec

pore across the cell membrane. To detect its effect on cutinase export, a

secretion reporter strain B. subtilis Marc1 was constructed containing an

integrated cassette expressing the cutinase fused to the signal peptide of

LipA. Intracellular overexpressed SecA in B. subtilis Marc1 improved the

cutinase export level five-fold most likely by functioning as an intracellular

chaperone protecting the proteolytically sensitive cutinase precursor. A secA

mutant library was generated by Directed Evolution and its screening for

secretion efficiency revealed the SecA variant A23, D809 showing an eight-

fold improved cutinase export. Furthermore, the amino acid histidine at

position 289 in the preprotein binding domain of SecA was shown to be critical

for the effective binding and export of the hybrid protein SP-LipA-cutinase

since the exchange to tyrosine (H809Y) had a severe export reduction as the

consequence.

Zusammenfassung

94

6. Zusammenfassung

Unter den Gram-positiven Bakterien befinden sich verschiedene Vertreter der

Gattung Bacillus, die schon seit Jahrzehnten für die industrielle Produktion von

zahlreichen Biokatalysatoren genutzt werden. Der bekannteste unter ihnen ist

sicherlich Bacillus subtilis, der aufgrund seines leistungsfähigen Sekretionsapparates

zwar in der Lage ist, große Mengen an homologen Exportproteinen ins Nähmedium

zu sekretieren, doch sind die Exportausbeuten an heterologen Proteinen oft

enttäuschend gering. Im Rahmen der vorliegenden Arbeit lag der Schwerpunkt auf

der Entwicklung und Anwendung neuer Strategien, um die häufig auftretenden

Engpässe bei der heterologen Proteinproduktion in B. subtilis zu umgehen. Der

Aspekt der industriellen Anwendung sollte dabei im Vordergrund stehen.

(1) Im Vergleich zu erhältlichen Expressionssystemen für E. coli besteht immer

noch ein Mangel an stabil replizierenden und anwenderfreundlichen

Expressionsplasmiden für B. subtilis. Aus diesem Grund wurde eine neue

Vektorserie pBSMuL1-3 für B. subtilis entwickelt. Diese erlaubt das Klonieren

von Zielgenen unter der Kontrolle von einem oder zwei starken konstitutiven

Promotoren. Die neue Plasmidserie enthält (i) eine großzügige „Multiple

Cloning Site“, sorgt für (ii) eine starke Überexpression ohne die Notwendigkeit

der Induktion, ermöglicht (iii) das Erstellen von Genbänken für HTS Prozesse,

sorgt für (iv) effiziente Sekretion von heterologen Proteinen und ermöglicht (v)

eine einfache Ein-Schritt Proteinaufreinigung aus dem Kulturüberstand von

B. subtilis. Die Funktionalität der Plasmide konnte im Laufe dieser Arbeit für

die beiden heterologen Enzyme Cutinase und EstCL1 gezeigt werden.

(2) Ein wichtiger limitierender Faktor für die Sekretion heterologer Proteine in

B. subtilis stellt die Wahl eines „falschen“ Signalpeptides (SP) dar, was zu

ineffizienter Sekretion und damit zu ungenügender Proteinausbeute führt. Um

dieses Sekretionshemmnis zu umgehen, wurde eine neue Strategie

entwickelt, das sogenannte SP-Screening. Zunächst wurde ein funktionelles

Sekretionsreporter-System für B. subtilis entwickelt, was auf der lipolytischen

Aktivität des heterologen, biotechnologisch relevanten Enzyms Cutinase

basiert. In dieser Arbeit wurde zum ersten Mal überhaupt ein „Pool“ aller

bisher bekannten SP aus B. subtilis erstellt, vor die Cutinase kloniert und auf

Zusammenfassung

95

Exporteffizienz der Cutinase getestet. Es ergab sich eine Rangliste unter den

verschiedenen Signalpeptiden, wobei sich das Signalpeptid von Epr als das

beste erwies. Es führte zu einer Sekretionsausbeute von 35 mg Cutinase/L.

Anhand von Pulse-Chase Experimenten wurde die Prozessierungskinetik des

Vorläuferproteins der Cutinase, fusioniert mit verschiedenen SP, analysiert. Es

stellte sich heraus, dass der Transport des Vorläuferproteins zur Membran der

kritische Schritt bei der Sekretion der Cutinase ist. Durch vergleichende

Analyse aller getesteter SP unter Einbeziehung des Computerprogrammes

SignalP und verschiedener Kriterien von SP konnte gezeigt werden, dass

aufgrund der sehr komplexen Interaktion des Fusionsproteines mit allen

beteiligten Komponenten des Sekretionsweges die in siliko-Vorhersage des

effizientesten SP für ein beliebiges Zielprotein bislang nicht möglich ist.

(3) Die gelungene Integration des Sekretionsreporter-Systems in einen

automatisierten Hochdurchsatz-Screening- (HTS-) Prozess für B. subtilis

erlaubte die Anwendung einer Ein-Schritt-Optimierungsmethode; um das

beste aller SP für die Sekretion eines beliebigen Zielproteins in einem

einzigen Klonierungs- und Screeningverfahrens zu ermitteln. Diese neue

Methode konnte erfolgreich für den Export der Cutinase als auch für den

Export eines weiteren heterologen Enzyms, das nicht sekretorische Protein

EstCL1, angewandt werden, von dem über 17 mg Protein/L sekretiert wurde.

(4) Desweiteren konnte das entwickelte HTS-System genutzt werden, um

verbesserte Varianten des SP von AmyE zu finden, die mittels ungerichteter

Mutagenese generiert wurden. Mehrere SP-Varianten von AmyE wurden

gefunden, die eine dreifach gesteigerte Exportfunktionalität für die Cutinase

zeigten.

(5) Fast alle Komponenten des Sec-Apparates sind essentiell für einen

funktionellen Proteinexport. Die Schlüsselrolle spielt das zentrale Motorprotein

SecA, welches für den aktiven Transport des Vorläuferproteins über die

Zellmembran sorgt. Um den Effekt von SecA auf den Export der Cutinase zu

untersuchen, wurde der Sekretionsreporter-Stamm B. subtilis Marc1

konstruiert, der eine ins Genom integrierte Kassette trägt, die für das

Fusionsprotein aus Cutinase und dem SP von LipA codiert. Die

Überexpression von SecA im Cytoplasma von B. subtilis Marc1 erhöhte die

Exporteffizienz der Cutinase um ein fünffaches. Hierbei wirkt SecA

Zusammenfassung

96

wahrscheinlich als intrazelluläres Chaperon, welches das Vorläuferprotein der

Cutinase vor proteolytischem Abbau schützt. Eine SecA-Genbank wurde

mittels ungerichteter Mutagenese hergestellt und auf verbesserte Sekretion

der Cutinase getestet. Es wurde die SecA-Variante A23, D809 gefunden, die

zu einer achtfachen Exportsteigerung führte. Desweiteren zeigte sich die

Aminosäure Histidin an Position 289 in der Präprotein-Bindedomäne von

SecA als kritisch für das effektive Binden und den Transport des

Vorläuferproteins der Cutinase zur Translokase, da der Aminosäureaustausch

zum Tyrosin (H809Y) einen erheblichen Exportrückgang zu Folge hatte.

