2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The objective of the project is to. 1)determine the metabolic, physiologic, and genetic fundamentals underlying stress tolerance of ethanologenic yeast strains and other microbes, and. 2)to use this fundamental knowledge to engineer improved strains and/or 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. Many microorganisms have been found via screens of diverse populations or have been designed via genetic engineering to have industrially useful functions, such as improved substrate range, improved or novel products, improved product yields and rates, and others. However, many of the industrially interesting microorganisms obtained thus far are not robust enough for low-cost commercial application to processes, such as the production of ethanol from lignocellulosic farm residues and energy crops. In this instance, more stress-tolerant microorganisms are needed that are able to withstand, survive, and function in the presence of stress factors common to fermentations of lignocellulose hydrolysates, 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. The new strains, technologies, and fundamental knowledge gained from this research are key to the design of a cost-effective, highly productive microbial bioprocess for converting lignocellulosic biomass to ethanol. Such processes have the potential to add tens to hundreds of billions of gallons of ethanol to our country's renewable energy supply as new energy crops are developed. This program is important to strengthening the security of our nation by lessening dependence on foreign sources of fuel, preserving our environment and natural resources, and boosting our economy (especially in rural America). It specifically addresses the fermentative conversion of sugars from renewable biomass to ethanol, a process that will be required to surpass the nation's $7.5 billion gallon ethanol/year production goal set for 2012. Under National Program 307, Bioenergy and Energy Alternatives, this research is expected to contribute to methods to increase the efficiency and significantly reduce the costs of conversion of biomass to liquid fuel and feedstock chemicals through the development of microbial catalysts capable of surviving a variety of industrial stresses which currently limit performance. Under National Program 306, Quality and Utilization of Agricultural Products, the research will contribute to new uses for agricultural products and byproducts through the development of the knowledge base and novel techniques needed for design and application of commercially viable microbial processes for bioconversion of agricultural products to new bioproducts. Research to determine how cells survive such stresses and how to engineer their survival will provide the knowledge to allow us great flexibility and power in the design of cost-effective, highly productive microbial bioprocesses for the next decade. Success with this project will be far reaching and have impact on improving the commercialization prospects of many bioproducts produced from agricultural crops and residues by microbial catalysis, such as feedstock chemicals, specialty products such as thickeners, sweeteners, as well as bioactive products, such as antibiotics and biological control inocula to control agricultural pests, including diseases, insects, and weeds. 2.List by year the currently approved milestones (indicators of research progress) Objective 1 - Determine genetic fundamentals of stress tolerance: Year 2004-2005 (FY 05) - 1.A.1. Identify phenotype differences in tolerance--screen natural ethanologenic yeasts. - 1.A.4. Identify phenotype differences in tolerance--screen Coniochaeta ligniaria cDNA library. Year 2005-2006 (FY 06) - 1.A.2. Identify phenotype differences in tolerance--develop adapted strains. - 1.A.3. Identify phenotype differences in tolerance--screen Saccharomyces cerevisiae disruption library. - 1.A.5. Identify phenotype differences in tolerance--screen cultivation environment. - 1.B.1. Identify genes/regulatory networks/function--develop quality-controlled arrays. Year 2006-2007 (FY 07) - 1.B.4. Identify genes/regulatory networks/function--characterize cDNA library stress genes. Year 2005-2008 (FY 08) - 1.B.2. Identify genes/regulatory networks/function--genomic expression of stress tolerance. Year 2006-2008 (FY 08) - 1.B.3. Identify genes/regulatory networks/function--verify disruption library stress genes. - 1.B.5. Identify genes/regulatory networks/function--characterize protective gene function. - 1.B.6. Identify genes/regulatory networks/function--generate tolerance-specific gene array. Objective 2 - Engineer improved strains/processes: Year 2007-2009 (FY 09) - 2.A. Engineer commercial yeast strains. Year 2008-2009 (FY 09) - 2.B. Optimize bioreactor design. 