Biofuels Production programs

Engineering a Yeast Strain that Efficiently Utilizes C5/C6 Sugars

The program team is working to engineer a yeast strain capable of efficiently utilizing C5/C6 sugars for the economical production of biofuels. The group’s efforts focus on Saccharomyces cerevisiae, the pre-eminent microorganism for industrial production of ethanol. They are studying the construction and engineering of novel pentose utilization pathways in S. cerevisiae; the discovery, characterization and engineering of pentose transporters; and the optimization of sugar utilization pathways for ethanol production through metabolic flux analysis and construction of an integrated pathway model for sugar utilization.

program Highlights

2014 Highlights

In 2014, we engineered a yeast strain capable of growing on glucose as the sole carbon source with ethanol production completely eliminated, which was used to produce enantiopure (2R, 3R)-butanediol at high titer. We also created a yeast strain with increased cellular acetyl-CoA levels, which can serve as a production host for many acetyl-CoA derived fuels. In addition, we successfully reversed the beta-oxidation cycle in yeast to produce n-butanol, medium-chain fatty acids, and medium-chain fatty acid ethyl esters. In parallel, we engineered a yeast strain to produce fatty alcohols, which are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We also engineered a novel pathway orthogonal to the endogenous fatty acid metabolism to produce fatty acid ethyl esters in yeast. Finally, we engineered in vivo biosensors for acetyl-CoA and malonyl CoA, which can be used to further improve the intracellular concentrations of acetyl-CoA and malonyl CoA using directed genome evolution approaches.

2013 Highlights

In 2013, we made great advances in our program that consist of two distinct yet closely related thrusts, including sugar co-utilization and advanced biofuels. We have met or exceeded most of the milestones we proposed in 2011. We published a total of 12 research papers and submitted four additional manuscripts. Briefly, we developed and applied novel combinatorial pathway engineering strategies to improve the efficiencies of a xylose utilization pathway and a cellobiose utilization pathway, respectively. In addition, we used metabolic flux analysis and transcriptomic analysis tools to investigate the biological mechanisms for incomplete and inefficient uptake and utilization of xylose and cellobiose. In parallel, we engineered a yeast strain capable of growing on glucose as the sole carbon source with ethanol production completely eliminated, which can serve as a platform host for synthesis of fuels and chemicals. We also engineered a yeast strain to produce fatty alcohols, which are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. Finally, we created a yeast strain with increased cellular acetyl-CoA levels, which can serve as a product host for many acetyl-CoA derived fuels.

2012 Highlights

Engineering microorganisms to produce chemicals and fuels by introducing heterologous metabolic pathways is a conceptually simple task, yet the challenge of balancing metabolic flux through these pathways in order to maximize product titers and productivities remains overwhelming. To address this key challenge in metabolic engineering, we developed two novel approaches.

 

The first is “Customized Optimization of Metabolic Pathways by Combinatorial Transcriptional Engineering (COMPACTER),” which optimizes the flux at the transcriptional level. A library of mutant pathways is generated by de novo assembly of promoter mutants with various strengths for each pathway gene and then optimized through high-throughput screening/selection. As proof of concept, we have successfully created yeast strains capable of fermenting xylose (or cellobiose) with near highest (or highest) consumption rate and ethanol productivity ever reported through a single round of COMPACTER. More importantly, we have conclusively shown that the optimized xylose or cellobiose utilizing pathways are host-specific.

 

The second approach is combinatorial pathway engineering at the translational level. We used the DNA assembler method to generate a library of pathways consisting of enzymes from different sources, followed by high-throughput screening or selection to identify optimized pathways. As proof of concept, a library of over 8,000 xylose utilization pathways was generated in both laboratory and industrial yeast strains. High-throughput screening identified a number of strain-specific combinations of the enzymes for efficient conversion to ethanol. The balancing of metabolic flux through the xylose utilization pathway was demonstrated by a complete reversal of the major product from xylitol to ethanol with a similar yield and total byproduct formation as low as 0.06 g/g xylose without compromising cell growth.

