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Title: METABOLIC ENGINEERING OF A LACTOBACILLUS PLANTARUM DOUBLE LDH KNOCKOUT STRAIN FOR ENHANCED ETHANOL PRODUCTION

Author
item Liu, Siqing
item Nichols, Nancy
item Dien, Bruce
item Cotta, Michael

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2005
Publication Date: 5/20/2005
Citation: 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.

Interpretive Summary: Commercial ethanol fermentation biotechnology is currently based on a relatively small number of microorganisms that are limited to fermenting hexose sugar derived from rather expensive corn starch. A cost-effective process for fuel ethanol production will depend on economic conversion of abundant and renewable lignocellulosic biomass to ethanol and other chemicals. There are no known naturally occurring microorganisms that can directly ferment agricultural biomass efficiently into ethanol. Therefore, it is important to search or engineer industrially robust strains that can ferment mixtures of sugars derived from renewable biomass to ethanol. In this study, a robust food-grade organism was engineered for the production of ethanol. The recombinant strain grew slightly faster than the parent and produced 90-158 mM ethanol. Results will be valuable to researchers designing improved biocatalysts for ethanol production.

Technical Abstract: Lactobacillus plantarum ferments glucose through the Embden-Meyerhof-Parnas pathway, the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A pyruvate decarboxylase gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into a LDH deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent and produced 90-158 mM ethanol. The simultaneous insertion of pdc and inactivation of ldh by direct gene replacement could be an alternative approach to facilitate growth and increase ethanol production by lactic acid bacteria.