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 effective bioconversion of agricultural materials to fuels and other valuable products has been encumbered by the relatively small number of organisms suitable for use under harsh (industrial) environments and by the gaps in our understanding of the biosynthetic pathways that produce these products. New microorganisms and enzymes are needed that utilize lignocellulosic biomass to produce the desired products in high yield under conditions of extreme temperature, pH, osmotic pressure, and concentrations of substrate or end-products. These new biocatalysts may be developed through the application of metabolic and molecular screens to identify novel organisms and genes for exploitation. Once identified, the candidate organisms, genes, or enzymes can be evaluated, new expression systems developed, and bioconversion strategies optimized for production of biofuels and other microbial products. The development of new biocatalysts that will function in industrial environments will benefit not only commercial partners but farmers as well by providing new markets for agricultural based feedstocks. Expansion of the production of fuel ethanol, in particular, would reduce the nation's dependence on foreign oil and improve the environment by developing alternate energy sources from renewable resources. The broad goal of this research project is to develop new microorganisms and biocatalysts that can be employed in the fermentative conversion of renewable agricultural materials to fuels and other value-added products. The research entails engineering existing fermentative microorganisms to possess desirable traits for industrial fermentation of lignocellulosic material, and searching for new microorganisms that possess these traits. The specific objectives of the project are as follows: . 1)create efficient xylose-fermenting Saccharomyces cerevisiae strains;. 2)engineer lactic acid bacteria to make ethanol, and. 3)determine the potential for microorganisms from extreme environments to serve as biotechnological agents in the fermentation industry. This research is expected to increase the efficiency of conversion of biomass to liquid fuel, and to discover new uses for agricultural by-products. Therefore, portions of the research fall under National Program 307 (Bioenergy and Energy Alternatives) and under National Program 306 (Quality and Utilization of Agricultural Products). Specifically, the research contributes to Component I (Ethanol) of the NP 307 Action Plan and to Problem Area 2b (New Uses for Agricultural By-products) of the NP 306 Action Plan. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) 1.1. Construct functional proteomic robotic workcell. 1.2. Cloning pyruvate decarboxylase gene from Sarcina ventriculi and Zymobacter palmae. Year 2 (FY 2006) 2.1. Construct library of shuffled XI genes. 2.2. Transformation of pdc shuttle vector constructs into lactic acid bacteria. 2.3. Confirmation of transformants and examination of enzymatic activities. 2.4. Screening G. stearothermophilus strains for growth on xylan substrates. Year 3 (FY 2007) 3.1. In vitro transcription of XI library. 3.2. Construct Pichia library. 3.3. Characterization of xylan utilization enzymes and cloning of respective genes. Year 4 (FY 2008) 4.1. Mass transformation of S. cerevisiae with Pichia library and high throughput screening. 4.2. Transform S. cerevisiae strains with shuffled XI clones. 4.3. Evaluate recombinant XI yeast strains and Pichia transformed strains for anaerobic growth on xylose and EtOH production. 4.4. Construct stable strains by chromosomal integration of plasmid-encoded genes from strains producing ethanol. 4.5. Inactivation of undesired genes like als, ldh, ack, and pdh if needed. 4.6. Application of new biocatalysts against natural xylan substrates (corn fiber). Year 5 (FY 2009) 5.1. Integration of optimized strains to large scale fermentations. 5.2. Analyze and evaluation of strains for ethanol and other value-added products production. 5.3. Genome shuffling and screening for best combinations of genes required for ethanol. 5.4. Integration of new biocatalysts in large scale fermentations. 4a.List the single most significant research accomplishment during FY 2006. Construction of a Proteomic Workcell. The Ethanol component of the NP307 Action Plan seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical and process engineering. One approach to identifying new biocatalysts is to use an automated high-throughput strategy to screen tens of thousands of candidates for improved variants. In collaboration with Hudson Control Group, Inc., Springfield, NJ, scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois have developed a plasmid-based proteomic workcell that integrates all of the molecular, microbiological, and biochemical techniques used for the high-throughput strategy into a single robotic platform. Mechanical construction of the workcell is complete, and operational protocols have been tested using a multiplexed mutagenesis strategy of the CelF endoglucanase enzyme from the anaerobic fungus Orpinomyces PC-2. This workcell will ultimately be used for identifying important xylose-utilization genes and for improving strains of yeast that ferment xylose to ethanol. 