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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

2010 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
Project 3620-41000-147-00D started October 2009 and continues from Project 3620-41000-123-00D. Gene expression and metabolic profiling of an evolved tolerant Saccharomyces cerevisiae strain NRRL Y-50049 and its parent revealed reprogrammed glucose metabolic pathways and interactive gene networks underlying tolerance to stress challenges by furfural and hydroxymethylfurfural (HMF). Genetic constructs suitable for transformation were developed, and initial xylose utilizing recombinants with inhibitor-tolerance background were generated for future gene expression profiling. Using evolutionary engineering, a more ethanol-tolerant strain (patent pending) was derived from inhibitor-tolerant Y-50049. Dynamic transcription analysis of tolerant derivative and parent identified candidate genes and key regulators enhancing ethanol tolerance. In collaborative studies, first-year improved strains of the native pentose-fermenting yeast Scheffersomyces (Pichia) stipitis NRRL Y-7124 were obtained through targeted adaption in stressed environments with reduced oxygen, elevated temperature, elevated ethanol concentration, and high solids hydrolyzates (20-25%). Comparative kinetics documented significant improvements in adapted strains over parents. Using directed enzyme evolution, we engineered the genetic code of a reductase gene commonly up-regulated during inhibitor stress. Clones with beneficial mutations established viable cultures under high HMF (30mM) stress and displayed engineered reductase activities toward HMF and furfural that were several fold higher compared with the parent gene. Derivatives of Y-50049 transformed with mutated genes showed modified enzyme cofactor preference and significantly higher levels of inhibitor detoxification. Several putative xylose transporter genes present in the native pentose-fermenting S. stipitis genome were sequenced and cloned. Transformation of Y-7124 and an inhibitor-tolerant derivative of Y-50049 with cloned genes allowed assessment of xylose utilization and identification of transporter genes useful for future engineering of inhibitor–tolerant xylose fermenting strains. Commercially preferred low-cost nitrogen sources were identified in laboratory media experiments for optimal ethanol production by Y-7124, inhibitor-tolerant Y-50049, and its parent. Studies to optimize nitrogen source composition for fermentation of dilute acid switchgrass hydrolyzates are underway and scheduled to finish by October 2010. Studies showed that priming Y-7124 on xylose was key to successful use of mixed sugars because specific enzymes for xylose metabolism could be induced before repressive levels of ethanol accumulated. Studies to apply this strategy and cell recycling to hydrolyzate fermentation (utilizing a separable pentose stream) are underway and scheduled to complete by October 2010.


4. Accomplishments


Review Publications
Ma, M., Liu, Z. 2010. Quantitative Transcription Dynamic Analysis Reveals Candidate Genes and Key Regulators for Ethanol Tolerance in Saccharomyces cerevisiae. Biomed Central (BMC) Genomics. 10:169.

Allen, S.A., Clark, W., Mccaffery, J.M., Cai, Z., Lanctot, A., Slininger, P.J., Liu, Z., Gorsich, S.W. 2010. Furfural Induces Reactive Oxygen Species Accumulation and Cellular Damage in Saccharomyces cerevisiae. Biotechnology for Biofuels. 3(2):1-10.

Liu, Z., Menggen, M., Song, M.J. 2009. Evolutionarily Engineered Ethanologenic Yeast Detoxifies Lignocellulosic Biomass Conversion Inhibitors by Reprogrammed Pathways. Molecular Genetics and Genomics. 282(3):233-244.

Liu, Z., Moon, J. 2009. A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from Lignocellulosic biomass conversion. Gene. 446(1):1-10. DOI: 10.1016/j.gene.2009.06.018

Ma, M., Liu, Z. 2010. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 87(3)829-845.

Last Modified: 10/16/2017
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