Biofuels Production programs

Optimal Yeast Strains for Producing Cellulosic Biofuels

This program addresses issues in developing optimal microbial strains for producing cellulosic biofuels: efficient fermentation of the mixed sugars present in cellulosic hydrolysates under the presence of fermentation inbitors. Cellulosic biomass mainly consists of two major sugars (glucose and xylose) but there is no optimal microorganism which has the capacity of fermenting xylose as well as glucose with high yield and productivity. Also, the generation of inhibitory compounds during pretreatment processes of biomass is one of the major barriers impeding the commercialization of biofuels from cellulosic biomass. The goal of the program is to develop engineered yeast strains capable of simultaneously fermenting mixed sugars (glucose, cellodextrin, and xylose) under the presence of fermentation inhibitors in lignocellulosic hydrolysates.  Genetic/genomics tools for engineering industrial yeast strains and genetic bases of desirable traits are being generated.

program Highlights

2014 Highlights

Our program aims to deal with, or solve, two outstanding problems in producing cellulosic biofuels: efficient and rapid fermentation of cellulosic sugars, and enhanced tolerance against fermentation inhibitors in cellulosic hydrolysates. To this end, we have constructed yeast strains capable of fermenting all carbon sources (cellobiose, xylose, and acetate) in cellulosic hydrolysates simultaneously. When we optimized the expression levels of key enzymes in the acetate reduction pathway, the resulting engineered strain was able to produce ethanol with a near theoretical yield from cellulosic hydrolysates. Also, we identified an overexpression gene target eliciting improved tolerance against acetate. We envision that integration of complete and simultaneous utilization of the cellulosic carbon source with acetate tolerance will result in optimal engineered yeast for producing cellulosic biofuels.

2013 Highlights

The long-term goal of this program is to develop robust yeast strains capable of converting cellulosic sugars into biofuels even under the presence of fermentation inhibitors. To this end, we have engineered a cellobiose transporter which has a good potential but with undesirable kinetic properties, identified genetic elements responsible for improved xylose fermentation by an engineered industrial yeast, and engineered sucrose utilization pathways in yeast for simultaneous co-fermentation of sucrose and xylose. While a cellobiose transporter (CDT-2) from Neurospora crassa is a facilitator transporting cellobiose without energy input, an engineered yeast with CDT-2 exhibited slow and inefficient cellobiose fermentation under acidic conditions. Therefore, we mutagenized CDT-2 and isolated a mutant CDT-2 facilitating improved cellobiose fermentation under acidic conditions. The mutant CDT-2 was able to transport xylodextrin as well, suggesting that direct fermentation of both cellodextrin and xylodextrin might be feasible using the CDT-2 mutant. In order to identify genetic elements eliciting superior sugar fermentation by industrial yeast strains, we isolated a haploid strain from a polyploidy industrial strain. An engineered strain for xylose fermentation using the haploid strain showed much faster xylose fermentation than an engineered yeast based on a laboratory strain, indicating that the haploid inherited genetic elements responsible for improved xylose fermentation. We identified structural genetic variations in the haploid strain through genome sequencing and will investigate putative mechanisms by which the genetic variations influence xylose fermentation. Native yeast use sucrose after hydrolyzing it into glucose and fructose by secreted invertase. However, this extracellular utilization of sucrose in yeast might cause slow and sequential utilization of xylose when a mixture of sucrose and xylose is fermented. Therefore, we constructed an engineered yeast which can hydrolyze sucrose intracellularly. As expected, the engineered yeast was able to ferment a mixture of sucrose and xylose simultaneously. We have acquired deeper understanding of sugar metabolism in yeast and built various parts and chassis which can be employed for constructing yeast strains with designed phenotypes.

2012 Highlights

We have developed various metabolic engineering strategies for improving biofuel production from cellulosic hydrolyzates containing cellulosic sugars (cellobiose and xylose) and fermentation inhibitors (acetate).

