Background• Biomass is rich in C5 sugars that are difficult to

ferment into fuels. • Increasing the ratio between C6 and C5 sugars

in bioenergy crops would make biofuel production more cost efficient.

Approach• Beta-1,4-galactan is a polysaccharide made

entirely of C6 sugars, but it is normally of low abundance. The approach is to increase the deposition of beta-1,4-galactan in secondary cell walls of plants.

Outcomes• Plants coexpressing Gals1, galactan synthase,

and UGE2, UDP-Gal epimerase, deposit beta-1,4-galactan in secondary cell walls.

• The galactose content in the biomass is increased about 2x.

• Expression of only one of the enzymes did not lead to increased galactan content.

A Gene Stacking Approach leads to Engineered Plants with Highly Increased Galactan Levels in Arabidopsis

Gondolf, V. M., Stoppel, R., Ebert, B., Rautengarten, C., Liwanag, A., Loque, D., & Scheller, H. V. (2014). " A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis”, BMC Plant Biol, 14(1), 344. doi, 10.1186/s12870-014-0344-x

Significance• This approach is a promising strategy to improve the

C6/C5 sugar ratio in biomass. Simultaneous overexpression of a UDP-Gal transporter may lead to even higher C6 content.

Beta-1,4-galactan is synthesized by GalS1, which is located in the Golgi lumen. Its substrate is UDP-Gal synthesized in the cytosol by UGE.

Galactose content in stems of plants overexpressing GalS1, UGE2 or both enzymes. Some constructs also incorporates the NST1 transcription factor, which results to increased biomass density.

Secondary cell walls do not contain beta-1,4-galactan (staining with LM5 antibody) in control plants. High staining in plants overexpressing UGE2 and GalS1.

-UDP

-UDP

UMP

UMP

Cytosol

UDP-Glucose UDP-Galactose

UGE Photosynthesis

URGT

GalS

β-(1,4)-Galactan

Golgi lumen

0

2

4

6

8

10

12

14

Galactose

Mol

ar p

erce

ntag

e

EVC

35S:GalS1

pIRX5:NST1-2A-UGE2

35S:UGE2 + 35S:GalS1

pIRX5:NST1-2A-UGE2 + 35S:GalS1

LM5 anti-galactan

UGEox

UGEox GalS1ox

Principal Component Analysis of Proteomics (PCAP) as a Tool to Direct Metabolic Engineering

Alonso-Gutierrez, Eun-Mi Kima, Tanveer S. Battha, Nathan Choa, Qijun Hua, Leanne Jade G. Chana, Christopher J. Petzold, Nathan J. Hillson, Paul D. Adams, Jay D. Keasling, Hector Garcia Martin, Taek Soon Lee (2015). "Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering”, Metabolic Engineering 28: 123-133

Background• Targeted proteomics has been used to determine enzyme expression and identify bottlenecks of the pathway, but an

organized method for quantitative analysis of proteomics data has not been explored yet

Approach• We applied Principal Component Analysis (PCA) to a collection of proteomics data for isoprenoid biofuel pathway. We

plotted this with target molecule production data to pinpoint specific enzymes that need to have their expression level adjusted to balance the pathway and maximize biofuel production

Outcomes• PCAP-guided metabolic pathway engineering resulted in over a 40% titer improvement in the production of two valuable

isoprenoid biofuels, limonene and bisabolene

Significance• PCAP has been proven as an efficient quantitative analysis method of proteomics data in isoprenoids biofuel synthesis

pathway, and it would be applicable to optimize other heterologous metabolic pathways, too

Design of Low-cost Ionic Liquids for Biomass Pretreatment

Outcomes• Ionic liquids developed in this study, are effective in hydrous conditions (20% H2O) and have a cost of $1.25 /kg versus $50 /kg for the

benchmark 1-ethyl-2-methylimidazolium acetate ([C2C1im][OAc]).• The highest performing ionic liquids in hydrous conditions were ~ 70% as effective as [C2C1im][OAc] in terms of hydrolysis efficiency.

Background• Ionic Liquids have been shown to be

excellent pretreatment solvents forenhancing biomass cellulosehydrolysis.

• The best ionic liquids for this purposehave historically been expensive, socost reduction strategies need to bedeveloped.

Approach• Identify, synthesize and test ionic

liquids that have dramaticallyreduced production costs but areeffective pretreatment solvents.

• Anions and cations were chosen thatcan be synthesized from H2SO4 andammonia, giving a final cost ofaround $1.25 /kg.