4a.List the single most significant research accomplishment during FY 2006. First demonstration of furfural's effects on yeast cellular physiology (NP 307 Bioenergy and Energy Alternatives, Component 1 - Ethanol; NP 306 Quality and Utilization of Agricultural Materials, Component 2 - New Processes, New Uses, and Value Added Foods and Biobased Products): Furfural is one of the strongest inhibitors produced during the dilute acid hydrolysis of lignocellulosic biomass, the most economical process currently applied to release fermentable sugars for subsequent conversion to ethanol. During our research, yeast cells were exposed to furfural concentrations between 25 and 50 mM which caused severe stress based on several cellular assays. Furfural induced the accumulation of free radicals, which are known to cause severe DNA, protein, and membrane damage. Consequently, mitochondria and vacuole membrane damage was observed as well as nuclear chromatin damage. At 25 mM furfural, the cells were able to recover by 24 hours as indicated by a resumption of growth, decreased free radical accumulation, and a restoration of intracellular membranes. This coincided with the cell's ability to detoxify furfural after 24 hours. At 50 mM furfural the cells were unable to resume growth, decrease free radical accumulation, restore intracellular membranes, or detoxify furfural. These findings were the first demonstrating furfural's effects on yeast cellular physiology. By providing basic information about how cells are damaged by furfural, this work is key to the successful engineering of yeast and fermentation processes to prevent and repair damage to cells, thereby allowing more efficient and cost-effective conversion of biomass substrates to ethanol. This work was presented at the Biotechnology for Fuels and Chemicals Symposium and is being submitted to Eukaryotic Cell. 4b.List other significant research accomplishment(s), if any. Identification of genes involved in tolerance to 5-hydroxymethylfurfural stress in bioethanol conversion (NP 307 Bioenergy and Energy Alternatives, Component 1 - Ethanol; NP 306 Quality and Utilization of Agricultural Materials, Component 2 - New Processes, New Uses, and Value Added Foods and Biobased Products): 5-Hydroxymethylfurfural (HMF) is one of major inhibitory compounds derived from dehydration of hexoses during biomass degradation using dilute acid hydrolysis. It inhibits yeast growth, reduces enzymatic activities, breaks down DNA and represses protein and RNA synthesis. We examined global transcriptome profiles of ethanologenic yeast S. cerevisiae and identified 440 genes potentially responsible for the HMF stress response. We further presented a concept of genomic adaptation to further improve tolerant strain development which was accepted for publication in Applied Microbiology and Biotechnology in 2006. We classified these preliminary identified genes into eight subsets by co-expression patterns over time using self organizing map method. Each subset of genes was mined for statistically over-represented DNA binding motifs in the promoter regions. Association between a transcription factor and a gene cluster was statistically tested using previously catalogued targets of the transcription factor. Searching for potential regulators, we found numerous significant relationships, among which, PDR3 and GCR1 appeared to be convincing regulators of significantly induced genes under the HMF stress. By providing basic information about what gene networks and regulators are involved in the yeast stress response to HMF, this work is key to the successful engineering of yeast and fermentation processes to resist damage by HMF, thereby allowing more efficient and cost-effective conversion of biomass substrates to ethanol. An abstract of this work was submitted and will be presented in an international meeting in September 2006. Demonstration that nitrogen-source composition of culture media reduces specific death rate of cells inhibited by furfural or ethanol (NP 307 Bioenergy and Energy Alternatives, Component 1 - Ethanol; NP 306 Quality and Utilization of Agricultural Materials, Component 2 - New Processes, New Uses, and Value Added Foods and Biobased Products): More stress tolerant microorganisms are needed that are able to withstand, survive, and function in the presence of stress factors common to fermentations of lignocellulose hydrolysates, including various chemical fermentation inhibitors (furans, phenolics, organic acids etc.) and high ethanol concentration. In previous studies (Slininger et al., 2005), the striking influence of nitrogen source and mineral composition on the achievement of ethanol yields up to 70 g/L from media supplied high xylose concentrations (150 g/L) was noted for the natural pentose-fermenting yeast P. stiptis. Nitrogen