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United States Department of Agriculture

Agricultural Research Service


Location: Crop Bioprotection Research

2009 Annual Report

1a.Objectives (from AD-416)
Determine the metabolic, physiologic, and genetic fundamentals underlying stress tolerance of ethanologenic yeast strains and other microbes. Using this fundamental stress tolerance knowledge, engineer improved strains and/or design process conditions that foster stress tolerance and functionality of microbes for production of ethanol and bioproducts from corn fiber and other lignocellulosic materials, despite exposure to harsh environments.

1b.Approach (from AD-416)
Screening for phenotype differences in stress tolerance of yeast strains will be the first step in the search for genes and gene systems fostering resilience to stress factors. Stress factors common to fermentations of lignocellulose hydrolzates will be the focus of this research, including various chemical fermentation inhibitors (furans, phenolics, organic acids, etc.), high ethanol concentration, wide pH and osmotic shifts, and the high temperatures needed for simultaneous saccharification-fermentation processes. Cultures screened will include natural ethanologenic yeasts capable of hexose and pentose fermentation, strains adapted to increasing levels of stress, a Saccharomyces cerevisiae disruption mutant library, and S. cerevisiae transformants prepared using a cDNA library from a furan-resistant/detoxifying yeast. Genes responsible for observed changes in stress tolerance will be identified and characterized with respect to sequence homology, protein structural domains, and protective function. Cultural conditions (nutrition, carbon and nitrogen source/ratio/concentration, physiological cell age, osmotic pressure, pH, temperature, dissolved oxygen, and others) will also be varied to screen for shifts in stress tolerance. Quality-controlled microarrays will be designed and applied to identify genes associated with stress responses and to study the genomic response of cultures to applied stress factors during fermentation time courses in order to identify gene networks and control mechanisms involved in stress tolerance and structural domain. Genetic tools and information gained will be applied to engineer improved strains and optimize the fermentation process to foster improved stress tolerance for lower cost production of ethanol from hydrolyzates of lignocellulosic biomass.

3.Progress Report
Objectives of this project were to develop inhibitor tolerant yeast strains and processes for efficient biomass conversion to biofuel. Several accomplishments in the past 5 years lay the foundation for the next project plan to be implemented October FY 2010. Furfural and hydroxymethylfurfural (HMF) are key toxic byproducts of dilute acid hydrolysis of lignocellulosic biomass. Certain strains of native yeasts Saccharomyces cerevisiae and pentose-utilizing Pichia stipitis were found to survive/adapt to furfural and HMF and to detoxify them, allowing development of better strains by repeated exposure to gradually increasing inhibitor challenges. A patent application for S. cerevisiae Y-50049 was filed. Another result of the research was the first rigorous chemical identification of the microbial HMF detoxification product, and a synthesis method for the compound that has been adopted by others. Exogenous universal controls were developed to standardize two different types of high-throughput gene expression assays (microarray and quantitative real-time polymerase chain reaction (qRT-PCR)) used to assess impact of inhibitor stress on yeast at the gene level. These were the first of such controls available since high-throughput gene expression analysis techniques appeared a decade ago and were pursued by the External RNA Controls Consortium (ERCC) to develop industry-wide standards. Using these techniques, over 200 genes were shown to be involved in genomic adaptation to allow inhibitor resistance and in situ detoxification. Modeling of gene regulatory networks in collaboration with New Mexico State University revealed significant genes in the regulation of HMF detoxification. Screening an S. cerevisiae library of disrupted genes demonstrated that pentose phosphate pathway genes play a critical role in protecting yeast against furfural stress perhaps via generation of nicotinamide adenine dinucleotide phosphate (NADPH), an important reducing cofactor necessary to support cell repair and reduce oxidative stress. In later studies, furfural and HMF detoxification were shown to be mediated by multiple genes involving both NADPH and NADH (nicotinamide adenine dinucleotide) cofactors. Findings indicated the coupling of a major pentose sugar metabolism pathway with efficient furfural detoxification. Finally, culture conditions were developed to optimize ethanol production efficiency from xylose and inhibitor tolerance of P. stipitis. Nitrogen source and mineral composition optimization allowed ethanol yields up to 70 g/L from media having 150 g/L xylose. Priming inocula with xylose was observed to induce faster fermentation rates and to eliminate diauxic lag during mixed sugar conversion, allowing an economically recoverable 66 g/L ethanol in 48 hours at a conversion efficiency of 88%--a vast improvement over “stuck” fermentations commonly observed during the switch from glucose to xylose utilization by yeasts. Nitrogen source optimization was also key for ethanol production by tolerant strain Y-50049. As a result of this progress, new strains, tolerance mechanisms, and processes enable lower cost biofuel from renewable biomass.