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Appendix

111

8. Appendix

Table 12: Oligonucleotides used for PCR-amplification of all Sec-dependent signal sequences from genomic DNA of B.subtilis 168. All upstream primers start with the consensus sequence 5´-ATATAAGCTTAAGGAGGATATTATG-3´ containing the EcoRI-restriction site, the Bacillus ribosome binding site and the ATG-start codon for optimal translation. All downstream primers possess the consensus sequence 3´-ATATGAATTCAGC-5´ containing the HindIII restriction site and the GCT-triplett coding for alanine at the +1 position of all signal sequences. In some primers silent mutations are introduced avoiding additional EcoRI or HindIII restriction sites. These base substitutions are highlighted in bold letters. The signal sequences are listed in alphabetical order.

Signal

sequence

Upstream primer (5'→→→→3')

Downstream primer (5'→→→→3')

AbnA ATATAAGCTTAAGGAGGATATTATGAAAAAGAAAAAAACATGG ATATGAATTCAGCTGCCTCTGCGGGAGCAGCAGA AmyE ATATAAGCTTAAGGAGGATATTATGTTTGCAAAACGATTCAAA ATATGAATTCAGCAGCACTCGCAGCCGCCGGTCC AprE ATATAAGCTTAAGGAGGATATTATGAGAAGCAAAAAATTGTGG ATATGAATTCAGCAGCCTGCACAGACATGTTGCT AspB ATATAAGCTTAAGGAGGATATTATGAAACTGGCAAAAAGAGTA ATATGAATTCAGCCGCTTTCGCTGTGATTGCCAG BglC ATATAAGCTTAAGGAGGATATTATGAAACGGTCAATCTCTATT ATATGAATTCAGCTGCTGATGCCGGCGAAGCTAT BglS ATATAAGCTTAAGGAGGATATTATGCCTTATCTGAAACGAGTG ATATGAATTCAGCAGCTGAGGCAGTAGCAGTGAC Bpr ATATAAGCTTAAGGAGGATATTATGAGGAAAAAAACGAAAAAC ATATGAATTCAGCTGCCCCGGCTGCTCCCGGAAA CccA ATATAAGCTTAAGGAGGATATTATGAAATGGAACCCGCTTATT ATATGAATTCAGCTCCTTTTACTGATAAAAAGAA CitH ATATAAGCTTAAGGAGGATATTATGGGAAATACTCGTAAAAAA ATATGAATTCAGCAACGTCTGCCAGCTCTTTTTG CotC ATATAAGCTTAAGGAGGATATTATGAAAAATCGGCTCTTTATT ATATGAATTCAGCAGATTTTGCGGCTTGGACAGT Csn ATATAAGCTTAAGGAGGATATTATGAAAATCAGTATGCAAAAA ATATGAATTCAGCCGCAAAAACCGTTTCGCTCAT CwlD ATATAAGCTTAAGGAGGATATTATGAGGAAAAAACTTAAATGG ATATGAATTCAGCATTGCTGAACTGATACTTGAA DacB ATATAAGCTTAAGGAGGATATTATGCGCATTTTCAAAAAAGCA ATATGAATTCAGCAGCATGTGCTGTATTCACATT DacF ATATAAGCTTAAGGAGGATATTATGAAACGTCTTTTATCCACT ATATGAATTCAGCTGCAAATGCAGACGGTGCAAA DltD ATATAAGCTTAAGGAGGATATTATGAAAAAGCGTTTTTTCGGT ATATGAATTCAGCTGCGATGGCGCCTGCGAATAG Epr ATATAAGCTTAAGGAGGATATTATGAAAAACATGTCTTGCAAA ATATGAATTCAGCCGCATGAGCGAGAGGGCCTAT FliL ATATAAGCTTAAGGAGGATATTATGAAGAAAAAGTTAATGATC ATATGAATTCAGCAGCCGCCGCCCCGAGAGCACC FliZ ATATAAGCTTAAGGAGGATATTATGAAAAAGAGTCAATATTTT ATATGAATTCAGCTGCCGCGGCAGCAGCAATCGG GlpQ ATATAAGCTTAAGGAGGATATTATGAGAAAAAATAGAATACTG ATATGAATTCAGCTGCCGACACTGGCGTTACCAT LipA ATATAAGCTTAAGGAGGATATTATGAAATTTGTAAAAAGAAGG ATATGAATTCAGCAGCGGCTTTTGCTGACGGCTG LipB ATATAAGCTTAAGGAGGATATTATGAAAAAAGTACTTATGGCA ATATGAATTCAGCAGCTTTTGCGCCAGACGGCGG LytB ATATAAGCTTAAGGAGGATATTATGAAATCTTGCAAACAATTG ATATGAATTCAGCTGCAAAAGAAACTGATGGAAT LytC ATATAAGCTTAAGGAGGATATTATGCGTTCTTATATAAAAGTC ATATGAATTCAGCGGCCAAAGCTGTTGGCACAAA LytD ATATAAGCTTAAGGAGGATATTATGAAAAAGAGACTAATCGCA ATATGAATTCAGCTGCCTGGGCAGAACCAGACAT LytE ATATAAGCTTAAGGAGGATATTATGAAAAAGCAAATCATTACA ATATGAATTCAGCTGCAAATAACGCTCCTAAAAC LytF ATATAAGCTTAAGGAGGATATTATGAAAAAGAAATTAGCAGCA ATATGAATTCAGCTGCTTCAGCTGGTGTCACTAC LytR ATATAAGCTTAAGGAGGATATTATGAGAAACGAACGCAGAAAA ATATGAATTCAGCAGCTGCTTTATGCCATAAGTA Mdr ATATAAGCTTAAGGAGGATATTATGGACACAACAACAGCAAAA ATATGAATTCAGCCGCGGTGGCAACAATCGTATT MotB ATATAAGCTTAAGGAGGATATTATGGCGAGAAAAAAGAAGAAG ATATGAATTCAGCGCTGCTCGCGTACAGCACAAT Mpr ATATAAGCTTAAGGAGGATATTATGAAATTAGTTCCAAGATTC ATATGAATTCAGCCGCTTTTGCCGGTACGCCAAA MreC ATATAAGCTTAAGGAGGATATTATGCCGAATAAGCGGTTAATG ATATGAATTCAGCCGAAAATCCAATCATAGCCAC NprB ATATAAGCTTAAGGAGGATATTATGCGCAACTTGACCAAGACA ATATGAATTCAGCAGCTGAGGCATGTGTTACAAA NprE ATATAAGCTTAAGGAGGATATTATGGGTTTAGGTAAGAAATTG ATATGAATTCAGCAGCCTGAACACCTGGCAGGCT NucB ATATAAGCTTAAGGAGGATATTATGAAAAAATGGATGGCAGGC ATATGAATTCAGCCGAAGATGCGCCTTGGATCTG Pbp ATATAAGCTTAAGGAGGATATTATGAAAAAAAGCATAAAACTT ATATGAATTCAGCGGCAAGCGCAGCCTGATGCAT PbpB ATATAAGCTTAAGGAGGATATTATGATTCAAATGCCAAAAAAG ATATGAATTCAGCTGCCATTCTCCCCAGGATGAC PbpD ATATAAGCTTAAGGAGGATATTATGACCATGTTACGAAAAATA ATATGAATTCAGCAGCGATAACTGTAAATGCAAA PbpX ATATAAGCTTAAGGAGGATATTATGACAAGCCCAACCCGCAGA ATATGAATTCAGCAGCATTCCAAATCGTAATGCA Pel ATATAAGCTTAAGGAGGATATTATGAAAAAAGTGATGTTAGCT ATATGAATTCAGCTGCGTTCGCGCCAGCTGGAGT PelB ATATAAGCTTAAGGAGGATATTATGAAACGACTTTGTTTATGG ATATGAATTCAGCACCTAATGCTTTTCCAGGCAA PenP ATATAAGCTTAAGGAGGATATTATGAAGTTGAAAACTAAAGCG ATATGAATTCAGCTGCTTCGGCATGTGTTGAGTT PhoA ATATAAGCTTAAGGAGGATATTATGAAAAAAATGAGTTTGTTT ATATGAATTCAGCAGCTCCGGCAAAGATTCCAGC PhoB ATATAAGCTTAAGGAGGATATTATGAAAAAATTCCCGAAGAAA ATATGAATTCAGCGGCGCTGGCTTCAGGCACACT PhrA ATATAAGCTTAAGGAGGATATTATGAAATCTAAATGGATGTCA ATATGAATTCAGCTGCATGAACCATCACCTGAGT PhrC ATATAAGCTTAAGGAGGATATTATGAAATTGAAATCTAAGTTG ATATGAATTCAGCCGCATTAGCAGAAACGCCAGC PhrF ATATAAGCTTAAGGAGGATATTATGAAATTGAAGTCTAAACTA ATATGAATTCAGCTGCAATAGTTGTTGCCACGAA PhrG ATATAAGCTTAAGGAGGATATTATGAAAAGATTTCTGATTGGC ATATGAATTCAGCCGCAATAAACCAACCTGATAA PhrK ATATAAGCTTAAGGAGGATATTATGAAAAAACTTGTGCTTTGC ATATGAATTCAGCAGCTACTCCACTTAAAATCAC RpmG ATATAAGCTTAAGGAGGATATTATGAGAAAAAAGATTACGTTA ATATGAATTCAGCCGCTGATGCAGAGCTCTTCAT SacB ATATAAGCTTAAGGAGGATATTATGAACATCAAAAAGTTTGCA ATATGAATTCAGCCGCAAACGCTTGAGTTGCGCC SacC ATATAAGCTTAAGGAGGATATTATGAAAAAGAGACTGATTCAA ATATGAATTCAGCTGCATCTGCCGAAAATGCCAT SleB ATATAAGCTTAAGGAGGATATTATGAAGTCCAAAGGATCGATT ATATGAATTCAGCCGAAAAGGCAGAGATCGTTTC SpoIID ATATAAGCTTAAGGAGGATATTATGAAACAATTCGCAATCACA ATATGAATTCAGCGGCCCCCGCTTCCTTATTATG SpoIIP ATATAAGCTTAAGGAGGATATTATGAGAAATAAACGCAGAAAC ATATGAATTCAGCCACACCGGATAGAACAAAAAT SpoIIQ ATATAAGCTTAAGGAGGATATTATGAGAGAGGAAGAAAAGAAA ATATGAATTCAGCAAGGACAGCTGTTAAAATGAC

