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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL CATALYSTS TO PRODUCE FUEL ETHANOL AND VALUE ADDED PRODUCTS Title: Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis

Authors
item LIU, SIQING
item DIEN, BRUCE
item NICHOLS, NANCY
item BISCHOFF, KENNETH
item HUGHES, STEPHEN
item COTTA, MICHAEL

Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 18, 2007
Publication Date: July 25, 2007
Citation: Liu, S., Dien, B.S., Nichols, N.N., Bischoff, K.M., Hughes, S.R., Cotta, M.A. 2007. Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis. FEMS Microbiolology Letters. 274(2):291-297.

Interpretive Summary: New biocatalysts and new microorganisms are needed for economic conversion of abundant and renewable lignocellulosic feedstock to bio-fuels and bio-chemicals. The food-grade Gram-positive lactic acid bacteria are stress tolerant and capable of growing under industrial fermentation conditions. This group of bacteria has great potential in converting lignocellulosic biomass derived sugars (including xylose and glucose) to biofuel and value-added products. In this study, a unique strain of lactic acid bacteria that can grow on xylose was examined and engineered. The results of this study will be valuable to researchers to develop improved robust biocatalysts for enhanced ethanol production from sugars derived from lignocellulosic biomass.

Technical Abstract: Lactobacillus brevis ATCC 367 is able to metabolize xylose into lactate and acetate but not ethanol. In an attempt to transform L. brevis into an ethanologen that uses xylose, a Gram-positive gene for pyruvate decarboxylase (PDC) was introduced. This enzyme catalyzes the decarboxylation of pyruvate to acetyaldehyde, which can then be converted to ethanol by an endogenous alcohol dehydrogenase (ADH). The engineered L. brevis strain bbc03 carrying a Sarcina ventriculi PDC gene (Svpdc) produced a recombinant polypeptide corresponding to the expected SvPDC, and this was confirmed by Western blot analyses using specific antiserum raised against a SvPDC derived oligo-peptide. However, fermentation analyses indicated that Svpdc alone was not enough to create a de novo ethanol pathway in L. brevis bbc03. An alcohol dehydrogenase gene from L. brevis (Bradh) was cloned behind the Svpdc gene to generate pTRKH2 769SvpdcBradh. Functionality of this plasmid construct was confirmed by its ability to restore anaerobic growth of E. coli NZN111 (a fermentative defective mutant strain that is incapable to grow under anaerobic conditions). The recombinant E. coli NZN111 produced detectable ethanol in the anaerobic culture medium. When pTRKH2 769SvpdcBradh was introduced into L. brevis to create L. brevis bbc04, two recombinant proteins of approximately 58 kDa (SvPDC) and 28 kDa (BrADH) were observed. However, flask fermentation indicated that only lactate and acetate were produced from xylose in L. brevis bbc04, suggesting that pyruvate is not converted to ethanol by SvPDC and BrADH in L. brevis bbc04. This could be due to competition for pyruvate by endogenous lactate dehydrogenases.

Last Modified: 9/10/2014
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