2005 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? What 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 $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 the milestones (indicators of progress) from your Project Plan. 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.What was the single most significant accomplishment this past year? Development of furan tolerant S. cerevisiae and Pichia stipitis strains with enhanced ability to convert 10 mM furfural and 5-hydroxymethylfurfural (HMF) combined to less toxic forms: A single most significant accomplishment successfully completed in FY 2005 was development of more tolerant strains of Saccharomyces cerevisiae and Pichia stipitis with enhanced ability to convert furfural and 5-hydroxymethylfurfural (HMF) into less toxic compounds using our directed adaptation method. This work was published in a book chapter and in a refereed journal in 2005. In the past, we were successful in developing tolerant strains to a single inhibitor of furfural or HMF at relatively higher concentrations. Due to the synergistic effect of combined inhibitors, development of strain tolerance to multiple inhibitors even at low concentrations has been more difficult. This year, we successfully developed strains able to tolerate combinations of both furfural and HMF in addition to single inhibitors. These improved strains grow normally in the presence of both furfural and HMF and produce normal ethanol yields. The inhibitor tolerance and enhanced inhibitor detoxification ability of these strains indicate the potential application of such adapted strains for efficient ethanol production from renewable lignocellulose. Currently, our improved strains of S. cerevisiae and P. stipitis tolerate and transform without lag time inhibitor concentrations typically found in biomass dilute acid hydrolyzates (10 mM each of both furfural and HMF). These strains are stable. Continued efforts will be made in the year 2006 to increase the strain tolerance levels to the combined inhibitors. 4b.List other significant accomplishments, if any. Identification of pentose phosphate pathway genes that influence furfural and 5-HMF tolerance: We identified 62 potential target genes involved in furfural tolerance by screening the S. cerevisiae disruption library. We had hypothesized that we could obtain target genes through this screen that when overexpressed would provide a growth advantage to S. cerevisiae in the presence of furfural. Of the 62 mutants, we initially focused on a group of pentose phosphate pathway mutants. Yeast cells lacking zwf1, gnd1, rpe1, or tkl1 failed to grow at furfural concentrations as low as 5-20 mM. Moreover, these mutants were also sensitive to another ferementation inhibitor, 5-HMF. As proof of principle of our hypothesis, we showed that by overexpressing ZWF1 a growth advantage was observed for S. cerevisiae in otherwise lethal furfural concentrations. We now propose that the inability of these mutants to survive in the presence of relatively low concentrations of furfural is linked to a lower abundance of reducing equivalents and also to a role in physiological stress protection. This study demonstrated a strong relationship between pentose phosphate genes and furfural tolerance and provided additional putative target genes to study as we begin to engineer inhibitor-tolerant yeast strains for efficient ethanol production from lignocellulose hydrolyzates. Development of a quality-controlled gene microarray for ethanologenic yeast study: In order to develop a quality microarray with high levels of reproducibility and reliability, we developed six external RNA controls this year and fabricated a yeast gene chip microarray with three replications on one slide. We also created a novel component of PreScan mini arrays to incorporate into the conventional microarray design. It provides a reference to adjust laser scanner voltage for both dye channels and avoids multiple scanning of the entire array and thereafter to reserve the quality and fidelity of microarray data acquisition. It serves as an unbiased reference for data normalization. This new design contributes to the microarray technology development. Furthermore, we developed the external RNA controls as a standard universal control for gene expression analysis across different platforms including microarray and real time quantitative RT-PCR. Previously there was no control of this nature available. This accomplishment constitutes an important scientific advancement of the methodology of gene expression analysis that will first benefit the development of stress tolerant microbes for production of low cost biofuels and bioproducts. This work was published in a book chapter and conference proceedings and will be presented in 2005. In 2006, we will further perfect the method and its applications. Identification of the pleiotropic drug resistance gene family having a significant role in HMF stress tolerance: During the course of study on genomic expression in response to inhibitors involved in biomass conversion, we identified and first reported that a group of genes belonging to the pleiotropic drug resistance gene family played a significant role to cope with HMF stress involved in biomass conversion. These genes function to transport ATP-binding cassette proteins and are encoded for plasma membrane proteins. Many PDR genes mediate membrane translocation of ions and have a wide range of substrates. It appeared that expression of multiple numbers of the PDR gene family are necessary for cell survival and detoxification of the biomass fermentation inhibitors such