 

Rapid xylose-fermenting yeast strains were constructed using both laboratory and industrial yeast strains through rational and combinatorial approaches. Specifically, we optimized expression levels of genes coding for the xylose metabolic pathway and mutant cellobiose transporters for enhanced co-fermentation of cellobiose and xylose. Cellobiose and xylose fermentation rates were further improved through laboratory evolution of rationally engineered strains under various culture conditions. Through collaboration with the Arkin group in Berkeley, we determined genome sequences of the evolved strains to discover beneficial genetic perturbations for improved cellobiose and xylose fermentation. As a result, we not only constructed optimal yeast strains for producing cellulosic ethanol, but also elucidated necessary genetic perturbations to ferment cellulosic hydrolyzates rapidly and efficiently.

 

We also collaborated with the Cate group in Berkeley for developing a redox coupling strategy to enable efficient xylose fermentation and simultaneous in situ detoxification of plant cell wall-derived feedstocks. By combining an NADH-consuming acetate consumption pathway and an NADH-producing xylose utilization pathway, engineered yeast converted cellulosic sugars and toxic levels of acetate together into ethanol under anaerobic conditions. This strategy resolves a key remaining bottleneck for producing cellulosic biofuels and will help to make cellulosic ethanol economically feasible.

We continued genetic and genomic experiments for domesticating industrial yeast strains for developing a platform EBI yeast strain for metabolic engineering and understanding genetic bases for outperforming phenotypes of industrial yeast strains.

2011 Highlights

Jin’s group identified an efficient xylose-fermenting strain, sequenced it and then dramatically improved that behavior by knocking out a key gene. That strain is now being evaluated for industrial use. Together with Jamie Cate’s group, Jin’s team identified a cellobiose transporter mutant that works well within the cellobiose-fermenting pathway, resulting in an 80 percent improvement in ethanol productivity as compared to the wild type of transporter. Jin’s and Arkin’s groups isolated a stable industrial yeast strain and uncovered the genetic basis of its useful industrial traits. They identified two strains with superior fermentation properties (cellobiose fermentation and xylose/glucose co-fermentation), one of which also has the best thermotolerance and cell viability after heatshock.

 

Publications

Published in 2014

2,3-Butanediol Production from Cellobiose by Engineered Saccharomyces cerevisiae, N. Hong, S. S. Seo, E. J. Oh, J. H. Seo, J. H. Cate, Y. S. Jin, Applied Microbiology and Biotechnology, V. 98, pp. 5757-5764, April 18, 2014. 

 

Overcoming Inefficient Cellobiose Fermentation by Cellobiose Phosphorylase in the Presence of Xylose, K. Chomvong, V. Kordic, X. Li, S. Bauer, A. E. Gillespie, S. J. Ha, E. J. Oh, J. M. Galazka, Y. S. Jin, J. H. Cate, Biotechnology for Biofuels, V. 7, pp. 85, June 7, 2014. 

 

Leveraging Transcription Factors to Speed Cellobiose Fermentation by Saccharomyces cerevisiae, Y. Lin, L. Chomvong, L. Acosta-Sampson, R. Estrela, J. M. Galazka, S. R. Kim, Y. S. Jin, J. H. Cate, Biotechnology for Biofuels, V. 7, pp. 126, August 27, 2014. 

 

Development and Physiological Characterization of Cellobiose-Consuming Yarrowia lipolytica, S. Lane, S. Zhang, N. Wei, C. Rao, Y. S. Jin, Biotechnology and Bioengineering, doi: 10.1002/bit.25499, November 25, 2014. 

 

Enhanced Hexose Fermentation by Saccharomyces cerevisiae Through Integration of Stoichiometric Modeling and Genetic Screening, J. Quarterman, S. R. Kim, P. J. Kim, Y. S. Jin, Journal of Biotechnology, doi: 10.1016/j.jbiotec.2014.11.017, November 27, 2014. 

 

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Using RNA-Guided Cas9 Nuclease, G. Zhang, I. Kong, H. Kim, J. Liu, J. H. Cate, Y. S. Jin, Applied and Environmental Microbiology, doi:10.1128/AEM.02310-14, Oct. 3, 2014

 

Published in 2012

Rational and Evolutionary Engineering Approaches Uncover a Small Set of Genetic Changes Efficient for Rapid Xylose Fermentation in Saccharomyces cerevisae, Soo Rin Kim, Jeffrey M. Skerker, Wei Kang, Anastashia Lesmana, Na Wei, Adam P. Arkin, Yong-Su Jin, PLoS One 8 (2), doi: 10.1371/journal.pone.0057048, February 262013.

