JBEI Highlights December 2014
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Transcript of JBEI Highlights December 2014
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.
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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.