as furfural and HMF. This work was presented and abstract published in the conference proceedings. In 2006, work will be continued to carry out for further illustration of these gene functions. Discovery of furan tolerance genes from C. ligniaria--a natural strain able to grow on furfural and HMF--that can provide S. cerevisiae the ability to grow in previously lethal concentrations of furfural: In efforts to engineer yeast strains that are tolerant to various fermentation inhibitors, we have looked beyond S. cerevisiae's genome for help. A wild growing fungus, C. ligniaria, has been identified that grows on furfural and HMF as its sole carbon source. We have attempted to find genes from C. ligniaria that, when placed in S. cerevisiae, will confer a growth advantage to S. cerevisiae when furfural or HMF is present. Currently, we have screened over 10,000 genes from C. ligniaria and have identified at least 3 genes that do indeed appear to allow S. cerevisiae to grow in otherwise lethal concentrations of furfural. This finding is a promising step toward engineering inhibitor tolerant yeast strains capable of lower-cost conversion of biomass sugars to bioproducts such as ethanol for biofuels. Optimization of a culture medium for kinetic evaluation of stress tolerant strains and design of nutritional and environmental conditions to foster the expression of tolerance during ethanol fermentation of sugars from lignocellulose: Pichia stipitis NRRL Y-7124 is a natural yeast able to convert xylose to ethanol, a capability not possessed by traditional Saccharomyces strains used to produce ethanol from corn starch. To investigate nutrition-based strategies for improving xylose to ethanol conversion by P. stipitis, kinetic studies were conducted on cultures provided a defined medium which was varied in nitrogen, vitamin, mineral, and purine/pyrimidine content. Mineral and nitrogen source optimization significantly enhanced D-xylose conversion to ethanol by the yeast P. stipitis. Certain amino acids, especially proline, arginine and histidine significantly improved both ethanol and cell yields. Results further showed that ethanol production from 150 g/L xylose was significantly enhanced by optimizing the combination of urea and amino acids to supply 50-80% nitrogen from urea and 50-20% from casamino acids. When either urea or casamino acids was used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/L, respectively, from the maximum of 53 g/L for the optimal nitrogen combination. Magnesium, iron, and zinc sulfates significantly improved ethanol production supported by casamino acids, urea, or their combination, while manganese chloride reduced it. The striking influence of nitrogen source and mineral composition on ethanol yield will be further investigated to study impact on yeast cell viability and inhibitor tolerance mechanisms. Efficient fermentation processes that foster inhibitor tolerance and allow ethanol production from both hexose and pentose sugars released by dilute acid hydroysis of low-cost lignocellulosic biomass are sought to support the expansion of the biofuels industry. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated February 6, 2002, as a result of the FY 2002 Appropriations Bill passed by Congress and signed by the President for research on Stress-Tolerant Microbes for Lower Cost Production of Bioenergy and Bioproducts. Research began after the staffing of two permanent SY positions in 2003 as a five-year plan was envisioned, and efforts were augmented with the staffing of two permanent technician positions in February 2004. The new research project plan developed has been certified by the Office of Scientific Quality Review as having completed NP 307 Bioenergy and Energy Alternatives Panel Review, and is continuing under the new project 3620-41000-123-00D entitled "Genomics and Engineering of Stress-Tolerant Microbes for Lower Cost Production of Biofuels and Bioproducts," which was implemented 8/18/04. Since 2002, significant research accomplishments have been made. Furfural and 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, and that these strains were able to convert these compounds to other apparently less toxic forms. We observed that adapted strains were better able to convert furfural into furfuryl alcohol via a pathway previously documented in the literature. In collaboration with Dr. Bruce Dien (NCAUR, Fermentation Biotechnology Research Unit) and Dr. Mark Berhow (NCAUR, New Crops Research Unit), Crop Bioprotection Research Unit scientists led the charge to isolate the HMF conversion product from yeast culture supernatant, purify the compound, and identify it as 2,5-bis-hydroxymethylfuran. Although previous literature reports have speculated on structure, our research provided the first rigorous chemical identification of the HMF conversion product. Description of the adaptive response allowing tolerance of the furans and the identification of the HMF conversion end product of 2,5-bis-hydroxymethylfuran 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. Building on these findings, we developed a directed adaptation method and applied it to efficiently enrich for HMF- and furfural-tolerant strains of S. cerevisiae (a traditional hexose-fermenting yeast) and P. stipitis (a natural pentose/hexose-fermenting yeast) able to grow and produce ethanol normally in the presence of 30 mM furfural and HMF, and showing enhanced biotransformation ability to reduce furfural into furfuryl alcohol and HMF into 2,5-bis-hydroxymethylfuran. Continued efforts using directed adaptation have yielded S. cerevisiae and P. stipitis strains able to tolerate 10 mM furfural and HMF in combination despite their lethal synergism. Such adapted strains have enhanced ability to convert these inhibitors to less toxic forms that do not interfere with the production of ethanol from sugars released by low-cost dilute acid hydrolysis of lignocellulose. To further explore natural furan and ethanol tolerance mechanisms, an S. cerevisiae disruption library was screened to identify mutants that failed to grow or grew better than wild type in the presence of furfural or ethanol. Over 100 disruption mutants were identified that grew better than wild type and another 100 that failed to grow in the presence of 15 mM/L furfural. Furthermore, this screen identified 40 disruption mutants that grew better and 55 that grew slower or not at all compared to wild type in 8 or 10% ethanol. 