Significance• A path to producing very inexpensive ionic liquids has been demonstrated.• With further optimization the cost of ionic liquids need not be a barrier to their use at industrial scales in

bio-refineries.

qsuB-7

Anthe George, Agnieszka Brandt, Kim Tran, Shahrul M. S. Nizan S. Zahari, Daniel Klein‐Marcuschamer, Ning Sun, NoppadonSathitsuksanoh, Jian Shi, Vitalie Stavila, Ramakrishnan Parthasarathi, Seema Singh, Bradley M. Holmes, Tom Welton,  Blake A. Simmons, Jason P. Hallett (2014) “Design of low‐cost ionic liquids for lignocellulosic biomass pretreatment”, Green Chemistry, DOI: 10.1039/C4GC01208A

Metatranscriptomic Analysis of LignocellulolyticMicrobial Communities involved in High-solids Decomposition of Rice Straw

Outcomes• The thermophilic community exhibited great endoglucanase and xylanase activity.• Actinobacteria cellulase enzymes from GH family 48 with CBM family 2 domains and polysaccharide monooxygenase

enzymes from CBM family 33 were significantly overexpressed in the thermophilic community1

1Simmons, C. W., Reddy, A. P., D’haeseleer, P., Khudyakov, J., Billis, K., Pati, A., Simmons, B. A., Singer, S. W., Thelen, M. P., & VanderGheynst, J. S. (2014). "Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high‐solids decomposition of rice straw”, Biotechnology for Biofuels, 7(1). doi, 10.1186/s13068‐014‐0180‐0

Background• Complex lignocellulolytic

microbial communities may be a source of robust, synergistic enzymes for industrial lignocellulose deconstruction.

• There is a need for cellulolytic enzymes that maintain optimal activity under high temperature and high solids conditions expected in industrial bioprocessing.

Significance• These enzymes are not currently represented in any protein database and could contribute

to increased cellulolytic activity under high temperature and high solids conditions.

Approach• Metatranscriptomes from thermophilic and mesophilic compost communities grown on rice straw under high solids

conditions were compared to identify differentially expressed lignocellulolytic enzymes in the thermophilic community.

Actinobacteria express the majority of deconstructive enzymes (DM) but a minority of the total enzymes (TM) in the thermophilic community, suggesting a major role in lignocellulose deconstruction.

Complete Genome Sequence of the Lignin-degrading Bacterium Klebsiella sp. strain BRL6-2

Outcomes• The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of

genes encoding lignocellulolytic carbohydrate active enzymes. It grows in relatively high concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44 mM and NaCl up to 1.5 M.

Woo, H., Ballor, N., Hazen, T., Fortney, J., Simmons, B., Davenport, K., Goodwin, L., Ivanova, N., Kyrpides, N., Mavromatis, K., Woyke, T., Jansson, J., Kimbrel, J., & DeAngelis, K. (2014). "Complete genome sequence of the lignin‐degrading bacterium Klebsiella sp. strain BRL6‐2”, Standards in Genomic Sciences, 9(1), 19. doi, 10.1186/1944‐3277‐9‐19 ]

Background• Lignin is an underutilized

feedstock of aromatics.• Few anaerobic bacteria are

known to degrade lignin in the presence of ionic liquid.

Approach• Bacterial strain, Klebsiella sp.

strain BRL6-2, was isolated from Puerto Rican tropical forest soil under anaerobic conditions using lignin as the sole carbon source.

• We sequenced its genome to understand its metabolic potential and performed physiological studies to understand its response to osmotic stressors.

Significance• Klebsiella sp. strain BRL6-2’s fast growth, facultative lifestyle, and tolerance to high ionic

strength conditions make it an attractive microbial host to bioengineer for industrial lignocellulose degradation and consolidated bioprocessing of biofuels

Comparison of Different Biomass Pretreatment Techniques and their Impact on Chemistry and Structure

Outcomes & Significance• Our results indicate that disruption of lignin-carbohydrate linkages of mainly polymeric lignin contribute to the efficiency of

AFEX pretreatment. • DA pretreatment appears to start with significant lignin de-polymerization, with 50% of the lignin re-condensed in the final

pretreated corn stover. DA pretreated corn stover was found to be thermally more stable, however, fiber width was measured to be significantly smaller than IL and AFEX pretreated corn stover.

• Disappearance of β-aryl ether units from lignin from IL confirms the depolymerization of β-aryl ether linkages during IL pretreatment. In addition, absence of X’

2 and X’3 cross peaks confirmed deacetylation of hemicelluloses during IL

pretreatment. Both NMR and SEC showed similar patterns of lignin depolymerization with highest degree of depolymerization observed for IL followed with DA and AFEX.

Seema Singh, Gang Cheng, Noppadon Sathitsuksanoh, Dong Wu, Patanjali Varanasi, Anthe George, Venkatesh Balan, Xiadi Gao, Rajeev Kumar Bruce E. Dale, Charles E. Wyman, and Blake A. Simmons (2014) “Comparison of Different Biomass Pretreatment Techniques and their Impact on Chemistry and Structure”, Frontiers in Energy Research, 2. doi, 0.3389/fenrg.2014.00062

Background• Pretreatment of lignocellulosic

biomass is a prerequisite to overcome recalcitrance and allow enzyme accessibility to cellulose.

