Source: NORTHERN REGIONAL RES CENTER submitted to
MICROBIAL CATALYSTS TO PRODUCE FUEL ETHANOL AND VALUE ADDED PRODUCTS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Accession No.
0408950
Grant No.
(N/A)
Project No.
3620-41000-121-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 19, 2004
Project End Date
Sep 18, 2009
Grant Year
(N/A)
Project Director
BISCHOFF K M
Recipient Organization
NORTHERN REGIONAL RES CENTER
(N/A)
PEORIA,IL 61604
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5111510104050%
5114010110030%
5114020110220%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria; 4020 - Fungi; 1510 - Corn;

Field Of Science
1100 - Bacteriology; 1102 - Mycology; 1040 - Molecular biology;
Goals / Objectives
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.
Project Methods
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. BSL-1 and Risk Group RG1 recertified April 17, 2008.

Progress 09/19/04 to 09/18/09

Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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. Technology Transfer Number of New/Active MTAs(providing only): 9 Number of Invention Disclosures submitted: 3

Impacts
(N/A)

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. 2008. Engineered Saccharomyces cerevisiae Strain for Improved Xylose Utilization with a Three-plasmid SUMO Yeast Expression System. Plasmid Journal. 61:22-38.
  • 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.
  • Liu, S., Skinner-Nemec, K.A., Leathers, T.D. 2008. Lactobacillus buchneri strain NRRL B-30929 converts a concentrated mixture of C5 and C6 sugars into ethanol and other products. Journal of Industrial Microbiology and Biotechnology. 35:75-81.
  • Hughes, S.R., Dowd, P.F., Hector, R.E., Panavas, T., Sterner, D.E., Qureshi, N., Bischoff, K.M., Bang, S.B., Mertens, J.A., Johnson, E.T., Li, X., Jackson Jr, J.S., Caughey, R.J., Riedmuller, S.B., Bartolett, S., Liu, S., Rich, J.O., Farrelly, P.J., Butt, T.R., Labaer, J., Cotta, M.A. 2008. Lycotoxin-1 Insecticidal Peptide Optimized by Amino Acid Scanning Mutagenesis and Expressed as a Co-product in an Ethanologenix Saccharomyces cerevisiae Strain. Journal of Peptide Science. 14(9):1039- 1050. Available: http://www3.interscience.wiley.com/cgi- bin/fulltext/119030240/PDFSTART.
  • Bischoff, K.M., Skinner-Nemec, K.A., Leathers, T.D. 2007. Antimicrobial susceptibility of Lactobacillus species isolated from commercial ethanol plants. Journal of Industrial Microbiology and Biotechnology. 34(11):739- 744.


Progress 10/01/06 to 09/30/07

Outputs
Progress Report 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. 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. Accomplishments 1. Construction of proteomic workcell. One approach to improving biocatalysts for the fermentation industry is to use an automated high-throughput strategy to screen tens of thousands of candidates. Scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Research (NCAUR), 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. In FY07, construction of the workcell was completed, and the unit has been delivered and installed at NCAUR. The workcell is facilitating the development of xylose-utilizing biocatalysts, improvement of enzyme function of xylanases, and mutagenesis of insecticidal proteins. This work contributes to the Ethanol component of the NP307 Action Plan, which seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical, and process engineering. 2. Isolation and characterization of novel biocatalyst strain 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 NCAUR, have identified a novel biocatalyst strain (Lactobacillus buchneri NRRL B30929) isolated from a survey of bacterial contaminants inhabiting commercial ethanol plants. The strain is tolerant to high concentrations of ethanol, and can ferment mixed sugars, like those derived from lignocellulosic biomass, to produce ethanol, lactate, and acetate. L. bucheri NRRL B30929 has potential industrial application in bio-based refinery platforms. This work contributes to the Ethanol component of the NP307 Action Plan, which seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical, and process engineering. 3. Cloning of a cellulase gene from a thermophilic bacterium. Thermophilic bacteria are a potential source of robust enzymes for use in the fermentation industry. Scientists at NCAUR identified a thermophilic strain of Bacillus licheniformis that produced endoglucanase, an enzyme that helps degrade cellulose. The gene for this enzyme was cloned, and a recombinant form of the enzyme produced in Escherichia coli for characterization. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial application in the conversion of biomass to glucose for production of fuel ethanol or other valuable fermentation products. This work contributes to the Ethanol component of the NP307 Action Plan, which seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical, and process engineering. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 1 Number of Invention Disclosures submitted: 2 Number of Non-Peer Reviewed Presentations and Proceedings: 11 Number of Newspaper Articles,Presentations for NonScience Audiences: 7