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 NADPH, a cofactor that is necessary for protein, nucleic acid, and lipid biosynthesis, and for protection from oxidative stress. Once again, members of our NCAUR Crop Bioprotection research team combined forces with Fermentation Biotechnology scientists to discover furan tolerance genes from C. ligniaria--a natural strain able to grow on furfural and HMF--that can provide S. cerevisiae the ability to grow in previously lethal concentrations of furfural. This finding is a promising step toward engineering inhibitor tolerant yeast strains capable of lower-cost conversion of biomass sugars to bioproducts such as ethanol for biofuels. 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 ethanol yield in the fermentation of high xylose concentrations to ethanol by the natural pentose-fermenting strain P. stipitis. The design of culture nutritional and environmental conditions is expected to be key in fostering stress tolerance and rapid fermentation rates needed for efficient conversion of biomass-derived sugars to ethanol and other bioproducts. Thus, according to our proposed plan, phenotype differences in natural and mutant yeast strains were demonstrated when inhibitor stress factors were applied. 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. 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. 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). Liu, Z.L. What's your top advice for designing a microarray analysis experiment to ensure that your statistical analysis, control for error, and normalization procedures will give you an accurate end answer? Genome Technology November/December 2004:38. Gorsich, S.W. The physiological damage in yeast caused by furfural and the hunt for furfural tolerant genes in S. cerevisiae and other organisms. Department of Chemical Engineering at Lund University, Lund Sweden, August 25, 2005. Liu, Z., Duboy, R.T., Press, C., Loper, J.E., Paulsen, I.T., Slininger, P.J. A new DNA oligo microarray for complete genome of Pseudomonas fluorescens pf-5. International Union of Microbiological Societies (IUMS 2005), San Francisco, CA, July 13-28, 2005. Liu, Z.L., Slininger, P.J. Universal external RNA controls for gene expression analysis using microbial gene microarray and real time quantitative RT-PCR. 8th International Meeting of the Microarray Gene Expression Data Society, Bergen, Norway, September 11-13, 2005. Gorsich, S.W., Slininger, P.J., Liu, Z.L. Physiological responses to furfural and HMF and the link to other stress pathways. 12th European Congress on Biotechnology, Copenhagen, Denmark, August 21-24, 2005. Review Publications Gorsich, S.W., Dien, B.S., Nichols, N.N., Slininger, P.J., Liu, Z. 2004. The Saccharomyces cerevisiae pentose phosphate pathway gene, rpe1, functions in furfural tolerance during fermentation [abstract]. Proceedings of the 11th International Congress on Yeasts in Science and Biotechnology. Paper No. PM24. Liu, Z., Slininger, P.J. 2005. Induction of pleiotropic drug resistance gene expression indicates important roles of pdr to cope with furfural and 5-hydroxymethylfurfural stress in ethanologenic yeast [abstract]. Biotechnology for Fuels and Chemicals Symposium Proceedings. Abstract No. 169. Liu, Z., Slininger, P.J., Gorsich, S.W. 2005. Enhanced biotransformation of furfural and 5-hydroxymethylfurfural by newly developed ethanologenic yeast strains. Applied Biochemistry and Biotechnology. 121-124: 451-460. Gorsich, S.W., Liu, Z., Slininger, P.J. 2005. The role of the pentose phosphate pathway in fermentation inhibitor tolerance. Biotechnology for Fuels and Chemicals Symposium Proceedings. Abstract No. 5-33. Liu, Z., Slininger, P.J. 2005. Transcriptome dynamics of ethanologenic yeast in response to 5-hydroxymethylfurfural stress related to biomass conversion to ethanol [abstract]. Abstract Book. p. 991. Liu, Z. 2005. Genomic adaptive response of yeast to biofuel fermentation inhibitors [abstract]. Society of Industrial Microbiology Annual Meeting. Abstract No. S37. Slininger, P.J., Dien, B.S., Gorsich, S.W., Liu, Z. 2005. Mineral and nitrogen source optimization enhance d-xylose conversion to ethanol by the yeast Pichia stipitis [abstract]. Society of Industrial Microbiology Annual Meeting. Paper No. P07. Gorsich, S.W. 2005. Stress tolerance in Saccharomyces cerevisiae cells overexpressing furfural-stress genes [abstract]. Gordon Research Conference Proceedings. Abstract No. 19. Liu, Z., Slininger, P.J. 2005. Development of genetically engineered stress tolerant ethanologenic yeasts using integrated functional genomics for effective biomass conversion to ethanol. In: Collins, K., Duffield, J., Outlaw, J., editors. Agriculture as a Producer and Consumer of Energy. Wallingford, UK: CAB International, p. 283-294.