2011 Highlights

Zhao’s group developed an approach called “Customized Optimization of Metabolic Pathways by Combinatorial Transcriptional Engineering (COMPACTER)” that can quickly tune gene expression in a complex pathway. Using COMPACTER, Zhao’s group successfully created yeast strains that can ferment xylose or cellobiose with among the highest consumption rates and ethanol productivity ever reported. The group created a library of xylose-utilization pathways, which use different enzymes and several yeast strains. Then, using high-throughput screening, the team identified a number of strain-specific combinations of the enzymes for efficient ethanol conversion. This technique also optimizes enzymes for minimal by-product production. Using this library, the group determined that optimal combinations of enzymes will vary, depending on both growth conditions and yeast strains. Using yeast data – from microarrays, 1,577 metabolic reactions and 4,210 regulatory interactions between 136 transcriptional factors and 904 metabolic genes – Zhao’s group created a model that integrates both networks. The model accurately predicted the lethality of gene knockouts, growth rates and metabolic flux changes under various conditions. This model is the first of its kind in eukaryotic cells. It provides a platform to guide rational strain design in the future. Zhao’s group also developed a set of new genetic tools to engineer the metabolism of industrial strains.

2010 Highlights

Two novel xylose-specific transporters and one arabinose-specific transporter were isolated and characterized. A recombinant yeast strain capable of co-utilizing cellobiose and xylose was engineered, which showed minimal glucose repression in mixed sugar fermentation. Additional 20 new putative cellobiose transporters and 30 putative xylose specific transporters were cloned. A DNA assembler based method was developed to optimize the metabolic flux through the fungal xylose utilizing pathway. A genome-scale model was used to investigate the effect of cofactor balance in pentose utilization and ethanol production involving the fungal pentose pathway.

2009 Highlights

We cloned and characterized several new enzymes involved in L-arabinose utilization and also used protein engineering to alter the cofactor specificity of select enzymes in the D-xylose/L-arabinose utilizing pathway to address the cofactor redox imbalance problem. We successfully constructed a full fungal L-arabinose/D-xylose utilizing pathway in Saccharomyces cerevisiae that enables the co-utilization of L-arabinose and D-xylose. We discovered two D-xylose specific transporters (i.e. no or very low glucose transport activity and low arabinose transport activity) and one L-arabinose specific transporter. We determined the type of all these three transporters and the cellular localization of these transporters using confocal microscopy and green fluorescent protein. We determined the kinetic parameters of these transporters using isotope labeled sugars. To facilitate our metabolic engineering studies, we also established a genome-scale model of S. cerevisiae. Using this model, we performed computational simulation studies on the impact of cofactor redox imbalance on L-arabinose/D-xylose utilization. The results conclusively showed that the cofactor-balanced fungal L-arabinose/D-xylose utilizing pathway led to increased L-arabinose and D-xylose consumption rates and ethanol productivity. Further flux sensitivity analysis revealed the genes and pathways that were impacted most by the cofactor imbalance.

 

Publications

Published in 2014

Protein Design for Pathway Engineering, D. Eriksen, J. Lian, and H. Zhao, Journal of Structural Biology, 185, pp. 234–242, February 2014. 

 

Metabolic Engineering of Saccharomyces cerevisiae Strain Capable of Simultaneously Utilizing Glucose and Galactose to Produce Enantiopure (2R, 3R)-Butanediol, J. Lian, R. Chao, and H. Zhao, Metabolic Engineering, 24, pp. 139-149, February 10, 2014. 

 

Directed Evolution of a Cellodextrin Transporter for Improved Biofuel Production Under Anaerobic Conditions in Saccharomyces cerevisiae, J. Lian, Y. Li, M. M. HamediRad, and H. Zhao, Biotechnology and Bioengineering, 111, pp. 1521-1531, March 11, 2014. 

