2009 Annual Report 1a.Objectives (from AD-416) The broad goal of the proposed research 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 or searching for new microorganisms that possess these traits. 1b.Approach (from AD-416) Novel microorganisms, genes, and enzymes will be sought that can be employed in the fermentative conversion of agricultural commodities into biofuels and bioproducts. Specific approaches include: (1) application of high throughput screening procedures to develop, by use of directed evolution and gene shuffling, microbial strains and enzymes with superior ability to convert agricultural materials to biofuels and bioproducts, (2) apply metabolic engineering and genetic manipulation methods to existing and newly discovered/developed microbial strains to improve on ability to perform in an industrial fermentation environment and to optimize production of desired products, and (3) determine the potential for use of microorganisms from extreme environments (e.g., extreme thermophiles, halophiles, acido/alkalophiles) as biotechnological agents. 3.Progress Report This report documents accomplishments for the research project 3620-41000-121-00D, entitled "Microbial Catalysts to Produce Fuel Ethanol and Value Added Products." Researchers are working 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. In FY 2009, ARS scientists have isolated new strains of microorganisms that may find application in the fermentation industry, including a thermophilic Bacillus strain for producing lactic acid from agricultural residues and a strain of Lactobacillus that secretes a unique antibacterial compound. Progress was made toward improving the xylose utilization of Saccharomyces cerevisiae, which will facilitate development of industrial yeast strains that make ethanol from lignocellulosic feedstocks. In ancillary work, new methods were developed to characterize bacterial contaminants that infect commercial fuel ethanol facilities. These methods may be used to develop new intervention methods to treat contamination. Progress achieved during FY 2009 has potential scientific impact for academic and government researchers and for industry representatives, and will facilitate development of efficient biocatalysts that lower the production costs of fuels and chemicals from lignocellulosic biomass. 4.Accomplishments 1. IMPROVEMENT OF XYLOSE UTILIZATION IN SACCHAROMYCES CEREVISIAE. The development of efficient microorganisms for producing fuels from lignocellulosic biomass requires improvements in the ability of yeasts to utilize the sugar xylose. Scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois screened a library of genes for improvement of xylose utilization in a strain of Saccharomyces cerevisiae that co-expressed the enzyme xylose isomerase. Eight genes were identified that improved anaerobic growth on xylose, and the products of five other genes were found to bind the xylose isomerase protein. S. cerevisiae strains that co-expressed these genes grew better anaerobically on xylose and produced more ethanol than background strains. This information has potential impact in the biofuels industry for the development of industrial yeast strains for biofuel production from agricultural residues. 2. A NEW STRAIN OF LACTOBACILLUS PARACASEI PRODUCES AN ANTIBACTERIAL PEPTIDE. 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 at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois have found that a bacterial strain of Lactobacillus paracasei produces a novel antibacterial compound (a peptide). The antibacterial peptide has a broad spectrum of activity against bacteria that contaminate ethanol plants and against some disease-causing bacteria. The peptide has potential application in the biofuels industry to treat infections of ethanol fermentations, and in the food industry to control contamination by food-borne pathogens. 3. ISOLATION OF A THERMOPHILIC MICROBIAL CATALYST FOR PRODUCTION OF LACTIC ACID. Improved microbial strains are needed to convert lignocellulosic biomass to fuels and chemicals. Scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois isolated a thermophilic xylose-fermenting bacterium from dairy manure compost. The strain used all the sugars available in hydrolysates of corn fiber and switchgrass to produce L lactate, a valuable chemical used in foods, cosmetics, and bio-based plastics. The strain has potential industrial application in bio-based refinery platforms that produce value-added bioproducts from agricultural commodities. 4. RAPID METHOD TO IDENTIFY BIOFILMS. New and improved methods are needed to rapidly identify microbial strains that produce biofilms and lead to contamination problems during commercial fuel ethanol production. Scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois have developed a rapid method to test bacteria for the capacity to form biofilms. The test was used to evaluate bacteria isolated as contaminants from commercial fuel ethanol facilities. This work has potential impact in the biofuels industry for development of new methods and approaches to control contamination of fuel ethanol fermentations. 6.Technology Transfer Number of the New/Active MTAs (providing only) 9 Number of Invention Disclosures Submitted 3 Review Publications Hughes, S.R., Sterner, D.E., Bischoff, K.M., Hector, R.E., Dowd, P.F., Qureshi, N., Bang, S.S., Grynavyski, N., Chakrabarty, T., Johnson, E.T., Dien, B.S., Mertens, J.A., Caughey, R.J., Liu, S., Butt, T.R., Labaer, J., Cotta, M.A., Rich, J.O. 2009. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system. Plasmid Journal. 61(1):22-38. Rich, J.O., Budde, C.L., Mcconeghey, L.D., Cotterill, I.C., Mozhaev, V.V., Singh, S.B., Goetz, M.A., Zhao, A., Michels, P.C., Khmelnitsky, Y.L. 2009. Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone. Bioorganic and Medicinal Chemistry Letters. 19:3059-3062. Bischoff, K.M., Wicklow, D.T., Jordan, D.B., De Rezende, S.T., Liu, S., Hughes, S.R., Rich, J.O. 2009. Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae. Current Microbiology. 58:499-503. Bischoff, K.M., Liu, S., Leathers, T.D., Worthington, R.E., Rich, J.O. 2009. Modeling bacterial contamination of fuel ethanol fermentation. Biotechnology and Bioengineering. 103(1):117-122. Liu, S., Bischoff, K.M., Hughes, S.R., Leathers, T.D., Price, N.P., Qureshi, N., Rich, J.O. 2009. Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri. Letters of Applied Microbiology. 48(3):337-342.