source composition was further investigated to study its influence on the ability of P. stipitis to survive in the presence of concentrations of furfural (100-150 mM/L) and ethanol (70-100 g/L) that were high enough to halt cell growth when spiked to cultures in logarithmic growth and early stationary phases. When spiked with ethanol, the observed specific cell death rate of cells was greater for cultures in stationary phase compared with those in logarithmic growth phase; but when spiked with furfural, the death rate was somewhat less for cultures in stationary phase compared with those in logarithmic growth phase. The inclusion of amino acids in addition to urea in the culture medium significantly reduced specific death rate in the presence of furfural or ethanol. This finding impacts the development of efficient fermentation processes that foster inhibitor tolerance and allow ethanol production from both the hexose and pentose sugars released by dilute acid hydrolysis of low-cost lignocellulosic biomass. Such process improvements support the expansion of the biofuels industry. Continued development of adapted yeast strains for studies of stress tolerance mechanisms (NP 307 Bioenergy and Energy Alternatives, Component 1 - Ethanol; NP 306 Quality and Utilization of Agricultural Materials, Component 2 - New Processes, New Uses, and Value Added Foods and Biobased Products): Biomass pretreatment using economic dilute acid hydrolysis generates furfural and 5-hydroxymethylfurfural (HMF) which inhibit yeast growth and fermentation. Remediation of the inhibitors increases cost and wastes for bioethanol production. We aim to develop tolerant strains that withstand inhibitors furfural and HMF, and produce ethanol without additional detoxification procedures. Using functional genomics approaches, we investigated transcriptome and metabolic profiling for ethanologenic yeast under the inhibitor challenged conditions. Based on previous development, we further improved strain tolerance to both furfural and HMF, each individually or in combination, under simulated laboratory conditions. These strains showed significantly enhanced biotransformation to convert furfural into furfural alcohol and HMF into 2,5-bis-hydroxymethylfuran (furan-2,5-di-methanol, FDM) and produce a normal yield of ethanol. The transformation dynamics of furfural and HMF was completed and ethanol produced in 48 hours for a tolerant strain. In contrast, a normal control strain was unable to grow and establish a culture in the presence of the inhibitors at 48 hours. Our study suggests that it is possible to in situ detoxify inhibitors generated by economic dilute acid hydrolysis pretreatment for cost-competitive bioethanol conversion. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. Furfural and hydroxymethylfurfural (HMF) are key toxic byproducts of the dilute acid hydrolysis of lignocellulosic biomass, the most economical method of releasing sugars for fermentation to ethanol biofuel. Currently, the lack of yeasts able to tolerate these toxic byproducts is a significant factor limiting commercial-scale biomass to ethanol conversion in the United States. As a result of our research, we showed that certain natural strains were better able to tolerate the presence of furfural and HMF than others. Our research has shown that natural strains of the yeasts Saccharomyces cerevisiae and Pichia stipitis can survive and adapt to the presence of furfural and HMF. We have also shown that exposure to gradually increasing levels of each inhibitor can lead to the development of strains able to tolerate relatively high levels of HMF and furfural (30 mM). Fermentation analysis of adapted strains has revealed that these strains are more efficient than their parent strains in reducing these inhibitors to their corresponding less toxic alcohol, i.e., furfural to furfuryl alcohol and HMF to 2,5-bis-hydroxymethylfuran, suggesting the role of in situ detoxification in the inhibitor-tolerant strains. Our description of the adaptive response of natural yeasts allowing the tolerance of furan inhibitors via a detoxification mechanism provides important groundwork and guidelines for the further development of industrial yeasts capable of in situ detoxification of HMF and furfural as a means of alleviating these stress factors in commercial dilute acid hydrolysates of lignocellulosic biomass. Our results show that it is possible for adapted yeast strains to in situ detoxify inhibitors generated by economic dilute acid hydrolysis pretreatment for cost-competitive bioethanol conversion. This body of work also suggests that an understanding of adaptation mechanisms can be utilized to either engineer new strains or to design inoculum production and fermentation process conditions to enhance and sustain tolerance during