Appendix

112

Signal

sequence



SpoIIR ATATAAGCTTAAGGAGGATATTATGAAAAAAACAGTAATCATT ATATGAATTCAGCTACGAGCGCTCCGGATAATAA TasA ATATAAGCTTAAGGAGGATATTATGGGTATGAAAAAGAAATTG ATATGAATTCAGCTGCCCATGTTCCTCCTCCAAC TyrA ATATAAGCTTAAGGAGGATATTATGAATCAAATGAAAGATACA ATATGAATTCAGCGGCTAGGGCAATCGAACCGCC Vpr ATATAAGCTTAAGGAGGATATTATGAAAAAGGGGATCATTCGC ATATGAATTCAGCAGCCGGAGCTGCCTGAACGCC WapA ATATAAGCTTAAGGAGGATATTATGAAAAAAAGAAAGAGGCGA ATATGAATTCAGCTGCTAGTACATCGGCTGGCAC WprA ATATAAGCTTAAGGAGGATATTATGAAACGCAGAAAATTCAGC ATATGAATTCAGCCGCTGCAGCTTTGGTTCCCGG XynA ATATAAGCTTAAGGAGGATATTATGTTTAAGTTTAAAAAGAAT ATATGAATTCAGCTGCAGAGGCGGTTGCCGAAAA YbbC ATATAAGCTTAAGGAGGATATTATGAGAAAAACAATATTCGCT ATATGAATTCAGCAGCAGAGGCAGCGGTTATCGT YbbE ATATAAGCTTAAGGAGGATATTATGAAAACAAAGACACTGTTC ATATGAATTCAGCTGCGAAGGTTTCATTTGGCGC YbbR ATATAAGCTTAAGGAGGATATTATGGATAAATTCTTAAACAAC ATATGAATTCAGCGCTGTTAACCGCCACATAAAG YbdG ATATAAGCTTAAGGAGGATATTATGAAAACATTATGGAAAGTC ATATGAATTCAGCCGAGACGGATACAAGCAAAAC YbdN ATATAAGCTTAAGGAGGATATTATGGTGAAAAAATGGCTTATT ATATGAATTCAGCAGCTGATGCCGAATACGTAAA YbfO ATATAAGCTTAAGGAGGATATTATGAAACGAATGATAGTGAGA ATATGAATTCAGCAGCACGGGCTGAGGCTGAAAA YbxI ATATAAGCTTAAGGAGGATATTATGAAAAAATGGATATATGTT ATATGAATTCAGCTGCGTGGACGGAGAAGCCGCC YckD ATATAAGCTTAAGGAGGATATTATGAAACGAATAACCATCAAC ATATGAATTCAGCGGCCTCAGCTGTACCGGTCAG YdbK ATATAAGCTTAAGGAGGATATTATGAAACTTTTTAATCGGAAG ATATGAATTCAGCACTGATGACAATCCGTTTAAT YddT ATATAAGCTTAAGGAGGATATTATGAGAAAGAAAAGAGTTATT ATATGAATTCAGCTGCAGAAGCGTAACCTGCAGG YdhT ATATAAGCTTAAGGAGGATATTATGTTTAAGAAACATACGATC ATATGAATTCAGCTGCTAAAACAGCAGACGCAAG YdjM ATATAAGCTTAAGGAGGATATTATGTTGAAGAAAGTCATTTTA ATATGAATTCAGCCGCACTGGCATCTGATGAAAA YdjN ATATAAGCTTAAGGAGGATATTATGAAAAAAAGAATCATATTA ATATGAATTCAGCCGCAACCCCCGCGGCAGCTGC YfhK ATATAAGCTTAAGGAGGATATTATGAAAAAGAAACAAGTAATG ATATGAATTCAGCAGCTTTTGCTGCGGGAGCGGA YfjS ATATAAGCTTAAGGAGGATATTATGAAGTGGATGTGTTCAATA ATATGAATTCAGCCGCCTGTGCTGCACCTCCGGC YfkD ATATAAGCTTAAGGAGGATATTATGATGAAAAAGCTATTTCAT ATATGAATTCAGCCGCGTGGATGGGCTGAACGCC YfkN ATATAAGCTTAAGGAGGATATTATGAGAATACAGAAAAGACGA ATATGAATTCAGCTGCATGAATGGGTGGTGTTGG YhaK ATATAAGCTTAAGGAGGATATTATGCGCACATGGAAACGTATA ATATGAATTCAGCTGCCAGTGCTGCGTAAAACAA YhcR ATATAAGCTTAAGGAGGATATTATGCTGTCTGTCGAAATGATA ATATGAATTCAGCAGCTTCGAACGTGTACATTAC YhdC ATATAAGCTTAAGGAGGATATTATGAAATCCCTGCCGTATACC ATATGAATTCAGCAGCCATTGACACGATAATCAA YhfM ATATAAGCTTAAGGAGGATATTATGAAAAAAATAGTGGCAGCC ATATGAATTCAGCCGCGTCTACCGATTGATACAC YhjA ATATAAGCTTAAGGAGGATATTATGAAAAAAGCGGCGGCGGTT ATATGAATTCAGCGGCTTCCGCCACATGGCCAGC YjcM ATATAAGCTTAAGGAGGATATTATGAAAAAAGAATTACTTGCT ATATGAATTCAGCTGCAAAAACTTCATTTGTTGA YjcN ATATAAGCTTAAGGAGGATATTATGAAAAAGAAAACTAAAATT ATATGAATTCAGCCGAAGAAGCTGTAAAGACAAC YjdB ATATAAGCTTAAGGAGGATATTATGAATTTCAAAAAAACGGTT ATATGAATTCAGCAGCTGAAGCTACCCCGCTGAC YjfA ATATAAGCTTAAGGAGGATATTATGAAAAGACTGTTTATGAAG ATATGAATTCAGCCGCCTTGGCGGGTGCACCTTT YjiA ATATAAGCTTAAGGAGGATATTATGGCAGCACAGACTGATTAC ATATGAATTCAGCAGAGAACACAAAGAGTACGAA YknX ATATAAGCTTAAGGAGGATATTATGAAAAAAGTCTGGATCGGA ATATGAATTCAGCGGCGCTGCCGCTTGTCGGAGC YkoJ ATATAAGCTTAAGGAGGATATTATGCTCAAGAAAAAATGGATG ATATGAATTCAGCGGCAAACGCGTTGTAGCTGAA YkvT