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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE

Location: Bioenergy Research

2012 Annual Report


1a. Objectives (from AD-416):
1) Determine the key metabolic, physiologic, transport, genetic, and regulatory mechanisms underlying stress tolerance and adaptation in ethanologenic yeast when they convert lignocellulosic hydrolyzates. 2) Via directed evolution, genetic engineering, and/or adaptation, create new commercially preferred yeast strains for converting lignocellulose hydrolyzates to ethanol. 3) In collaboration with Cooperative Research and Development (CRADA) partner(s), optimize fermentation process conditions so as to (1) leverage advantages of stress tolerant strains developed in Objective 2 and (2) minimize the cost of fermenting lignocellulosic hydrolyzates to fuel-grade ethanol.


1b. Approach (from AD-416):
The lignocellulose-to-ethanol process involves pretreatment of biomass to predispose it to chemical and enzymatic hydrolysis, saccharification of sugar polymers to simple sugars, and fermentation of the sugars to ethanol. The hydrolysis product is difficult to ferment because inhibitory byproducts are produced, and the resulting sugar mixture contains both hexose and pentose sugars, the latter not fermentable by traditional brewing yeasts. Needed are improved yeast strains which will ferment both types of sugars and are able to withstand, survive, and function in the presence of inhibitors (including furfural and hydroxymethyl furfural), high ethanol concentration and osmotic pressure, and sufficiently elevated temperatures for simultaneous saccharification-fermentation processes. In the research proposed, fermentation hurdles will be overcome by combining process optimization strategies and strain improvements aided by new molecular biology tools allowing high throughput screening of whole genomes to identify key genes and gene networks involved in stress tolerance and sugar utilization. Products of the research will be stress-tolerant yeasts capable of resisting and detoxifying inhibitors and efficiently fermenting hexose and pentose sugars to ethanol, a genetic blueprint describing tolerance mechanisms and metabolic pathways, and optimal culture conditions and process configurations to lower costs by maximizing yeast stress resistance, ethanol productivity and yield.


3. Progress Report:
The overall goal of this project is to develop stress tolerant microbes for lower cost production of ethanol from renewable biomass. By determining key mechanisms of tolerance, evolving and engineering new strains, and optimizing key process conditions, we have made significant progress during FY12. Research studies on genomic mechanisms of yeast tolerance in Saccharomyces cerevisiae were expanded under a National Research Initiative project in collaboration with New Mexico State University scientists. This past year significant time effort has focused on the in-depth analysis of genomics data to understand the biological relevance of our findings and uncover inhibitor tolerance mechanisms through modeling approaches. The experimental phase of a non-funded cooperative agreement with the Institute of BioEnergy and Bioprocessing Technology, Chinese Academy of Sciences, Qingdao, China has recently finished and provided complete sequencing data needed for a genomic comparison of an adapted inhibitor-tolerant strain and its parent. Collaborative data analysis for a manuscript is ongoing. Our prior studies have indicated that the native yeast pentose metabolism may compete for reductase activities needed to reduce toxic furan aldehyde inhibitors to less toxic alcohols, so it is important to integrate the engineering of pentose and tolerance traits in S. cerevisiae to improve hydrolyzate sugar utilization. Our inhibitor-tolerant evolved S. cerevisiae strain was engineered with xylose isomerase and other enzymes to allow conversion of xylose to ethanol. Then cloning of new xylose transporter genes from the S. stipitis genome into S. cerevisiae allowed us to verify the function of each transporter gene and at the same time develop new advanced strains (patent application to be filed FY12). Evolved tolerant S. stipitis strains were developed which exhibited reduced diauxic lag from glucose to xylose uptake and improved production rate and yield of ethanol from ammonia fiber explosion pre-treated corn stover hydrolyzates at 18% solids, and an invention disclosure on top strains was approved for patent application. Additionally, S. stipitis serial micro-batch and continuous cultures have been challenged with concentrated ethanol and dilute acid switchgrass hydrolyzates to enrich for tolerant variants. This past year over 500 single-cell colonies were recovered from enrichment cultures via selective plating and screened to rank strains based on ethanol production kinetics on concentrated switchgrass hydrolyzates with varied nutrient levels. Culture nutrition was key to performance of both native and inhibitor-tolerant S. cerevisiae and S. stipitis strains on switchgrass hydrolyzates, and commercially useful supplements are being developed to maximize performance. Interestingly, nutrient requirements to maximize performance of adapted versus parent strains were often very different, indicating a need for attention to nutrient supplies during both strain selection and process development.


4. Accomplishments


Review Publications
Ma, M., Liu, Z., Moon, J. 2012. Genetic engineering of inhibitor-tolerant Saccharomyces cerevisiae for improved xylose utilization in ethanol production. Bioenergy Research. 5:459-469.

Slininger, P.J., Schisler, D.A. 2012. Biological control agents for suppression of post-harvest diseases of potatoes: strategies on discovery and development. In: Dhanasekaran, N., Panneerselvam, A., editors. Fungicides for Plant and Animal Diseases. Rijeka, Croatia: In-Tech. p. 141-166.

Moon, J., Liu, Z. 2012. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. Enzyme and Microbial Technology. 50:115-120.

Zhang, L., Li, J., Li, S., Liu, Z. 2011. Challenges of cellulosic ethanol production from xylose-extracted corncob residues. BioResources. 6(4):4302-4316.

Liu, Z., Weber, S.A., Cotta, M.A., Li, S. 2012. A new beta-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation. Bioresource Technology. 104:410-416.

Schisler, D.A., Slininger, P.J., Boehm, M.J., Paul, P.A. 2011. Co-culture of yeast antagonists of Fusarium head blight and their effect on disease development in wheat. Plant Pathology Journal. 10:128-137. DOI: 10.3923/ppj.2011.128.137.

Hughes, S.R., Bischoff, K.M., Gibbons, W.R., Bang, S.S., Pinkelman, R., Slininger, P.J., Qureshi, N., Liu, S., Saha, B.C., Jackson, J.S., Cotta, M.A., Rich, J.O., Javers, J. 2012. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars. Journal of Industrial Microbiology and Biotechnology. 39(1):163–173.

Liu, Z. 2011. Genomics of yeast tolerance and in situ detoxification. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 1-28.

Liu, Z. 2011. Unification of gene expression data for comparable analyses under stress conditions. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 279-299.

Ma, M., Liu, Z. 2012. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels: Systems Biology. Microbiology Monographs 22. Berlin, Germany: Springer-Verlag. p. 77-115.

Matsushika, A., Liu, Z., Sawayama, S., Moon, J. 2011. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 137-160.

Liu, Z., Weber, S.A., Cotta, M.A. 2013. Isolation and characterization of a ß-glucosidase from a Clavispora strain with potential applications in bioethanol production from cellulosic materials. Bioenergy Research. 6:65-74.

Last Modified: 10/20/2017
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