 

Design and Construction of Acetyl-CoA Overproducing Saccharomyces cerevisiae Strains, J. Lian, T. Si, N.U. Nair, and H. Zhao, Metabolic Engineering, 24, pp. 139-149, July 2014. 

 

Recent Advances in DNA Assembly Technologies, R. Chao, Y. Yuan, and H. Zhao, FEMS Yeast Research, doi: 10.1111/1567-1364.12171, June 23, 2014. 

 

Reversing the beta-Oxidation Cycle in Saccharomyces cerevisiae for Synthesis of Fuels and Chemicals, J. Lian and H. Zhao. ACS Synthetic Biology, doi: 10.1021/sb500243c, June 24, 2014. 

 

Recent Advances in Biosynthesis of Fatty Acids Derived Products in Saccharomyces cerevisiae, J. Lian, H. Zhao, Journal of Industrial Microbiology and Biotechnology, doi: 10.1007/s10295-014-1518-0, October 12, 2014. 

 

Metabolic Engineering of Saccharomyces Cerevisiae to Improve 1-Hexadecanol Production, Xueyang Feng, Jiazhang Lian, Huimin Zhao, Metabolic Engineering, doi: 10.1016/j.ymben.2014.10.001, November 2014.

Published in 2013

Directed Evolution as a Powerful Synthetic Biology Tool, R. E. Cobb, N. Su, H. Zhao, Methods, 15:60(1), pp. 81-90,  doi: 10.1016/j.ymeth.2012.03.009, March 13, 2013.

 

Pathway Engineering as an Enabling Synthetic Biology Tool (book chapter), D. T. Ericksen, L. Jiazhang, H. Zhao, Synthetic Biology: Tools and Applications (edited by Huimin Zhao), 2013.

 

Protein Design for Pathway Engineering, D. T. Ericksen, J. Lian, H. Zhao, Journal of Structural Biology, 185(2), pp. 234-242, doi/10.1016/j.jsb.2013.03.01, April 1, 2013.

 

Investigation of the Functional Role of Aldose 1-Epimerase in Engineered Cellobiose Utilization (2013), S. Li, S. J. Ha, H. J. Kim, J. M. Galazka, J. H. Cate, Y. S. Jin, H. Zhao, Journal of Biotechnology 168, pp. 1-6.

 

Directed Evolution of a Highly Efficient Cellobiose Utilizing Pathway in an Industrial Saccharomyces cerevisiae Strain, Y. Yuan, D. T. Ericksen, H. Zhao, Biotechnology and Bioengineering, 110, pp. 2874-2881, doi: 10.1002/bit.24946, April 25, 2013.

 

Investigating Glucose and Xylose Metabolism in Saccharomyces cerevisiae and Scheffersomyces stipitis Via 13c Metabolic Flux Analysis, Xueyang Feng, Huimin Zhao, AIChE Journal, 59, pp. 3195-3202, doi: 10.1002/aic.14182, June 17, 2013.

 

Directed Evolution of a Cellobiose Utilization Pathway in Saccharomyces cerevisiae by Simultaneously Engineering Multiple Proteins, D. T. Ericksen, P. C. Hsieh, P. Lynn, H. Zhao, Microbial Cell Factories, 12, pp. 61, doi: 10.1186/1475-2859-12-61, June 28, 2013

 

Investigating Host Dependence of Xylose Utilization in Recombinant Saccharomyces cerevisiae Strains Using Rna-Seq Analysis, X. Y. Feng, H. M. Zhao, Biotechnology for Biofuels, 6, pp. 96, doi: Artn 96 Doi 10.1186/1754-6834-6-96, July 6, 2013.

 

DNA Assembly Techniques for Next-Generation Combinatorial Biosynthesis of Natural Products, Ryan E. Cobb, Jonathon C. Ning, Huimin Zhao, Journal of Industrial Microbiology and Biotechnology, doi: 10.1007/s10295-013-1358-3, October 15, 2013.