ATATAAGCTTAAGGAGGATATTATGACAACGAAATTCACTGCT ATATGAATTCAGCAATTTTGGCTGCAGGCATGAA YkvV ATATAAGCTTAAGGAGGATATTATGTTGACGAAGCGCTTGCTT ATATGAATTCAGCAGCTTGTGCCGCACCTGGAAA YkwD ATATAAGCTTAAGGAGGATATTATGAAGAAAGCATTTATTTTA ATATGAATTCAGCCGCTGATGCTTGCTGTACGCC YlaE ATATAAGCTTAAGGAGGATATTATGAAGAAAACATTTGTAAAA ATATGAATTCAGCTGCGCTGGCTGCATCAGGACC YlbL ATATAAGCTTAAGGAGGATATTATGCTACGTAAAAAACATTTT ATATGAATTCAGCCGCTTCTCCCGGTTTTGTAAT YlqB ATATAAGCTTAAGGAGGATATTATGAAAAAAATCGGTTTATTG ATATGAATTCAGCAGCGTCTGCTTGCTGTGCCGG YlxF ATATAAGCTTAAGGAGGATATTATGTCCGGCAAAAAGAAAGAA ATATGAATTCAGCACCAGCAGCCCAAAGAACAAT YlxW ATATAAGCTTAAGGAGGATATTATGAGGGGCAAATCAGCAGTC ATATGAATTCAGCAGCCGCGCTTTTGTTGTTTTC YlxY ATATAAGCTTAAGGAGGATATTATGTACAAAAAATTTGTACCG ATATGAATTCAGCTGCCCCTATATAATCAAGTGC YncM ATATAAGCTTAAGGAGGATATTATGGCGAAACCACTATCAAAA ATATGAATTCAGCAGCGTCTGCCGCGGGTAAACC YndA ATATAAGCTTAAGGAGGATATTATGAGATTCACTAAGGTAGTT ATATGAATTCAGCTGCTTGTGCCGTTAATGGAAA YnfF ATATAAGCTTAAGGAGGATATTATGATTCCACGCATAAAAAAA ATATGAATTCAGCTGCCAAAACTTCAGTAGCGCC YngK ATATAAGCTTAAGGAGGATATTATGAAGGTTTGCCAAAAGTCG ATATGAATTCAGCCGCATTGGCCATAAACGGAAC YnzA ATATAAGCTTAAGGAGGATATTATGGAATTGAGCTTCACAAAA ATATGAATTCAGCGCCAAGCGCCGGCAGTTTATC YoaW ATATAAGCTTAAGGAGGATATTATGAAAAAGATGTTGATGTTA ATATGAATTCAGCGGCCGACGCTTCCCCTACATG YobB ATATAAGCTTAAGGAGGATATTATGAAGATTAGGAAAATCTTA ATATGAATTCAGCAGCCAATGCGGGAACCGCGGA YobV ATATAAGCTTAAGGAGGATATTATGAAACTGGAACGTTTGTTA ATATGAATTCAGCAGCCTGCACTTGTTTTTTGCT YocA ATATAAGCTTAAGGAGGATATTATGAAGAAAAAGAGAAAAGGC ATATGAATTCAGCGGCGATGACAAAGACAAAAAT YocH ATATAAGCTTAAGGAGGATATTATGAAGAAGACGATTATGTCC ATATGAATTCAGCAGCGTGAGCTCCGAATGCAGT YodV ATATAAGCTTAAGGAGGATATTATGAAGGTTCCAAAAACAATG ATATGAATTCAGCAGCCGACACCGAGGTTGCTGT YojL ATATAAGCTTAAGGAGGATATTATGAAAAAGAAGATTGTAGCC ATATGAATTCAGCTGCTTCCGCGGGTGCTGCGGC YolA ATATAAGCTTAAGGAGGATATTATGAAGAAGAGAATTACATAT ATATGAATTCAGCCGCTTTTGCTTTTGATGAATC YolC ATATAAGCTTAAGGAGGATATTATGAAGAAGAGGCTAATCGGA ATATGAATTCAGCTGCTTCGATTAAAGTAATACC YolI ATATAAGCTTAAGGAGGATATTATGAAAAAGTGGATTGTTTTA ATATGAATTCAGCTTCGCTACCTGTAGAAACATA YomL ATATAAGCTTAAGGAGGATATTATGAGAAAGAAAAGAGTTATT ATATGAATTCAGCTGCAGTAGCGTAACCTGCAGG YopL ATATAAGCTTAAGGAGGATATTATGAAAAAACTTATTATGGCT ATATGAATTCAGCTGCACTTATGTAAGAAGTGCC YoqH ATATAAGCTTAAGGAGGATATTATGAAACGATTTATTTTAGTT ATATGAATTCAGCGGCGTTTGTTTGAATGGGATA YoqM ATATAAGCTTAAGGAGGATATTATGAAATTAAGAAAAGTATTG ATATGAATTCAGCAGCGAATGCAGGAGAAGCAGA YpbG ATATAAGCTTAAGGAGGATATTATGAAGCTATCAGTGAAAATT ATATGAATTCAGCTGCTGTTGCATACATTTTTGC YpcP ATATAAGCTTAAGGAGGATATTATGAATAATAATAAACTATTG ATATGAATTCAGCGGCCGTGGCAAAAAAGGCCCG YpjP ATATAAGCTTAAGGAGGATATTATGAAATTGTGGATGAGAAAG ATATGAATTCAGCCGCCATAAGGGCAGCCGGAGG YpmB ATATAAGCTTAAGGAGGATATTATGAGAAAAAAAGCATTAATA ATATGAATTCAGCTGCCATGGCTGATTTATAAAT YpmS ATATAAGCTTAAGGAGGATATTATGAATAAGTGGAAGCGACTG ATATGAATTCAGCCACCTGAGCCTGTTCCCCCGG YpuA ATATAAGCTTAAGGAGGATATTATGAAGAAAATTTGGATTGGA ATATGAATTCAGCGGCATCCGCGAGACTGACCTT YpuD ATATAAGCTTAAGGAGGATATTATGGGAAGAATAAAAACCAAG ATATGAATTCAGCTCCAATTGCTGTCGGAACATC YqfZ ATATAAGCTTAAGGAGGATATTATGAAGCGTCTCACCTTAGTA ATATGAATTCAGCTGCTGATGCTTCATAGACAGG YqgA ATATAAGCTTAAGGAGGATATTATGAAGCAAGGAAAATTTTCT ATATGAATTCAGCAGCCTCTGCTTTTCCTTTAGG