1. REPROGRAMMED METABOLIC PATHWAYS FOR INHIBITOR DETOXIFICATION FOUND IN ADAPTED TOLERANT YEAST. Using newly developed enhanced rigorous gene expression technology, we discovered that the tolerant ethanologenic yeast Y-50049 has reprogrammed pathways that allow the yeast to in situ detoxify biomass conversion inhibitors while producing ethanol. Inhibitory compounds generated during hydrolysis pretreatment of lignocellulosic biomass inhibit cell growth and interfere with subsequent fermentation of fermentative microbes. Currently, there is no tolerant yeast available for cellulosic ethanol production at an industrial scale. Development of tolerant yeast to overcome inhibitor stress is needed for a sustainable biomass-to-ethanol industry. Using tolerant yeast strain Y-50049 developed at NCAUR (Crop BioProtection Unit, Peoria, IL), we identified numerous genes, new elements, and alternative pathways responsible for the detoxification of the inhibitors and ethanol production. The tolerant yeast can be applied to ferment hydrolyzates without additional inhibitor removal treatment such as overliming. Thus, processing can be simplified and cost reduced. Knowledge obtained from this study contributes understanding of mechanisms of stress tolerance and guides future more robust strain development in the bioenergy field.

2. NOVEL GENE CONTRIBUTES TO DETOXIFICATION OF LIGNOCELLULOSE FERMENTATION INHIBITORS. Fermentation inhibitors pose a significant challenge for a sustainable lignocellulose-to-ethanol industry. Before fermentation to ethanol can begin, numerous different inhibitors, many classified as aldehydes, are generated during the chemical hydrolysis of lignocellulosic biomass to simple sugars. These interfere with subsequent microbial growth and fermentation. In situ detoxification of many aldehyde inhibitors was shown by a tolerant ethanologenic yeast strain that we obtained by adaptation. During the studies of tolerance mechanisms of this strain, we discovered a novel gene coding for an aldehyde reductase enzyme with strong reduction activity toward at least 14 toxic aldehydes, many common to biomass hydrolysis. Identification and understanding of this new gene and its enzyme product impacts tolerant strain development for low-cost bioethanol production and contributes to the descriptive literature on the genome of the tolerant yeast strain in which it was found.

3. CULTURE NUTRITION CRITICAL TO INHIBITOR TOLERANT YEAST PERFORMANCE. Currently, there is no process available for cellulosic ethanol production at an industrial scale. Inhibitory compounds generated during acid hydrolysis pretreatment of lignocellulosic biomass inhibit cell growth and interfere with subsequent fermentation. A tolerant yeast strain Y-50049 has recently been developed in our laboratory, but process conditions to maximize its ethanol production potential are needed. Data were collected on the parent yeast and the tolerant strain Y-50049 considering the impact of nitrogen sources, vitamins, and minerals on ethanol production potential. A striking difference in nutritional requirements of the two strains was observed. Although cultures supplied biomass hydrolyzates will be fairly nutrient rich, they may not supply the best nutrient mix for both ethanol production and inhibitor tolerance. Since the right “mix” is critical, these results are expected to impact the design of industrial fermentation processes with optimal nutrient supplementation for efficient production of ethanol from biomass at minimal cost.

4. MASTER EQUATION FOR ENHANCED GENE EXPRESSION ANALYSIS. Real time quantitative reverse transcription polymerase chain reaction (qRT- PCR) is currently the assay of choice among available techniques for quantification of gene expression levels. Gene expression varies in response to different conditions and environmental stimuli. By convention, housekeeping genes have been applied as internal reference for data normalization since the technology appeared. However, all housekeeping genes vary under environmental stimuli and there is no commonly accepted housekeeping gene reference available. Accurate data acquisition and data reproducibility remain challenging, and it is difficult to compare results from different experimental sets. We developed a robust standardization system using a fixed threshold based on a sole exogenous gene transcript called a “universal control” and a master equation derived from assaying this universal control at various known concentrations. Providing a constant reference for simple, accurate, absolute quantitation of gene expression, this new technology impacts not only development of improved yeast for lower cost biofuel but also the larger gene expression research community.

6.Technology Transfer

Number of the New/Active MTAs (providing only)2
Number of Invention Disclosures Submitted1
Number of New Patent Applications Filed1

Review Publications
Liu, Z., Moon, J., Andersh, B.J., Slininger, P.J., Weber, S.A. 2008. Multiple Gene Mediated NAD(P)H-Dependent Aldehyde Reduction is a Mechanism of in situ Detoxification of Furfural and HMF by Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 81:743-753.

Slininger, P.J., Gorsich, S.W., Liu, Z. 2009. Culture Nutrition and Physiology Impact the Inhibitor Tolerance of the Yeast Pichia stipitis NRRL Y-7124. Biotechnology and Bioengineering. 102(3):788-790.

Balan, V., Rogers, C.A., Chundawat, S.P., Sousa, L.D., Slininger, P.J., Gupta, R., Dale, B.E. 2008. Conversion of Extracted Oil Cake Fibers into Bioethanol Including DDGS, Canola, Sunflower, Seasame, Soy, and Peanut for Integrated Biodiesel Processing. Journal of the American Oil Chemists' Society. 86:157-165.

Song, M., Ouyang, Z., Liu, Z. 2008. Discrete dynamical system modelling for gene regulatory networks of 5-hydroxymethylfurfural tolerance for ethanologenic yeast. IET Systems Biology. 3:203-218.

Liu, Z., Palmquist, D.E., Ma, M., Liu, J., Alexander, N.J. 2009. Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR. Journal of Biotechnology. 143:10-16.

Last Modified: 8/30/2014
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