growth and ethanol fermentation. (These findings contribute to NP 307 Bioenergy and Energy Alternatives, Component 1-Ethanol; NP 306 Quality and Utilization of Agricultural Products, Component 2 – New Processes, New Uses, and Value Added Foods and Biobased Products.) Screening a S. cerevisiae disruption library identified 40 genes involved in tolerance of 8-10% ethanol and 65 genes involved in tolerance of 15 mM furfural. Over-expression of a subset of these genes enhances S. cerevisiae’s tolerance to furfural. The identified gene mutants implicated several pathways in ethanol tolerance, including macromolecule modification and biosynthesis, organelle dynamics, plasma membrane and cell wall maintenance, fermentation, cell cycle, endocytosis, metabolite biosynthesis, and membrane transport mechanisms. Genes influencing furfural tolerance have been verified to be involved in pathways including transcription, translation, metabolite biosynthesis, cytoskeletal dynamics, and organelle function (vacuole, mitochondria, and peroxisome). Notably, our work provides the first evidence that the pentose phosphate pathway (long known for converting xylose to ethanol) plays a critical role in protecting yeast against furfural stress perhaps via generation of nicotinamide adenine dinucleotide phosphate (NADPH), a cofactor that is necessary for protein, nucleic acid, and lipid biosynthesis, and for protection from oxidative stress. (These findings contribute to NP 307 Bioenergy and Energy Alternatives, Component 1-Ethanol; NP 306 Quality and Utilization of Agricultural Products, Component 2 – New Processes, New Uses, and Value Added Foods and Biobased Products.) Furfural's effects on yeast cellular physiology were demonstrated for the first time. Furfural was found to induce the accumulation of free radicals, which are known to cause severe DNA, protein, and membrane damage. Consequently, mitochondria and vacuole membrane damage were observed as well as nuclear chromatin damage. Given low concentrations of furfural (25 mM), the cells were able to recover by 24 hours as indicated by a resumption of growth, decreased free radical accumulation, and a restoration of intracellular membranes. This coincided with the cell's ability to detoxify furfural after 24 hours. At high concentrations of furfural (50 mM), the cells were unable to resume growth, decrease free radical accumulation, restore intracellular membranes, or detoxify furfural. By providing basic information about how cells are damaged by furfural, this work is key to the successful engineering of yeast and fermentation processes to prevent and repair damage to cells, thereby allowing more efficient and cost-effective conversion of biomass substrates to ethanol. (These findings contribute to NP 307 Bioenergy and Energy Alternatives, Component 1-Ethanol; NP 306 Quality and Utilization of Agricultural Products, Component 2 – New Processes, New Uses, and Value Added Foods and Biobased Products.) Microarray studies comparing HMF-treated wild-type and adapted strain cultures have shown that tolerant strains have distinct expression profiles of selected genes compared with that of the parent strain. Genes in all categories of biological process, cellular component, and molecular function were involved; some were HMF-specific while others could be associated with a core set of stress genes, such as those belonging to the pleiotropic drug resistance gene family. Our development of a unique quality-controlled gene microarray for ethanologenic yeast study has now allowed these phenotype differences to be exploited to track down key genes and gene systems for planned genomics studies that will elucidate stress tolerance mechanisms and how they are regulated and networked. For example, we have identified the pleiotropic drug resistance gene family as having a significant role in HMF stress tolerance. We examined global transcriptome profiles of ethanologenic yeast S. cerevisiae and identified 440 genes potentially responsible for the HMF stress response. We classified these preliminarily identified genes into eight subsets by co-expression patterns over time using self-organizing map method. Each subset of genes was mined for statistically over-represented DNA binding motifs in the promoter regions. Association between a transcription factor and a gene cluster was statistically tested using previously catalogued targets of the transcription factor. Searching for potential regulators, we found numerous significant relationships, which revealed two genes that appeared to be convincing regulators of significantly induced genes under the HMF stress. By providing basic information about what gene networks and regulators are involved in the yeast stress response to HMF, this work is key to the successful engineering of yeast and