2008 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, an S. 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 The following milestones are currently in progress and expected to be met as planned in FY 09: 2.A. Engineer commercial yeast strains (FY 09); 2.B. Optimize bioreactor design (FY 09). Sets of genes and a mechanism responsible for tolerance and detoxification of furfural and hydroxymethylfurfural (HMF) by Saccharomyces cerevisiae were identified and characterized. A tolerant ethanologenic yeast S. cerevisiae NRRL Y-50049 was developed by directed evolution, and a provisional patent application was filed. Process conditions fostering inhibitor tolerance are under investigation. In collaboration with Michigan State University scientists, process technologies developed in Peoria, IL, were applied which showed that the native yeast strain Pichia stipitis (able to ferment xylose-glucose mixtures) could efficiently produce ethanol from edible oil cakes. Cell recycle experiments of P. stipitis were initiated to investigate techniques to eliminate dauxic inhibition of xylose update by glucose and process designs needed to maintain viable cells. Enrichment of P. stipitis for corn stover hydrolyzate tolerant variants was begun. This research addresses NP 307 and NP 306, Component 2. 4.Accomplishments 1. CULTURE CONDITIONS FOR INHIBITOR TOLERANCE AND RAPID ETHANOL PRODUCTION BY PICHIA STIPITIS. To expand the biomass to fuel ethanol industry, process strategies are needed to foster the production and utilization of microorganisms which can survive and ferment hexose and pentose sugars while exposed to inhibitors (such as ethanol, furfural, and hydroxymethylfurfural, or HMF). Furfural and HMF are key byproducts of dilute acide hydrolysis of biomass which is currently the most economical pretreatment process to allow efficient enzymatic release of fermentable sugars. The conversion of xylose to ethanol and the inhibitor tolerance of the natural pentose-fermenting yeast Pichia stipitis were optimized using a culture medium that supplied sufficient minerals and nitrogen as a mixture of urea and amino acids. Priming inocula with a high xylose concentration was observed to induce faster fermentation rates in ethanol production fermentors and to eliminate diauxic lag during mixed sugar conversion by P. stipitis NRRL Y-7124. Using both nutrition and culture priming technologies, P. stipitis Y-7124 yields an economically recoverable 66 grams per liter (g/L) ethanol in 48 hours at a conversion efficiency of 88% (0.44 g ethanol/g sugar) from 95 g/L of glucose and 55 g/L xylose. This performance is a vast improvement in efficiency over “stuck” fermentations commonly observed during the switch from glucose to xylose utilization by yeasts. Use of these process strategies in industrial plant designs has the potential to lower the cost of ethanol from biomass. This research addresses NP 307, Component 1--Process Efficiencies; and NP 306, Component 2, Problem Area 2c. 2. DETOXIFICATION MECHANISM INVOLVES MULTIPLE GENE-MEDIATED COFACTOR-DEPENDENT ALDEHYDE REDUCTION. Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated from biomass pretreatment using dilute acid hydrolysis that interfere with yeast growth and subsequent fermentation. Few yeast strains tolerant to inhibitors are available largely due to a lack of understanding of the mechanism of the tolerance to the inhibitors. We found that individual genes encoding enzymes possessing aldehyde reduction activities demonstrated cofactor preference. However, protein extract from whole yeast cells showed equally strong aldehyde reduction activities coupled with either cofactor, and deletion of a single candidate gene did not affect yeast growth in the presence of the inhibitors. These results suggest that detoxification of furfural and HMF by the ethanologenic yeast S. cerevisiae strain Y-50049 likely involves multiple gene mediated cofactor-dependent aldehyde reduction. Our findings elucidate the genetic and metabolic basis of the tolerance mechanism and contributes to development of desirable strains for lower-cost production of biofuels and bioproducts. This research addresses NP 307, Component 1--Process Efficiencies; and NP 306, Component 2; Problem Areas 2c. 3. ROBUST STANDARD REFERENCE FOR QUANTITATIVE GENE EXPRESSION ANALYSIS USING QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (qRT-PCR). The qRT-PCR has been widely accepted as the assay of choice for quantification of gene expression. By conventional practice, housekeeping genes have been applied as internal reference for data normalization and process since the technology appeared; but all housekeeping genes vary under environmental conditions, and there is no commonly accepted housekeeping gene reference available. We developed a robust standard system using a fixed threshold messenger ribonucleic acid (mRNA) and a master equation derived from the universal ribonucleic acid (RNA) control as a constant reference for simplified and efficient absolute mRNA quantification. Using yeast and a Fusarium fungus, we demonstrated independent performance of a sole reference gene as a constant reference of manual threshold for data acquisition and analysis under varied conditions. A master equation with highly fitted linear relationship and PCR amplification efficiency was obtained for reactions in varied RNA background of Saccharomyces cerevisiae and Fusarium sporotrichioides, respectively. This newly developed quality control system simplifies conventional qRT-PCR procedures and increases data reliability, reproducibility, and throughput of the assay. This research addresses NP 307, Component 1, Problem Area Process Efficiencies; and NP 306, Component 2, Problem Area 2c. 4. COMPUTATIONAL MODELING FOR STRESS TOLERANCE GENE NETWORKS. Yeast tolerance to biomass conversion inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are important to a sustainable low-cost, biomass-to-ethanol industry; but the development of tolerant strains is hampered due to a lack of understanding of genetic mechanisms underlying stress tolerance for ethanologenic yeast. We investigated global transcriptome profiling of yeast under challenge of HMF and identified more than 300 genes significantly involved in regulation of HMF tolerance and detoxification; and we described a data-driven discrete dynamic system modeling methodology to detect gene regulatory interactions and to predict system dynamic behavior based on large-scale microarray data sets. Using our model system, we identified 12 potentially significant regulatory interactions for HMF tolerance in yeast. The discrete dynamic system model to detect interactions in a network is novel and has potential for broad applications in biology. These results contribute to our understanding of mechanisms of yeast stress tolerance and are expected to expedite research and development of more tolerant strains for biomass conversion to ethanol. This research addresses NP 307, Component 1, Problem Area Process Efficiencies; and NP 306, Component 2, Problem Area 2c. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of the New MTA (providing only) 3 Number of New Commercial Licenses Executed 3 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Liu, Z., Saha, B.C., Slininger, P.J. 2008. Lignocellulosic biomass conversion to ethanol by Saccharomyces. In: Wall, J., Harwood, C., Demain, A., editors. Bioenergy. Chapter 4. Washington, DC: ASM Press. p. 17-36. Bai, D., Li, S., Liu, Z., Cui, Z. 2007. Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate. Applied Biochemistry and Biotechnology. 144:79-85.