• The three BRCs are collaborating to gain a better understanding of biomass recalcitrance and impact of pretreatment on structure and chemistry.

Approach• High resolution AFM imaging, NMR, XRD, SANS, size exclusion chromatography

(SEC) and TGA was used to understand Physical, chemical, and thermal characteristics of corn stover solids from Ionic Liquid (IL), Dilute Acid (DA), and AFEX pretreatments to understand induced morphological and chemical changes incorporated to corn stover.

Pretreatment Comparison ‐ AFM

Pretreatment Comparison ‐ NMR

Modifying Plants for Biofuel and Biomaterial Production: The challenge of sustainable biofuel production and the key role of biomass composition

Summary & Outlook• Plants have been domesticated mainly for food, fibre and feed applications. Biofuel production may require the breeding of very different

forms of these species or the domestication of new species better suited to biofuel production.• Key options to modify and improve biomass for biofuel and biomaterial production include the reduction in lignin content and modification

of the carbohydrate components to maximise the recovery of glucose in biochemical conversions• Reducing cross linking in the cell wall will play an important role in development of improved biomass composition. • Use of transgenic approaches will allow direct modification of biomass composition.• Genomic analysis will help to define loci for conventional selection in plant improvement or for

targeted mutation. • Advances in the analysis of the genomes of key bioenergy species will accelerate these developments.

Furtado A, Lupoi JS, Nam H, Healey A, Singh S, Simmons BA and Henry RJ (2014) “Modifying plants for biofuel and biomaterial production". Plant Biotechnol J, 12(9), 1246‐1258. doi, 10.1111/pbi.12300

Background• Development of genetically improved

plants for biofuel production is a key factor in improving the efficiency and viability of sustainable biofuel and biomaterial production.

• Plants that deliver high yields of biomass that can be easily converted to end products in high yield will greatly facilitate the replacement of oil with biomass.

• This work reviewed the options and research direction for improving plant biomass for fuels and chemicals via biochemical conversion.

Plant phylogeny: The evolution of different plant groups has involved divergence in biomass composition. Major plant groups may be associated with differences in cell wall composition 

Improving Olefin Tolerance and Production in E. coli Using Native and Evolved AcrB

Outcomes• Styrene and 1-hexene are highly toxic to E. coli. The AcrAB-TolC efflux pump is involved in

the tolerance towards them.• For styrene we identified that the presence of this pump was essential for optimal production,

but the negative selection imposed by the compound confounded efforts to obtain improved pump variants.

• For 1-hexene, we were able to obtain mutants of the AcrAB-TolC pump that resulted in improved tolerance.

Background• E. coli can be engineered for the

production of bioplastic precursors: styrene or 1-hexene.

• Toxicity of these compounds on E. coli and tolerance mechanisms are unknown.

Approach• We identified the efflux pump

involved in the tolerance towards these compounds.

• We created a library of this pump to generate variants able to improve the survival of E. coli in presence of these chemicals.

Significance• Efflux pumps are an important target to improve tolerance towards chemicals and improved

tolerance is a key step to optimizing production level.

Impact of AcrAB expression on styrene production. The gray line represents the styrene production level in E. coli expressing the pump (pABs-placSty) and the black one in the strain without the pump (p0c-placSty).

Impact of AcrB mutation(s) on 1-hexene tolerance. A: Survival test of E. coli in presence of 1-hexene with either the AcrB wild type or variants, B: Localization of the six beneficial mutations represented in red on the structure of the AcrB trimer or C: on the schematic representation of the AcrB trimer.

This project was funded by a CRADA between JBEI and Total New Energies. 

Mingardon F, Clement C, Hirano K, Nhan M, Luning EG, Chanal A, Mukhopadhyay A (2014) “Improving olefin tolerance and production in E. coli using native and evolved AcrB”, Biotechnology and Bioengineering, doi: 10.1002/bit.25511

Narrowing the Gap between the Promise and Reality of Polyketide Synthases as a Synthetic Biology Platform

Outcomes and Impacts• Steps to improve each step in

the design-build-test-learn cycle were proposed and discussed

Poust, S., Hagen, A., Katz, L., & Keasling, J. D. (2014). "Narrowing the gap between the promise and reality of polyketide synthases as a synthetic biology platform”, Curr Opin Biotechnol, 30C(0), 32-39. doi, 10.1016/j.copbio.2014.04.011

Background• Engineering modular

polyketide synthases (PKSs) has the potential to be an effective methodology to produce existing and novel chemicals. However, this potential has only just begun to be realized.

Approach• We propose the adoption of

an iterative design–build–test–learn paradigm to improve PKS engineering.