 

Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces Stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisae, S. J. Ha, H. Kim, Y. Lin, M. U. Jang, J. M. Galazka, T. J. Kim, J. H. Cate, Y. S. Jin, Metabolic Applied and Environmental Microbiology 79 (5): pp. 1500-1507, doi: 10: 1128/AEM.03253-12, March 2013.

 

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae,  N. Wei, H. Xu, S. R. Kim, Y. S. Jin, Applied and Environmental Microbiology 79 (10): pp. 3193-3201, doi: 10.1128/AEM.00490-13, March 8, 2013.

 

Strain Engineering of Saccharomyces cerevisiae for Enhanced Xylose MetabolismS. R. Kim, Y. C. Park, Y. S. Jin, J. H. Seo, Biotechnology Advances, 31(6), pp. 851-861, doi: 10.1016/j.biotechadv.2013.03.004, March 21, 2013.

 

Combinatorial Genetic Perturbation to Refine Metabolic Circuits for Producing Biofuels and Biochemicals, H. J.  Kim, T. L. Turner, Y. S. Jin, Biotechnology Advances 31, pp. 976-985, doi: 10.1016/j.biotechadv.2013.03.010, April 5, 2013.

 

Simultaneous Saccharification and Fermentation by Engineered Saccharomyces cerevisiae Without Supplementing Extracellular Beta-Glucosidase, W. H. Lee, H. Nan, H. J. Kim, Y. S. Jin, Journal of Biotechnology, 167, pp. 316-322, doi: 10.1016/j.jbiotec.2013.06.016, July 10, 2013.

 

Analysis of Cellodextrin Transporters from Neurospora Crassa in Saccharomyces Cerevisiae for Cellobiose Fermentation, H. Kim, W. H. Lee, J. M. Galazka, J. H. Cate, Y. S. Jin, Applied Microbiology and Biotechnology, doi: 10.1007/s00253-013-5339-2, June 2013.

 

Continuous Co-Fermentation of Cellobiose and Xylose by Engineered Saccharomyces cerevisiae, Suk-Jin Ha, Soo Rin Kim, Heejin Kim, Jing Du, Jamie H.D. Cate, Yong-Su Jin, Bioresource Technology, 149, pp. 525-531, doi: 10.1016/j.biortech.2013.09.082, September 27, 2013.

 

Two-Stage Acidic-Alkaline Hydrothermal Pretreatment of Lignocellulose for the High

Recovery of Cellulose and Hemicellulose Sugars, (2013), B. Guo, Y. Zhang, G. Yu, W. H. Lee, Y. S. Jin, E. Morgenroth, Applied Biochemistry and Biotechnology 169, pp. 1069-1087.

 

Feasibility of Xylose Fermentation by Engineered Saccharomyces cerevisiae Overexpressing Endogenous Aldose Reductase (GRE3), Xylitol Dehydrogenase (XYL2), and Xylulokinase (XYL3) from Scheffersomyces stipites (2013), S. R. Kim, N. R. Kwee, H. Kim, Y. S. Jin, FEMS Yeast Research 13(3), pp. 312-321.

 

Construction of an Efficient Xylose-Fermenting Diploid Saccharomyces cerevisiae Strain Through Mating of Two Engineered Haploid Strains Capable of Xylose Assimilation (2013), S. R. Kim, K. S. Lee, Il Kong, A. Lesmana, W. H. Lee, J. H. Seo, D. H. Kweon, Y. S. Jin, Journal of Biotechnology 164, pp. 105-111.

 

Enhanced Biofuel Production Through Coupled Acetic Acid and Xylose Consumption by Engineered Yeast, N. Wei, J. Quarterman, S.R. Kim, J. H. Cate, and Y.S. Jin, Nature Communications 4, pp. 2580, doi: 10.1038/ncomms3580, October 8, 2013.

