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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 Unit

2011 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
Inhibitor and ethanol tolerant strains of Saccharomyces cerevisiae and Scheffersomyces (formerly Pichia) stipitis have been developed by adaptation. Molecular mechanisms, including important genes, pathways, and regulatory genes responsible for ethanol and furan aldehyde hydrolyzate inhibitor tolerance were identified in the yeast S. cerevisiae using high-throughput gene expression analyses. Gene expression dynamics of tolerant S. cerevisiae strains in response to the furfural and hydroxymethylfurfural (HMF) inhibitor complex were also examined. Evolved tolerant S. stipitis strains exhibited reduced diauxic lag and improved production rate and yield of ethanol from ammonia fiber explosion pre-treated corn stover hydrolyzate with high solids loading. High temperature and low oxygen adaptation of S. stipitis strains will be continued to address nutritional requirements in hydrolyzate to support improved traits observed thus far. Inhibitor-tolerant S. cerevisiae has been genetically engineered with genes supporting xylose fermentation. The new tolerant strain was able to consume both glucose and xylose and to make moderate, but significant amounts of ethanol from xylose. Pentose fermentation and reduction of toxic aldehydes both compete for reductase activities which convert toxic furan aldehyde inhibitors to less toxic alcohol forms such that engineering of both traits needed to be integrated. Our ethanol and inhibitor tolerant S. cerevisiae strain was engineered with xylose isomerase and other enzymes to balance the conversion of xylose to ethanol and also with new transporter genes from the sequenced P. stipitis genome. The cloning of the new xylose transporter genes into S. cerevisiae allowed us to verify the function of each transporter gene and at the same time develop a new advanced strain (patent application in process). Nutrient and culture conditions impacting the viability of growing and recycled S. stipitis and S. cerevisiae strains were examined. Culture nutrition was found to be key to performance of both inhibitor-tolerant S. cerevisiae and S. stipitis strains on switchgrass hydrolyzates. A new view of how ethanol impacts the induction of xylose utilizing enzymes in native pentose fermenting yeasts, such as S. stipitis, points to optimal process strategies for converting biomass to ethanol — produce cells and ethanol on xylose from pretreatment; then saccharify cellulose and feed the glucose to enhance ethanol accumulation; then potentially recycle cells to preserve cell growth investment.


4.Accomplishments
1. New yeast resists inhibitors, ferments more sugar (xylose and glucose) to ethanol biofuel. Unlike gasoline which is a fossil fuel, ethanol is a renewable fuel that can be produced from agricultural biomass such as new energy crops like switchgrass or crop residues such as corn stover and wheat straw. Biomass can be hydrolyzed, or broken down, chemically and with enzymes to its simplest sugars, predominantly glucose and xylose which could be economically fermented to ethanol by yeast if two obstacles can be overcome: the inhibitory effects of hydrolysis byproducts and poor fermentation of the xylose (about one-third of the available sugar). Saccharomyces cerevisiae is the traditional yeast used to produce ethanol biofuel from the corn starch-derived sugar, i.e., glucose, but in its natural form is unable to ferment xylose. Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, developed an inhibitor tolerant strain of Saccharomyces through adaptation and then identified and engineered new genes to improve the yeast’s arsenal of xylose transporters and enzymes needed to efficiently consume and metabolize xylose to ethanol. The new more resilient, more efficient yeast and associated know-how will foster lower cost production of renewable ethanol from agricultural biomass. An ARS patent application is currently in process.

2. Hydrolyzate-tolerant yeast strains allow enhanced ethanol production from biomass. Unlike gasoline which is a fossil fuel, ethanol is a renewable fuel that can be produced from agricultural biomass such as new energy crops like switchgrass or crop residues such as corn stover and wheat straw. Biomass can be hydrolyzed, or broken down, chemically and with enzymes to its simplest sugars, predominantly glucose and xylose, but byproducts inhibitory to the subsequent sugar fermentation are also formed in the process, and xylose is fermentable by only a few native yeasts. Among the few, Scheffersomyces (Pichia) stipitis holds the greatest commercial promise. In collaboration with Michigan State University, scientists in the Agricultural Research Service (ARS) Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, IL, adapted S. stipitis to grow in concentrated corn stover hydrolyzates (i.e., 18% solids). The evolved strains show excellent hydolyzate survival with enhanced fermentation rates and ethanol accumulations due to lag-free transition from glucose to xylose utilization. The evolved yeast will support lower cost production of renewable ethanol from agricultural biomass reducing United States dependence on foreign petroleum.

3. Key regulatory mechanisms and genes identified for yeast tolerance to biomass hydrolyzates. Unlike gasoline which is a fossil fuel, ethanol is a renewable fuel that can be produced from agricultural biomass such as new energy crops like switchgrass or crop residues such as corn stover and wheat straw. Biomass can be hydrolyzed, or broken down, chemically and with enzymes to its simplest sugars, predominantly glucose and xylose, but byproducts inhibitory to fermentation are also formed in the process. Following prolonged exposure, Saccharomyces cerevisiae is slowly able to adapt and detoxify several inhibitors commonly found in biomass hydrolyzates, but the mechanisms of tolerance remain unknown. An Agricultural Research Service (ARS) Bioenergy Research Unit scientist at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, identified at least 3 key regulatory mechanisms and 365 genes potentially forming a network enabling yeast to withstand and detoxify inhibitory byproducts of biomass hydrolysis. These findings help to develop the genetic blueprint of the ideal robust yeast which will guide efforts to engineer the optimal strains needed for a commercially successful lignocellulose-to-ethanol process and reduced United States dependence on foreign oil.


Review Publications
Liu, Z. 2011. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates. Applied Microbiology and Biotechnology. 90(3):809-825.

Liu, Z. 2010. Unification of gene expression data applying standard mRNA quantification references for comparable analyses. Journal of Microbial and Biochemical Technology. 2(5):124-126.

Ma, M., Liu, Z. 2010. Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae. Biomed Central (BMC) Genomics. 11:660. Available: http://www.biomedcentral.com/1471-2164/11/660.

Slininger, P.J., Thompson, S.R., Weber, S.A., Liu, Z., Moon, J. 2011. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag. Biotechnology and Bioengineering. 108(8):1801-1815.

Adiyaman, T., Schisler, D.A., Slininger, P.J., Sloan, J.M., Jackson, M.A., Rooney, A.P. 2011. Selection of biocontrol agents of pink rot based on efficacy and growth kinetics index rankings. Plant Disease. 95(1):24-30.

Johnson, E.T., Dowd, P.F., Liu, Z., Musser, R.O. 2011. Comparative transcription profiling analyses of maize reveals candidate defensive genes for seedling resistance against corn earworm. Molecular Genetics and Genomics. 285(6):517-525.

Last Modified: 12/21/2014
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