4b.List other significant research accomplishment(s), if any. Co-expression of ethanol production genes in lactic acid bacteria. The Ethanol component of the NP307 Action Plan seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical, and process engineering. Lactic acid bacteria are well suited to industrial fermentation environments and have the potential to be developed into biocatalysts for the production of ethanol from agricultural feedstocks. Scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois have genetically modified a strain of Lactobacillus brevis to express genes required for ethanol production. Genes for pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) were successfully cloned and expressed in L. brevis, but ethanol production in recombinant strains did not increase. Significant optimization and engineering of metabolic pathways in lactic acid bacteria remains to be explored for the development of new biocatalysts to convert agricultural materials to biofuels. Purification and characterization of thermophilic cellulase. The Ethanol component of the NP307 Action Plan seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology and in chemical, biochemical, and process engineering. Thermophilic bacteria are a potential source of robust enzymes for use in the fermentation industry. Scientists in the Bioproducts and Biocatalysis Research Unit examined a collection of thermophilic bacteria for enzymes that degrade cellulose and hemicellulose. An endoglucanase enzyme from a thermophilic strain of Bacillus licheniformis was purified, identified, and characterized. The broad pH range and thermophilic properties of this enzyme may prove suitable for application in the conversion of biomass to glucose for production of fuel ethanol or other valuable fermentation products. Antimicrobial susceptibility of bacterial contaminants from fuel ethanol plants. The Ethanol component of the NP307 Action Plan emphasizes the need for technologies to reduce the cost of producing ethanol from cornstarch. Bacterial contamination of commercial fermentation cultures is a common and costly problem to the fuel ethanol industry. Scientists in the Bioproducts and Biocatalysis Research Unit have examined bacterial species isolated from a wet-mill and from a dry-grind ethanol plant for susceptibility to penicillin and virginiamycin, two antimicrobial agents commonly used to control contamination. Isolates from the dry-grind plant were less susceptible to this virginiamycin than isolates from the wet-mill plant, but most isolates had minimum inhibitory concentrations lower than the maximal application rate used for virginiamycin. This information is important to ethanol plant managers and will help guide intervention strategies that control bacterial contamination. 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. The plasmid-based proteomic workcell developed by scientists in the Bioproducts and Biocatalysis Research Unit represents an advancement in the field of laboratory automation. The robotic integration of microbiological, molecular, and biochemical techniques will facilitate the development of improved biocatalysts for converting biomass to fuel and valuable products. The workcell will also find broad application in other fields of agricultural and pharmaceutical biotechnology. 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? Information derived from this research has been disseminated to producers, industry representatives, and other scientists through the presentation of data at national and international scientific meetings. Two provisional patents covering the workcell and a third covering its associated biology have been written and will be placed in conjunction with the completion of the workcell. Presently, our collaborative partner has constructed a second integrated robotic platform that has been sold for use in the pharmaceutical industry. 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). None. Review Publications Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Bischoff, K.M., Cotta, M.A., Farrelly, P. 2005. Development of a liquid handler component for a plasmid-based functional proteomic robotic workcell. Journal of the Association for Laboratory Automation. 10(5):287-300. Liu, S., Nichols, N.N., Dien, B.S., Cotta, M.A. 2006. Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production. Journal of Industrial Microbiology and Biotechnology. 33(1):1-7. Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Li, X., Bischoff, K.M., Qureshi, N., Cotta, M.A., Farrelly, P.J. 2006. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell. Proteome Science. 4:10. Liu, S., Dien, B.S., Cotta, M.A., Bischoff, K.M., Hughes, S.R. 2005. Lactobacillus brevis: a potential biocatalyst for lignocellulosic biomass to ethanol [abstract]. Society of Industrial Microbiology. Paper #P05. Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Qureshi, N., Bischoff, K.M., Jordan, D.B., Cotta, M.A., Farrelly, P. 2005. Plasmid-based functional proteomic robotic workcell process for high-throughput screening of multiplexed libraries of mutagenized clones [abstract]. Optimization high-throughput Cultures for Bioprocessing 2005. p. 3. Hughes, S.R., Riedmuller, S., Li, X., Qureshi, N., Liu, S., Bischoff, K.M., Cotta, M.A., Farrelly, P. 2006. Mass transformation of plasmid libraries of cdna or mutagenized clone sets into yeast or bacteria using a functional proteomic robotic workcell [abstract]. PepTalk 2006. p. 10. 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. Liu, S. 2006. A simple method to generate chromosomal mutations in Lactobacillus plantarum strain TF103 to eliminate undesired fermentation products. Applied Biochemistry and Biotechnology. 129-132:854-863. Bischoff, K.M., Skinner-Nemec, K., Leathers, T.D., Hughes, S.R. 2005. Antimicrobial susceptibility of bacterial contaminants from a wet-mill ethanol plant [abstract]. Society for Industrial Microbiology. Poster P42. Bischoff, K.M., Li, X., Rooney, A.P., Liu, S., Hughes, S.R. 2005. Characterization of carboxymethylcellulase activity from Geobacillus stearothermophilus [abstract]. Society for Industrial Microbiology. Paper #P38. Bischoff, K.M., Skinner, K.A., Leathers, T.D. 2006. Antimicrobial susceptibility of lactobacillus species isolated from fuel-ethanol [abstract]. American Society for Microbiology. Poster No. O-038. Liu, S. 2006. Genetically engineered lactic acid bacteria for the production of fuels and chemicals [abstract]. 10th International Symposium on the Genetics of Industrial Microorganisms. Paper #015.