Appendix

113

Signal

sequence



YqxI ATATAAGCTTAAGGAGGATATTATGTTTAAGAAATTACTTTTA ATATGAATTCAGCAGCTTTGGCATGTCCATCCAA YqxM ATATAAGCTTAAGGAGGATATTATGTTTCGATTGTTTCACAAT ATATGAATTCAGCAGCGCTTGTATCATCGGAAAA YqzC ATATAAGCTTAAGGAGGATATTATGACAAAACGGGGCATTCAG ATATGAATTCAGCAGCAGCGGCCTGGTCTTCGTC YqzG ATATAAGCTTAAGGAGGATATTATGATGATCAAACAATGTGTG ATATGAATTCAGCTGCGTGAGCGGCTGTGGTGCC YraJ ATATAAGCTTAAGGAGGATATTATGACATTGACTAAACTGAAA ATATGAATTCAGCGGCAAGTGCCTGACTGGAAAA YrrL ATATAAGCTTAAGGAGGATATTATGTATATCAATCAGCAAAAA ATATGAATTCAGCTAAAAATGCCCCGCCAATGAT YrrR ATATAAGCTTAAGGAGGATATTATGAAGATATCGAAACGAATG ATATGAATTCAGCGATTTCTGCAAGTCTCAGCAG YrrS ATATAAGCTTAAGGAGGATATTATGAGCAATAATCAATCTCGT ATATGAATTCAGCCGCTGCTACTACGACAATTAG YrvJ ATATAAGCTTAAGGAGGATATTATGAACAAGAAATACTTTGTC ATATGAATTCAGCAGCTGTAACGGATGAAAAGGT YuaB ATATAAGCTTAAGGAGGATATTATGAAACGCAAATTATTATCT ATATGAATTCAGCTTCAGCCGCGAAAGAAGCTGT YunA ATATAAGCTTAAGGAGGATATTATGATTACTGACATTTTTAAG ATATGAATTCAGCTGCAAACAAAAGGAGAAGAAG YunB ATATAAGCTTAAGGAGGATATTATGCCAAGATATCGCGGCCCT ATATGAATTCAGCAAGGCTGACCGTTGTTGACAG YurI ATATAAGCTTAAGGAGGATATTATGACAAAAAAAGCATGGTTT ATATGAATTCAGCCGCGCTTGCTGAAGCTGCTGG YusW ATATAAGCTTAAGGAGGATATTATGCATTTGATCAGAGCAGCC ATATGAATTCAGCTCCTTCTGCCTGATGCTGGTC YvbX ATATAAGCTTAAGGAGGATATTATGAAAAAATGGCTGATCATA ATATGAATTCAGCTGCCTTCGCTTCTCCTTTTGT YvcE ATATAAGCTTAAGGAGGATATTATGAGAAAGAGTTTAATTACA ATATGAATTCAGCCGCCGATGCAGTTTTACTTGT YveB ATATAAGCTTAAGGAGGATATTATGAACTATATAAAAGCAGGC ATATGAATTCAGCTAAGTCGATAAACAGCAATAT YvgO ATATAAGCTTAAGGAGGATATTATGAAACGTATTCGTATCCCA ATATGAATTCAGCAGCGGAAGCAAAGGACAAAGG YvgV ATATAAGCTTAAGGAGGATATTATGAAAAAGAAACAGCAGTCT ATATGAATTCAGCGACAATGGCTGCTAGCAATAC YvnB ATATAAGCTTAAGGAGGATATTATGAGGAAATACACGGTTATT ATATGAATTCAGCGCCGCCAGACAAAACGGAAAG YvpA ATATAAGCTTAAGGAGGATATTATGAAAAAAATCGTGTCTATC ATATGAATTCAGCTGCAAAAACGGTTGATGGCTG YvpB ATATAAGCTTAAGGAGGATATTATGAAAACACTGCGAACTCTA ATATGAATTCAGCTGCATCTATTTTAAGTCCGAA YwaD ATATAAGCTTAAGGAGGATATTATGAAAAAGCTGTTGACTGTC ATATGAATTCAGCAGCGTGCGCGGCAGGGGTGAC YwcI ATATAAGCTTAAGGAGGATATTATGAAACGTTTGCTTGTATCT ATATGAATTCAGCGGCACGCGCAGTTTTTCTGTC YwdK ATATAAGCTTAAGGAGGATATTATGAAAGTTTTTATCATATTA ATATGAATTCAGCGCCGAATGCTCCGAGTCCCAC YweA ATATAAGCTTAAGGAGGATATTATGCTAAAAAGAACTTCATTC ATATGAATTCAGCTGCATGAGCTTGGCCCGAAGG YwfM ATATAAGCTTAAGGAGGATATTATGAAGGGGAATATTTACAGC ATATGAATTCAGCTGCCTGAACGGTTCCCGTGGT YwgB ATATAAGCTTAAGGAGGATATTATGAAAATGAAATCAGGAATG ATATGAATTCAGCAGCCTGTACCGGCAGCCGTGA YwjE ATATAAGCTTAAGGAGGATATTATGAAGGTATTTATCGTGATT ATATGAATTCAGCGGCACGGCCCATGAAGATATC YwmB

ATATAAGCTTAAGGAGGATATTATGAAGAAAAAACAAGTAAGC

ATATGAATTCAGCCGCATGAATGGTATGGAAAACAGCAATGACGAAGCTCAACA

YwmC ATATAAGCTTAAGGAGGATATTATGAAGAAAAGATTTTCACTG ATATGAATTCAGCTGCAAAAGCAGGTGAAGTTAA YwmD ATATAAGCTTAAGGAGGATATTATGAAAAAATTGCTGGCTGCC ATATGAATTCAGCGGCAAAAGACGGGGAGGCAAT YwoF ATATAAGCTTAAGGAGGATATTATGAGAAAATGGTATTTTATT ATATGAATTCAGCCGCTTTTGTCTTATCATAAAC YwqC ATATAAGCTTAAGGAGGATATTATGGGAGAATCTACGAGCTTA ATATGAATTCAGCAGCAGTGGCAGCCGCGGTTAC YwqO ATATAAGCTTAAGGAGGATATTATGAAGTTTTTGCTGTCGGTC ATATGAATTCAGCGTCCAGCCTCCTGTTTGGATA YwsB ATATAAGCTTAAGGAGGATATTATGAACAAACCAACAAAACTA ATATGAATTCAGCAGCATGGATCGTGCCAGCGCC YwtC ATATAAGCTTAAGGAGGATATTATGAAATTTGTCAAAGCCATC ATATGAATTCAGCAGCTGACATAAACATGAACAC YwtD ATATAAGCTTAAGGAGGATATTATGAACACACTGGCAAACTGG ATATGAATTCAGCAGCTTCCGCAATCTCCGCTTT YwtF ATATAAGCTTAAGGAGGATATTATGGAAGAACGATCACAGCGC ATATGAATTCAGCAGCATAGGCGCCGACAGAGCC