 

Enhanced Xylitol Production Through Simultaneous Co-utilization of Cellobiose and Xylose by Engineered Saccharomyces cerevisiae (2013), E. J. Oh, S. J. Rin, S. Kim, W. H. Lee, J. M. Galazka, J. H. Cate, Y. S. Jin, Metabolic Engineering 15, pp. 226-234.

 

Marine Macroalgae: an Untapped Resource for Producing Fuels and Chemicals (2013), N. Wei, J. Quarterman, Y. S. Jin,  Trends in Biotechnology 31, pp. 70-77

 

Published in 2012

Single Amino Acid Substitutions of HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae, S. J. Ha, H. Kim, Y. Lin, M. U. Jang, J. M. Galazka, T. J. Kim, J. H. Cate, Y. S. Jin. Applied Environmental Microbiology, December 21, 2012.

 

Marine Macroalgae: An Untapped Resource for Producing Fuels and Chemicals, Na Wei, Josh Quarterman, Yong-Su Jin, Trends in Biotechnology, http://dx.doi.org/10/1016/j.tibtech.2012.10.009, December 12, 2012.

 

Enhanced Xylitol Production Through Simultaneous Co-Utilization of Cellobiose and Xylose by Engineered Saccharomyces cerevisiae, E. J. Oh, S. J. Ha, Soo Rin Kim, W. H. Lee, J. M. Galazka, J. H. Cate, Y. S. Jin, Metabolic Engineering, doi: 10.1016/j.ymben.2012.09.003, October 24, 2012.

 

High Expression of XYL2 Coding for Xylitol Dehydrogenase Is Necessary for Efficient Xylose Fementation by Engineered Saccharomyces cerevisiae, Soo Rin Kim, Suk-Jin Ha, In Iok Kong, Yong-Su Jin, Metabolic Engineering, doi: 10.1016/j.ymben.2012.04.001, April 13, 2012.

 

Energetic Benefits and Rapid Cellobiose Fermentation by Saccharomyces cerevisiae Expressing Cellobiose Phosphorylase and Mutant Cellodextrin Transporters, S. J. Ha, J. M. Galazka, Oh E. Joong, V. Kordić, H. Kim, Y.S. Jin, J. H. Cate, Metabolic Engineering 15:134-43, doi: 10.1016/j.ymben.2012.11.005. November 22, 2012.

 

Model-Guided Strain Improvement: Simultaneous Hydrolysis and Co-Fermentation of Cellulosic Sugars, Y. S. Jin, J. H. Cate, Biotechnology Journal 7(3):328-9, doi: 10.1002/biot.201100489.

 

Simultaneous Co-Fermentation of Mixed Sugars: A Promising Strategy for Producing Cellulosic Ethanol, Soo Rin Kim, Suk-Jin Ha, Na Wei, Eun Joong Oh, Yong-Su Jin, Trends in Biotechnology, http://dx.doi.org/10.1016/j.tibntech.2012.01.005, February 20, 2012.

 

Combined Biomimetic and Inorganic Acids Hydrolysis of Hemicellulose in Miscanthus for Bioethanol Production, B. Guo, Y. Zhang, S. J. Ha, Y. S. Jin, E. Morgenroth, Bioresource Technology 110:278-87. doi: 10.1016/j.biortech.2012.01.133, February 8, 2012.

 

Published in 2011

Cofermentation of Cellobiose and Galactose by an Engineered Saccharomyces cerevisiae Strain, Suk-Jin Ha, Qiaosi Wei, Soo Rin Kim, Jonathan Galazka, Jamie Cate, Yong-Su Jin, Applied and Environmental Microbiology, 77(16), pp. 5822-5825, doi:10.1128/AEM.05228-11, August 2011.

 

Tuning Structural Durability of Yeast-Encapsulating Alginate Gel Beads with Interpenetrating Networks for Sustained Bioethanol Production, Chaenyung Cha, Soo Rin Kim, Yong-Su Jin, Hyunjoon Kong, Biotechnology and Bioengineering, doi: 10.1002/bit.23258, July 5, 2011.

 


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