 

Investigating Xylose Metabolism and Recombinant Saccharomyces cerevisiae via 13C Metabolic Flux Analysis (2013), X. Feng, H. Zhao, Microbial Cell Factories. 12, pp. 114.

Published in 2012

Combinatorial Design of a Highly Efficient Xylose Utilizing Pathway for Cellulosic Biofuels Production in Saccharomyces cerevisiae, Byoungjin Kim, Jing Du, Dawn Eriksen, Huimin Zhao, Applied and Environmental Microbiology, doi: 10.1128/AEM.02736-12, November 2012.

 

Strain Improvement Via Evolutionary Engineering (Chapter 4), Byoungjin Kim, Jing Du, and Huimin Zhao,  Engineering Complex Phenotypes in Industrial Strains, John Wiley & Sons, preview of 2013 book chapter.

 

Customized Optimization of Metabolic Pathways by Combinatorial Transcriptional Engineering, Jing Du, Yongbo Yuan, Tang Si, Jiazhang Lian, Huimin Zhao, Nucleic Acids Research, doi: 10.1093/nar/gks549, June 19, 2012.
 

Challenges and Opportunities in Synthetic Biology for Chemical Engineers, Yunzi Luo, Jung-Kul Lee, Huimin Zhao, Chemical Engineering Science, http://dx.doi.org/10.1016/j.ces.2012.06.013, June 15, 2012.

 

Directed Evolution: An Evolving and Enabling Synthetic Biology Tool, Ryan Cobb, Tong Si, Huimin Zhao, Current Opinion in Chemical Biology, http://dx.doi.org/10.1016/j.cbpa.2012.05.186, June 4, 2012.

 

Directed Evolution As a Powerful Synthetic Biology Tool, Ryan Cobb, Ning Sun, Huimin Zhao, Methods, http://dx.doi.org/j.ymethod.2012.03.009, March 23, 2012.

Published in 2011

Genome-Scale Consequences of CoFactor Balancing in Engineering Pentose Utilization Pathways in Saccharomices cerevisiae, Amit Ghosh, Huimin Zhao, Nathan Price, PLoS One, 6(11), e27316; doi: 10.1371/journal.pone.0027316, November 4, 2011.

Engineering Microbial Factories for Synthesis of Value-Added Products, Jing Du, Zengyi Shao, Huimin Zhao, Journal of Industrial Microbiology and Biotechnology, doi:10.1007/s10295-011-0970-3, April 28, 2011.

Published in 2010

Synthetic Biology: Putting Synthesis into Biology, Jing Liang, Yunzi Luo, Huimin Zhao, Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 3(1), pp. 7-20, July 13, 2010.

Overcoming Glucose Repression in Mixed Sugar Fermentation by Co-Expressing a Cellobiose Transporter and a beta-Glucosidase in Saccharomyces Cerevisiae, Yanglin Li, Jing Du, Jie Sun, John Galazka, Louise Glass, Jamie Cate, Xiang Yang, Huimin Zhao, Molecular Biosystems, (publication ahead of print), September 27, 2010. 

Discovery and Characterization of Novel d-xylose-specific Transporters from Neurospora crassa and Pichia stipitis, Jing Du, Sijin Li, Huimin Zhao, Molecular BioSystems, DOI: 10.1039/COMB00007H, August 11, 2010.

Cloning, Characterization, and Engineering of Fungal L-arabinitol Dehydrogenases, Byoungjin Kim, Ryan Sullivan, Huimin Zhao, Applied Microbiology and Biotechnology, 87(4): pp. 1407-1414, April 23, 2010.

Published in 2009

Protein Engineering in Designing Tailored Enzymes and Microorganisms for Biofuels Production, Fei Wen, Nakhil U Nair, Huiman Zhao, Current Opinion in Biotechnology, 20(4): pp. 412-419, August 2009. 

Industrial Biotechnology: Tools and Applications, Weng Lin Tang, Huimin Zhao, Biotechnology Journal, 4: pp. 1725-1739, Oct. 20, 2009.

 


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