YxaK ATATAAGCTTAAGGAGGATATTATGGTCAAGTCATTTCGTATGAAA ATATGAATTCAGCCGCATAAGCAGCTGTCGGCTG

YxiA ATATAAGCTTAAGGAGGATATTATGTTCAACCGATTGTTCCGT ATATGAATTCAGCTGCATAGACGGAGTTTGGGAG YxiT ATATAAGCTTAAGGAGGATATTATGAAATGGAATAACATGCTG ATATGAATTCAGCCGCTTGAACTGCTTTTAAAGA YyaB ATATAAGCTTAAGGAGGATATTATGGTTTATCAAACGAAGAGA ATATGAATTCAGCGGCATCTGCTTGTATCAGTAA YybN ATATAAGCTTAAGGAGGATATTATGAATAAATTCCTGAAGAGC ATATGAATTCAGCGGCTTTTATAAAATTACTACT YycP ATATAAGCTTAAGGAGGATATTATGAAAAAGTGGATGATTACC ATATGAATTCAGCGCTTTTGAGAGGAGAAATGAA

Table 13: Comparison of all screened signal sequences used for export of heterologous cutinase in B. subtilis. The results represent data of 12 independent experiments.

No.

Name

Signal sequence


a

Charge

N-region b

Hydro-

phobicity [% ]

c

D-

Score d

1 Epr MKNMSCKLVVSVTLFFSFLTIGPLAHA 4.67 2 62.96 0.919 2 YncM MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADA 4.12 4 76.19 0.507 3 YjfA MKRLFMKASLVLFAVVFVFAVKGAPAKA 3.84 3 78.57 0.924 4 YfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA 3.67 3 68.97 0.906 5 Csn MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA 3.35 3 62.86 0.689 6 LytD MKKRLIAPMLLSAASLAFFAMSGSAQA 3.33 3 70.37 0.87 7 Bpr MRKKTKNRLISSVLSTVVISSLLFPGAAGA 2.97 5 56.67 0.936 8 WapA MKKRKRRNFK RFIAAFLVLA LMISLVPADVLA 2.88 8 65.63 0.918 9 BglC MKRSISIFITCLLITLLTMGGMIASPASA 2.87 2 65.52 0.839

10 LytB MKSCKQLIVCSLAAILLLIPSVSFA 2.83 2 64.00 0.916 11 LipA MKFVKRRIIALVTILMLSVTSLFALQPSAKA 2.79 4 64.52 0.874 12 YckD MKRITINIITMFIAAAVISLTGTAEA 2.79 2 65.38 0.839 13 Pel MKKVMLATALFLGLTPAGANA 2.67 2 76.19 0.843 14 YnfF MIPRIKKTICVLLVCFTMLSVMLGPGATEVLA 2.61 3 68.75 0.699

Appendix

114

No.

Name

Signal sequence


a

Charge N-region

b

Hydro-phobicity

[% ]c

D-Score

d

15 PhrK MKKLVLCVSILAVILSGVA 2.53 2 73.68 0.669 16 YbdN MVKKWLIQFAVMLSVLSTFTYSASA 2.51 2 60.00 0.853 17 YobB MKIRKILLSSALSFGMLISAVPALA 2.49 3 72.00 0.904 18 YddT MRKKRVITCVMAASLTLGSLLPAGYASA 2.41 4 60.71 0.901 19 YhfM MKKIVAAIVVIGLVFIAFFYLYSRSGDVYQSVDA 2.30 2 64.71 0.286 20 BglS MPYLKRVLLLLVTGLFMSLFAVTATASA 2.25 2 71.43 0.911 21 Vpr MKKGIIRFLLVSFVLFFALSTGITGVQAAPA 2.20 3 74.19 0.877 22 AprE MRSKKLWISLLFALTLIFTMAFSNMSVQA 2.19 3 62.07 0.886 23 YjdB MNFKKTVVSALSISALALSVSGVASA 2.10 2 61.54 0.928 24 YbbE MKTKTLFIFSAILTLSIFAPNETFA 2.09 2 60.00 0.825 25 PhrC MLKSKLFVICLAAAAIFTAAGVSANA 2.00 3 69.23 0.862 26 GlpQ MRKNRILALFVLSLGLLSFMVTPVSA 1.96 3 69.23 0.921 27 SacC MKKRLIQVMIMFTLLLTMAFSADA 1.95 3 66.67 0.86 28 YurI MTKKAWFLPLVCVLLISGWLAPAASASA 1.94 2 75.00 0.871 29 PhoB MKKFPKKLLPIAVLSSIAFSSLASGSVPEASA 1.92 4 62.50 0.926 30 PenP MKLKTKASIKFGICVGLLCLSITGFTPFFNSTHAEA 1.92 4 55.56 0.708 31 YfkD MMKKLFHSTLIVLLFFSFFGVQPIHA 1.87 2 69.23 0.735 32 YvpA MKKIVSILFMFGLVMGFSQFQPSTVFA 1.87 2 70.37 0.717 33 YdjM MLKKVILAAFILVGSTLGAFSFSSDASA 1.83 2 67.86 0.832 34 AbnA MKKKKTWKRFLHFSSAALAAGLIFTSAAPAEA 1.82 6 59.38 0.898 35 YwjE MKVFIVIMIIVVIFFALILLDIFMGRA 1.80 1 88.89 0.638 36 YqgA MKQGKFSVFLILLLMLTLVVAPKGKAEA 1.64 2 71.43 0.807 37 LipB MKKVLMAFIICLSLILSVLAAPPSGAKA 1.62 2 75.00 0.914 38 FliZ MKKSQYFIVFICFFVLFSVHPIAAAAA 1.61 2 70.37 0.684 39 DacB MRIFKKAVFVIMISFLIATVNVNTAHA 1.57 3 66.67 0.876 40 SacB MNIKKFAKQATVLTFTTALLAGGATQAFA 1.51 3 62.07 0.866 41 YrvJ MNKKYFVLIVCIIFTSALFPTFSSVTA 1.49 2 59.26 0.762 42 YlaE MKKTFVKKAMLTTAAMTSAALLTFGPDAASA 1.47 4 61.29 0.93 43 Pbp MKKSIKLYVAVLLLFVVASVPYMHQAALA 1.46 3 68.97 0.733 44 YbxI MKKWIYVVLVLSIAGIGGFSVHA 1.44 2 73.91 0.612 45 YolA MKKRITYSLLALLAVVAFAFTDSSKAKA 1.44 3 57.14 0.834 46 YqxI MFKKLLLATSALTFSLSLVLPLDGHAKA 1.41 2 71.43 0.837 47 YoaW MKKMLMLAFTFLLALTIHVGEASA 1.36 2 70.83 0.821 48 NprB MRNLTKTSLLLAGLCTAAQMVFVTHASA 1.33 2 57.14 0.85 49 YlxF MSGKKKESGKFRSVLLIIILPLMFLLIAGGIVLWAAG 1.33 4 75.68 0.563 50 YbfO MKRMIVRMTLPLLIVCLAFSSFSASARA 1.31 3 64.29 0.86 51 YlqB MKKIGLLFMLCLAALFTIGFPAQQADA 1.30 2 74.07 0.838 52 SpoIID MKQFAITLSVLCALILLVPTLLVIPFQHNKEAGA 1.28 1 67.65 0.718 53 YwmC MKKRFSLIMMTGLLFGLTSPAFA 1.17 3 69.57 0.912 54 YvbX MKKWLIIAVSLAIAIVLFMYTKGEAKA 1.09 2 70.37 0.627 55 YkvV MLTKRLLTIYIMLLGLIAWFPGAAQA 1.02 2 76.92 0.808 56 YlxY MYKKFVPFAVFLFLFFVSFEMMENPHALDYIGA 0.98 2 69.70 0.488 57 XynA MFKFKKNFLVGLSAALMSISLFSATASA 0.95 3 64.29 0.913 58 SleB MKSKGSIMACLILFSFTITTFINTETISAFS 0.94 2 51.61 0.651 59 YbbC MRKTIFAFLTGLMMFGTITAASA 0.92 2 69.57 0.861 60 YxiT MKWNNMLKAAGIAVLLFSVFAYAAPSLKAVQA 0.90 2 71.88 0.707 61 LytC MRSYIKVLTMCFLGLILFVPTALA 0.90 2 70.83 0.786 62 PhrA MKSKWMSGLLLVAVGFSFTQVMVHA 0.86 2 68.00 0.74 63 YkvT MTTKFTALAVFLLCFMPAAKI 0.77 1 71.43 0.715 64 CotC MKNRLFILICFCVICLFLSFGQPFFPSMILTVQAAKS 0.68 2 64.86 0.787 65 AmyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA 0.67 3 78.79 0.904 66 NprE MGLGKKLSVAVAASFMSLSISLPGVQA 0.62 2 70.37 0.832 67 YolC MKKRLIGFLVLVPALIMSGITLIEA 0.58 3 76.00 0.727 68 YqzG MMIKQCVICLSLLVFGTTAAHA 0.57 1 63.64 0.829 69 YndA MRFTKVVGFLSVLGLAAVFPLTAQA 0.52 2 76.00 0.895 70 YfjS MKWMCSICCAAVLLAGGAAQA 0.49 1 71.43 0.845 71 YvcE MRKSLITLGLASVIGTSSFLIPFTSKTASA 0.43 2 56.67 0.891 72 YkwD MKKAFILSAAAAVGLFTFGGVQQASA 0.42 2 73.08 0.688 73 Mdr MDTTTAKQASTKFVVLGLLLGILMSAMDNTIVATA 0.41 1 60.00 0.595 74 YwfM MKGNIYSLFVLIAAFFWGTTGTVQA 0.36 1 68.00 0.669 75 NucB MKKWMAGLFLAAAVLLCLMVPQQIQGASS 0.34 2 72.41 0.746 76 YqxM MFRLFHNQQKAKTKLKVLLIFQLSVIFSLTAAICLQFSDDTSA 0.33 5 51.16 0.457 77 YkoJ MLKKKWMVGLLAGCLAAGGFSYNAFA 0.30 3 73.08 0.712 78 Mpr MKLVPRFRKQWFAYLTVLCLALAAAVSFGVPAKA 0.30 4 70.59 0.886 79 YpuA MKKIWIGMLAAAVLLLMVPKVSLADA 0.29 2 80.77 0.782 80 TasA MGMKKKLSLGVASAALGLALVGGGTWA 0.29 3 77.78 0.782 81 YwmD MKKLLAAGIIGLLTVSIASPSFA 0.26 2 73.91 0.789 82 YwtD MNTLANWKKFLLVAVIICFLVPIMTKAEIAEA 0.25 2 68.75 0.763 83 YdbK MKLFNRKVTLVSLILMAVFQFFMALIIKRIVIS 0.22 3 69.70 0.699 84 YfkN MRIQKRRTHVENILRILLPPIMILSLILPTPPIHA 0.21 3 62.86 0.654 85 YwaD MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHA 0.16 2 67.74 0.883 86 YpjP MKLWMRKTLVVLFTIVTFGLVSPPAALMA 0.15 3 75.86 0.842