Impacts
(N/A)

Publications

  • Bischoff, K.M., Rooney, A.P., Li, X., Liu, S., Hughes, S.R. 2006. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnology Letters. 28:1761-1765.
  • Bischoff, K.M., Liu, S., Hughes, S.R. 2007. Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361. Process Biochemistry. 42:1150-1154.
  • Hughes, S.R., Dowd, P.F., Hector, R.E., Riedmuller, S.B., Bartolett, S., Mertens, J.A., Qureshi, N., Liu, S., Bischoff, K.M., Li, X., Jackson Jr, J. S., Sterner, D., Panavas, T., Cotta, M.A., Farrelly, P.J., Butt, T. 2007. High-throughput fully automated construction of a multiplex library of mutagenized open reading frames for an insecticidal peptide using a plasmid-based functional proteomic robotic workcell with improved vacuum system. Journal of Laboratory Automation. 12(4):202-212.


Progress 10/01/05 to 09/30/06

Outputs
Progress 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. 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.

Impacts
(N/A)

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.


Progress 10/01/04 to 09/30/05

Outputs
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 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 the milestones (indicators of progress) from your Project Plan. 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 pyruvate decarboxylase (pdc) shuttle vector constructs into lactic acid bacteria. 2.3. Confirmation of transformants and examination of enzymatic activities. 2.4. Screening Geobacillus stearothermophilus strains for growth on xylan substrates. Year 3 (FY 2007) 3.1. In vitro transcription of xylose isomerase (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 Saccharomyces 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 ethanol 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. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Construction of a functional proteomic robotic workcell will be completed. Milestone Substantially Met 2. Genes for pyruvate decarboxylase from Sarcina ventriculi and Zymobacter palmae will be cloned. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 2.1. Construct library of shuffled XI genes. This library will provide a set of hybrid XI enzymes which can be screened for superior activity using in vitro transcription assays. 2.2. Transformation of pdc shuttle vector constructs into lactic acid bacteria. This will provide strains of lactic acid bacteria that will putatively produce ethanol. 2.3. Confirmation of transformants and examination of enzymatic activities. This will provide strains of lactic acid bacteria that will putatively produce ethanol. 2.4. Screening G. stearothermophilus strains for growth on xylan substrates. This information will be used to identify thermophilic bacteria for further development as biotechnological agents. FY 2007 3.1. In vitro transcription of XI library. This will identify clones of XI with superior traits for insertion into Saccharomyces to produce a xylose-utilizing and ethanol producing strain. 3.2. Construct Pichia library. This library will provide a set of genes to potentially enhance xylose utilization in ethanol-producing Saccharomyces strains. 3.3. Characterization of xylan utilization enzymes and cloning of respective genes. This will provide candidate genes and enzymes for potential use in the fermentation industry. FY 2008 4.1. Mass transformation of S. cerevisiae with Pichia library and high throughput screening. This will select for strains that utilize xylose. 4.2. Transform S. cerevisiae strains with shuffled XI clones. This will select for strains that utilize xylose. 4.3. Evaluate recombinant XI yeast strains and Pichia transformed strains for anaerobic growth on xylose and EtOH production. This will identify xylose-utilizing strains that produce ethanol. 4.4. Construct stable strains by chromosomal integration of plasmid- encoded genes from strains producing ethanol. This will result in strains of Lactobacillus that contain permanent insertion of ethanol producing genes. 4.5. Inactivation of undesired genes like als, ldh, ack, and pdh if needed. This is contingnent on results of milestone 4.4. Inactivation of these genes may be needed to increase the levels of ethanol produced from constructed strains. 4.6. Application of new biocatalysts against natural xylan substrates (corn fiber). This will identify biocatalysts from extremophiles that have superior activity against natural substrates, such as corn fiber. 4a What was the single most significant accomplishment this past year? CONSTRUCTION OF ETHANOL PRODUCING LACTOBACILLUS STRAIN. Lactic acid bacteria are a group of bacteria that survive and function well in industrial fermentation environments, and they have the potential to be developed into new biocatalysts for the efficient production of ethanol and other value-added products from agricultural feedstocks. Scientists in the BBC Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, have genetically modified a strain of Lactobacillus plantarum to express a gene encoding PDC, which is involved in the production of ethanol. Evaluation of this modified strain in flask fermentations demonstrated that the PDC enzyme was expressed in an active form and that the modified strain produced increased levels of ethanol in comparison to the parent strain. Although significant optimization of the recombinant strain remains to be explored, these results demonstrate that metabolic engineering of lactic acid bacteria is a viable strategy for the development of new biocatalysts to convert agricultural materials to biofuels. 4b List other significant accomplishments, if any. CONSTRUCTION OF PROTEOMIC WORKCELL. One approach to identifying new microbial strains that produce fuel ethanol from biomass is to employ an automated high-throughput strategy capable of screening tens of thousands of candidates. In collaboration with Hudson Control Group, Inc., Springfield, NJ, scientists in the BBC Research Unit have designed a plasmid-based proteomic robotic workcell. Protocols for the workcell were validated in a 96-well plate format by identifying clones of a cellulase enzyme with optimal activity at low pH from a library of mutagenized genes. This workcell and high-throughput strategy will ultimately be used for identifying strains of ethanologenic yeast capable of fermenting xylose. SCREENING THERMOPHILIC BACTERIA FOR CELLULOLYTIC ENZYMES. Thermophilic bacteria are a potential source of robust enzymes for use in the fermentation industry. Scientists in the BBC Research Unit have initiated a survey of thermophilic bacteria maintained within the NCAUR Culture Collection for enzymes that degrade cellulose and hemicellulose. To date, one strain of G. stearothermophilus has been identified that produces a cellulolytic enzyme. This is the first report of this type of activity from a member of this species and will be evaluated for use in industrial applications. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Methods have been developed to introduce recombinant DNA into lactic acid bacteria on plasmids for high level expression or on the chromosome of the recipient strains for stable expression. These methods are used to engineer strains that are capable of producing ethanol from xylose, one of the most abundant sugars derived from lignoceullosic biomass. 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. A patent application is in progress for the plasmid-based proteomic robotic workcell and its associated biology.

