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United States Department of Agriculture

Agricultural Research Service


Location: Bioenergy Research Unit

2013 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:
Relating to Objective 1 on determining stress tolerance mechanisms: Since industrial yeast strains are generally more robust than typical haploid laboratory strains, comparative genome expression experiments were conducted to uncover genetic elements that are responsible for tolerance to fermentation inhibitors. Important regulatory networks of Saccharomyces cerevisiae were identified when lab and tolerant industrial yeast strains were exposed to toxic compounds present in lignocellulosic hydrolysates. In collaboration with scientists from Chinese Academy of Sciences, we discovered that the industrial yeast more strongly expressed the mitogen-activated protein kinase (MAPK) signaling pathway and the phosphatidylinositol signaling pathway than did the model lab strain when challenged by inhibitors. These pathways are the most important signaling transduction pathways regulating a wide variety of cellular activities. Genome analyses also revealed sequence variations for many genes in these pathways. This research provided the first insight into the genetic background of industrial yeast tolerance and suggests that the more robust signaling transduction pathways of the industrial yeast may be attributed to sequence variations in its genome compared with the model lab strain. In collaborative research with New Mexico State University, we further defined globally rewired pathways for a tolerant industrial yeast strain after its adaptation to inhibitory compounds found in lignocelluloses hydrolyzates. Pertinent to Objective 2 to develop improved yeast strains for lignocellulose bioconversion: Superior hydrolyzate tolerant isolates of the xylose-fermenting strains of S. stipitis were characterized relative to their improved ability to grow and ferment in the presence of xylose and on mixed hexose and pentose sugars in the presence of ethanol and a range of acetic acid concentrations. Additionally, the most promising isolates were characterized relative to their improved ability to resist and grow on lignocellulosic sugars in the presence of furfural and hydroxymethylfurfural (HMF) inhibitors formed during lignocellulose pretreatment. Relative to process development in Objective 3: Since culture nutrition was key to performance of both native and inhibitor-tolerant S. cerevisiae and S. stipitis strains on switchgrass hydrolyzates, low cost commercially available nutrient supplements and nitrogen release processes were designed for use within the switchgrass pretreatment, saccharification and fermentation process train. Along with nutrient process considerations, pH and temperature interactive impacts on cell viability and fermentation were investigated for evolved strains in separate hydrolysis and fermentation (SHF) style processing. Based on isolate characterizations and preliminary process findings, the most promising strains and process strategies will be assembled and investigated in the next few months. Several manuscripts and a patent application are currently in preparation.

1. Stronger signaling gene networks may explain industrial yeast robustness. Ethanol can be produced using yeast to biologically convert cellulosic agricultural biomass, but the conversion process is obstructed by inhibitory compounds generated during the required pretreatment process, especially when dilute acid is used. For ease of genetic engineering, model laboratory strains of the yeast Saccharomyces cerevisiae are commonly used in lignocellulose-to-ethanol research. However, laboratory strains are often less robust than industrial strains, and new strains with engineered traits consequently do not meet the rigors of commercial application. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with scientists from Chinese Academy of Sciences compared the genome composition and expression of an industrial yeast and a model lab strain. When challenged by the toxic compounds liberated from lignocellulose biomass pretreatment, the industrial yeast displayed improved response in regulating a wide variety of cell functions during stress—especially mitogen-activated protein kinase (MAPK) and phosphatidylinositol pathways. In support of United States energy independence, the findings of this research revealed potential mechanisms of yeast tolerance and provided fundamental knowledge for developing the new biocatalysts needed for lower cost production of renewable ethanol biofuel from agricultural biomass.

2. Low cost nutrients and optimal processes for ethanol biofuel from switchgrass hydrolyzates. Unlike gasoline which is a fossil fuel, ethanol biofuel is renewable and can be produced using yeast to biologically convert cellulosic agricultural biomass, including new energy crops such as switchgrass. The process that prepares biomass for producing ethanol unfortunately also releases compounds that interfere with conversion process. To overcome this, tolerant yeast strains of S. cerevisiae and S. stipitis have recently been developed by Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, Illinois. It was discovered that culture nutrition is key to switchgrass hydrolyzate tolerance and performance of both yeasts, and low cost nutrient supplements and nitrogen release processes were designed for most economical use within the switchgrass pretreatment, saccharification and fermentation process train. The interaction of nutrients, pH and temperature was optimized for rapid and economical ethanol production by the tolerant yeast strains. This new technology will support lower cost production of renewable ethanol from agricultural biomass, reduce United States dependence on foreign petroleum and stimulate the rural economy.

3. Computational analysis points to rewired gene regulatory networks of inhibitor tolerant yeast. High throughput genome expression is a tool being used to explore and design the blue print of new inhibitor tolerant yeast strains for low cost ethanol biofuel production from renewable agricultural biomass. Interpretation of genome expression data is difficult because of its massiveness and complexity. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, Illinois, and scientists at New Mexico State University, Las Cruces, New Mexico, developed a computation program and compared the gene regulatory networks for the genomes of a parent yeast strain and an inhibitor-tolerant evolved strain. Application of the program revealed significant alterations of gene interaction and regulatory networks when the yeasts were challenged by inhibitors. These alterations involved at least 44 pathways, including important central metabolic pathways. The basic knowledge discovered will guide continued efforts as more robust strains are developed for the production of advanced biofuels in support of United States energy independence.

Review Publications
Moon, J., Liu, Z., Ma, M., Slininger, P.J., Weber, S.A. 2013. New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production. Biocatalysis and Agricultural Biotechnology. 2:247-254.

Yang, J., Ding, M., Li, B., Liu, Z., Wang, X., Yuan, Y. 2012. Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol. Omics: A Journal of Integrative Biology. 16(7-8):374-386.

Slininger, P.J., Schisler, D.A. 2013. High-throughput assay for optimizing microbial biological control agent production and delivery. Biocontrol Science and Technology. 23(8):920-943.

Last Modified: 12/19/2014
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