Appendix

115

No.

Name

Signal sequence


a

Charge

N-region b

Hydro-

phobicity [% ]

c

D-

Score d

87 RpmG MRKKITLACKTCGNRNYTTMKSSASA 0.14 3 30.77 0.536 88 DacF MKRLLSTLLIGIMLLTFAPSAFA 0.14 2 73.91 0.909 89 TyrA MNQMKDTILLAGLGLIGGSIALA 0.13 0 73.91 0.466 90 LytF MKKKLAAGLTASAIVGTTLVVTPAEA 0.13 3 65.38 0.744 91 WprA MKRRKFSSVVAAVLIFALIFSLFSPGTKAAA 0.12 4 67.74 0.941 92 YbbR MDKFLNNRWAVKIIALLFALLLYVAVNS 0.11 1 67.86 0.612 93 YhjA MKKAAAVLLSLGLVFGFSYGAGHVAEA 0.09 2 74.07 0.577 94 YjiA MAAQTDYKKQVVGILLSLAFVLFVFS 0.08 1 65.38 0.567 95 PbpD MTMLRKIIGWILLLCIIPLFAFTVIA 0.08 1 80.77 0.777 96 YjcM MKKELLASLVLCLSLSPLVSTNEVFA 0.08 1 57.69 0.776 97 YhaK MRTWKRIPKTTMLISLVSPFLLITPVLFYAALA 0.08 4 66.67 0.725 98 PelB MKRLCLWFTVFSLFLVLLPGKALG 0.07 2 75.00 0.857 99 SpoIIQ MREEEKKTSQVKKLQQFFRKRWVFPAIYLVSAAVILTAVL 0.07 5 52.50 0.408