Impacts
(N/A)

Publications

  • Liu, S., Saha, B.C., Cotta, M.A. 2005. Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis. Applied Biochemistry and Biotechnology. 121-124:391-402.
  • Liu, S., Dien, B.S., Cotta, M.A. 2005. Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in lactic acid bacteria. Current Microbiology. 50:1-6.
  • Liu, S., Nichols, N.N., Dien, B.S., Cotta, M.A. 2005. Ethanol fermentation by overexpressing a Gram-positive PDC gene in Lactobacillus plantarum strain TF103 [abstract]. Biotechnology for Fuels and Chemicals Symposium Proceedings, May 1-4, 2005, Denver, Colorado. p. 158.
  • Liu, S., Dien, B.S. 2005. Metabolic engineering of Lactobacillus brevis for ethanol production [abstract]. The World Congress on Industrial Biotechnology and Bioprocessing, April 20-22, 2005, Orlando, Florida. p. 23.
  • Hughes, S.R., Li, X., Cotta, M.A., Bischoff, K.M., Riedmuller, S., Farrelly, P. 2004. A plasmid-based functional proteomic workcell used to optimize open reading frames and generate improved strains for commercial use [abstract]. 36th Great Lakes Regional American Chemical Society Symposium, October 17-20, 2004, Peoria, Illinois. Poster 361.
  • Hughes, S.R., Li, X., Cotta, M.A., Bischoff, K.M., Riedmuller, S., Farrelly, P. 2004. A plasmid-based functional proteomic workcell assay designed to screen Orpinomyces cellulase F NNY mutagenized libraries in high throughtput [abstract]. 36th Great Lakes Regional American Chemical Society Symposium, October 17-20, 2004, Peoria, Illinois. Poster 361.
  • Hughes, S.R., Li, X., Cotta, M.A., Mertens, J.A., Bischoff, K.M., Riedmuller, S., Farrelly, P., Patel, M., Brown, L., Carter, L. 2005. A plasmid-based functional proteomic workcell used to optimize yeast for fuel ethanol production [abstract]. CHI's Annual PEP Talk Meeting, January 10-14, 2005, San Diego, California. p. 3.