fermentation processes to resist damage by HMF. Genomics data will help to provide a genetic blueprint that will let us develop and deploy viable strategies for engineering industrial yeast strains and processes that foster inhibitor tolerance and enhance the profitability of biomass to ethanol conversion. (These findings contribute to NP 307 Bioenergy and Energy Alternatives, Component 1-Ethanol; NP 306 Quality and Utilization of Agricultural Products, Component 2 – New Processes, New Uses, and Value Added Foods and Biobased Products.) Our optimization of culture nutritional factors has revealed another lead to follow in our investigation of inhibitor stress tolerance mechanisms and maintenance of yeast cell viability during fermentation. The striking influence of nitrogen source and mineral composition on the achievement of ethanol yields up to 70 g/L from media-supplied high-xylose concentrations (150 g/L) was noted for the natural pentose-fermenting yeast P. stiptis. Nitrogen source composition was further investigated and found to influence the ability of P. stipitis to survive in the presence of concentrations of furfural (100-150 mM/L) and ethanol (70-100 g/L) that were high enough to halt cell growth when spiked to cultures in logarithmic growth and early stationary phases. The inclusion of amino acids in addition to urea in the culture medium significantly improved ethanol yields and resistance of cells to furfural and ethanol. This finding impacts the development of efficient fermentation processes that foster inhibitor tolerance and allow rapid ethanol production from both the hexose and pentose sugars released by dilute acid hydrolysis of low-cost lignocellulosic biomass. Such process improvements support the expansion of the biofuels industry. (These findings contribute to NP 307 Bioenergy and Energy Alternatives, Component 1-Ethanol; NP 306 Quality and Utilization of Agricultural Products, Component 2 – New Processes, New Uses, and Value Added Foods and Biobased Products.) 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Our findings on stress-tolerant genes and pathways have been transferred to the scientific community through peer-reviewed journal articles, book chapters, abstracts, and presentations at relevant scientific meetings. This early dissemination of our results has already proven fruitful at stimulating interest in our research and establishing future collaborations. We anticipate that as new technologies are developed, the public will be informed by patents and popular articles. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Slininger, P.J. Engineering stress-tolerant microbes for lower-cost production of biofuels and bioproducts. Presented to researchers in the Department of Applied Microbiology, Lund University, Lund, Sweden, May 26, 2006. Review Publications Slininger, P.J., Liu, Z., Gorsich, S.W. 2005. Engineering stress-tolerant microbes for lower cost production of biofuels and bioproducts [abstract]. American Institute of Chemical Engineers Annual Meeting. Paper No. 303a. Gorsich, S.W., Slininger, P.J., Liu, Z. 2005. Physiological responses to furfural and HMF and the link to other stress pathways [abstract]. European Congress on Biotechnology. Paper No. G.4.4. Liu, Z., Slininger, P.J. 2006. In situ detoxification of fermentation inhibitors by stress tolerant ethanologenic yeast for low-cost biomass conversion to ethanol [abstract]. World Bio-Energy 2006 Proceedings. p. 121. Slininger, P.J., Dien, B.S., Gorsich, S.W., Liu, Z. 2006. Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124. Applied Microbiology and Biotechnology. 72(6):1285-1296. Gorsich, S.W., Slininger, P.J., McCaffery, J. 2006. The fermentation inhibitor furfural causes cellular damage to Saccharomyces cerevisiae [abstract]. Biotechnology for Fuels and Chemicals Symposium Proceedings. Paper No. 4-17. Liu, Z., Members of ERCC. 2005. Proposed methods for testing and selecting the ERCC external RNA controls. Biomed Central (BMC) Genomics. 6:150. Gorsich, S.W., Dien, B.S., Nichols, N.N., Slininger, P.J., Liu, Z., Skory, C.D. 2005. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPEL, and TKL1 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 71(3):339-349. Gorsich, S.W. 2005. Novel genes that provide increased stress tolerance in Saccharomyces cerevisiae [abstract]. Southern Great Lakes Local Section of the Society for Industrial Microbiology. Paper No. 2. Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Li, X., Bischoff, K.M., Liu, S., Qureshi, N., Cotta, M.A., Skory, C.D., Gorsich, S.W., Farrelly, P.J. 2006. Functional proteomic plasmid-based integrated workcell for high-throughput transformation of BL21 DE3 E. coli for expression in vivo with piromyces strain xylose isomerase [abstract]. Midwest Laboratory Robotics Information Group. p. 2.