100 MotB MARKKKKKHEDEHVDESWLVPYADILTLLLALFIVLYASS 0.07 6 50.00 0.307 101 YdhT MFKKHTISLLIIFLLASAVLA 0.06 2 71.43 0.701 102 YbdG MKTLWKVLKIVFVSLAALVLLVSVS 0.06 2 72.00 0.783 103 LytE MKKQIITATTAVVLGALFA 0.05 2 68.42 0.502 104 PhrF MKLKSKLLLSCLALSTVFVATTIA 0.05 3 58.33 0.807 105 YhcR MLSVEMISRQNRCHYVYKGGNMMRRILHIVLITALMFLNVMYTFEA 0.04 4 54.35 0.359 106 CccA MKWNPLIPFLLIAVLGIGLTFFLSVKG 0.03 1 81.48 0.745 107 CitH MGNTRKKVSVIGAGFTGATTAFLIAQKELADV 0.02 3 59.38 0.43 108 AspB MKLAKRVSALTPSTTLAITAKA 0.02 3 54.55 0.897 109 YknX MKKVWIGIGIAVIVALFVGINIYRSAAPTSGSA 0.01 2 72.73 0.585 110 YhdC MKSLPYTIALLFCGLIIVSMA 0 1 71.43 0.673 111 YlbL MLRKKHFSWMLVILILIAVLSFIKLPYYITKPGEA 0 3 65.71 0.501 112 YlxW MRGKSAVLLSLIMLIAGFLISFSFQMTKENNKSAA 0 2 60 0.614 113 YngK MKVCQKSIVRFLVSLIIGTFVISVPFMANA 0 3 66.67 0.878 114 YnzA MELSFTKILVILFVGFLVFGPDKLPALG 0 0 78.57 0.468 115 YobV MKLERLLAMVVLLISKKQVQA 0 1 61.9 0.623 116 YocH MKKTIMSFVAVAALSTTAFGAHA 0 2 65.22 0.88 117 YodV MKVPKTMLLSTAAGLLLSLTATSVSA 0 2 61.54 0.927 118 YojL MKKKIVAGLAVSAVVGSSMAAAPAEA 0 3 73.08 0.8 119 YomL MRKKRVITCVMAASLTLGSLLPAGYATA 0 4 60.71 0.884 120 YoqH MKRFILVLSFLSIIVAYPIQTNA 0 2 65.22 0.744 121 YoqM MKLRKVLTGSVLSLGLLVSASPAFA 0 3 68 0.911 122 YpbG KLSVKIAGVLTVAAAAMTAKMYATA 0 2 68 0.707 123 YpcP MNNNKLLLVDGMALLFRAFFATA 0 1 69.57 0.57 124 YpmS NKWKRLFFILLAINFILAAGFVALVLLPGEQAQV 0 3 76.47 0.701 125 YpuD MGRIKTKITILLVLLLLLAGGYMYINDIELKDVPTAIG 0 3 65.79 0.409 126 YqzC MTKRGIQAFAGGIILATAVLAAVFYLTDEDQAAA 0 2 67.65 0.518 127 YraJ MTLTKLKMLSMLTVMIASLFIFSSQALA 0 2 64.29 0.898 128 YuaB MKRKLLSSLAISALSLGLLVSAPTASFAAE 0 3 63.33 0.909 129 YusW MHLIRAAGAVCLAVVLIAGCRFNEDQHQAEG 0 1 61.29 0.421 130 YvgO MKRIRIPMTLALGAALTIAPLSFASA 0 3 73.08 0.89 131 YvgV MKKKQQSSAKFAVILTVVVVVLLAAIV 0 3 66.67 0.682 132 YvnB MRKYTVIASILLSFLSVLSGG 0 2 61.9 0.669 133 YvpB MKTLRTLCVLMILSGVIFFGLKIDA 0 2 68 0.775 134 YwcI MKRLLVSLRVWMVFLMNWVTPDRKTARA 0 2 60.71 0.577 135 YwdK MKVFIILGAINALLAVGLGAFG 0 1 90.91 0.553 136 YweA MLKRTSFVSSLFISSAVLLSILLPSGQAHA 0 2 60 0.923 137 YwgB MKMKSGMEQAVSVLLLLSRLPVQA 0 2 62.5 0.656 138 YwmB MKKKQVSHAIIISVMLSFVIAVFHTIHA 0 3 60.71 0.598 139 YwoF MRKWYFILLAGVLTSVILAFVYDKTKA 0 2 62.96 0.667 140 YwqC MGESTSLKEILSTLTKRILLIMIVTAAATA 0 -1 56.67 0.576 141 YwqO MKFLLSVIAGLLILALYLFWKVQPPVWI 0 1 82.14 0.569 142 YwsB MNKPTKLFSTLALAAGMTAAAAGGAGTIHA 0 2 70 0.662 143 YwtC MKFVKAIWPFVAVAIVFMFMSA 0 2 86.36 0.718 144 YwtF MEERSQRRKKKRKLKKWVKVVAGLMAFLVIAAGSVGAYA 0 9 56.41 0.675 145 YxaK MVKSFRMKALIAGAAVAAAVSAGAVSDVPAAKVLQPTAAYA 0 3 73.17 0.679 146 YxiA MFNRLFRVCFLAALIMAFTLPNSVYA 0 2 69.23 0.873 147 YybN MNKFLKSNFRFLLAAALGISLLASSNFIKA 0 3 63.33 0.81 148 YycP MKKWMITIAMLILAGIALFVFISPLKS 0 2 77.78 0.725 a The lipolytic activity assay was performed at 30°C; the enzymatic activity was calculated using a molar

absorbtion coefficient of 15000 M-1

·cm-1

. b The net charge of the N-region was calculated with amino acids aspartate and glutamate defined as -1; arginine

and lysine defined as +1 and any other amino acid defined as 0. c The percentage of hydrophobic amino acids of the each signal sequence was calculated with amino acids G, A,

V, L, I, M, F, W and P defined as hydrophobic, any other amino acid being characterized as hydrophilic. d D-score calculated by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/)

Appendix

116

pW TsecA

10000 bps

A) ep-PCR of PPX-domain

pW TsecA/

pMutsecA

C) DpnI digestion of wildtype plasmid

pMutsecA

10000 bps

pW TsecApW TsecA//pMutsecA

PPX-cassetteas primer pair

B) PCR of whole plasmid

pW TsecA

10000 bps

A B

Fig.35: Overview of the complete Gemini pipetting Script ”Ulf_PnPP1_10_tiptouch_bis_10PL”. The script was generated using software Gemini Version 4.2 (Tecan, Crailsheim, Germany) and integrated in the Process Manager Script “Ulf_PnPP_1_10V_Trog2000” providing an automated HTS process using the pipetting robot “TECAN workstation Genesis 200 Freedom (Tecan, Crailsheim, Germany. (A) First part of the pipetting script providing a 1:10 dilution of B. subtilis culture supernatants in one MTP (96 wells) using fresh LB-medium. (B) Second part of the pipetting script providing the filling of the assay plate with assay solution and diluted supernatants.

Fig. 34: Cassette mutagenesis to generate a library of the PPX-domain of SecA. A) ep-PCR of the PPX-domain with primers ppx_up and ppx_down using methylated plasmid DNA (pBSMuL3secA) from E.coli strain JM109 as the template lead to 350 bp DNA fragments containing the library of the PPX-domain of SecA. The used PCR conditons resulted in an error rate of 5-8 bases per kb DNA. B) The DNA of gel extracted PPX-fragments were taken as the primer pair in a second PCR amplifiying the whole plasmid pBSMuL3secA resulting in a plasmid mix of methylised wildtype plasmid and generated plasmid variants. C) To eliminate methylised wildtype secA (template) the PCR sample was incubated with endonuclease DpnI. An aliquote of this sample was directly used for electrotransformation into E.coli JM109 for plasmid library construction described before.

Lebenslauf

Lebenslauf

Name: Brockmeier Vorname: Ulf Geburtsdatum: 18.11.1973 Geburtsort: Münster Familienstand: ledig Schulausbildung: 1980-1982 Marienschule, kathol. Grundschule, Ochtrup 1982-1984 St.-Josef-Schule, kathol. Grundschule, Ahaus 1984-1988 Alexander-Hegius-Gymnasium in Ahaus 1988-1993 Gymnasium am Ostring, Bochum

Abschluss: Allgemeine Hochschulreife Zivildienst: 1993-1994 St. Josef Hospital Bochum Hochschulausbildung: 10/1994-04/2001 Diplomstudiengang der Fachrichtung Biologie an der Ruhr-Universität Bochum 04/2001-04/2002 Diplomarbeit am Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum

Thema: Die Sekretionswege der lipolytischen Enzyme LipA und LipB von Bacillus subtilis

seit 08/2002 Promotion am Institut für Molekulare Enzymtechnologie der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich

Thema: New strategies to optimize the secretion capacity for heterologous proteins in Bacillus subtilis

Anstellungen: 06/2001-04/2002 Studentische Hilfskraft an der Ruhr-Universität Bochum (Lehrstuhl Biologie der Mikroorganismen) 08/2005-04/2006 Wissenschaftlicher Angestellter an der Heinrich-Heine Universität

Düsseldorf (Institut für Molekulare Enzymtechnologie) Stipendien: 08/2002-07/2005 Stipendium des Europäischen Graduiertenkollegs der Ruhr-

Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications

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