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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: Functional Expression of the Thiolase Gene thl from Clostridium beijerinckii p260 in Lactococcus lactis and Lactobacillus buchneri

Authors
item Liu, Siqing
item Bischoff, Kenneth
item Qureshi, Nasib
item Hughes, Stephen
item Rich, Joseph

Submitted to: New Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 26, 2010
Publication Date: July 23, 2010
Citation: Liu, S., Bischoff, K.M., Qureshi, N., Hughes, S.R., Rich, J.O. 2010. Functional expression of the thiolase gene THl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri. New Biotechnology. 27(4):283-288.

Interpretive Summary: In this research, we isolated a gene from a butanol producing bacterium Clostridium beijerinckii and introduced this gene into lactic acid bacteria. New and improved microbial organisms are needed to convert agricultural residues into fuels and chemicals, such as butanol. The gene was active in the lactic acid bacteria and represents the first step for converting alternative microbial hosts for butanol production. Results will be valuable to researchers who use genetic engineering to develop new microbes for biofuels.

Technical Abstract: The first step of the butanol pathway involves an acetyl-CoA acetyltransferase (ACoAAT), which controls the key branching point from acetyl-CoA to butanol. ACoAAT, also known as thiolase (EC 2.3.1.9), is encoded by the thl gene and catalyzes ligation of 2 acetyl-CoA into acetoacetyl-CoA. Bioinformatics analyses suggest there are no thl in the genomes of lactic acid bacteria (LAB), in this study we aimed to introduce the thl gene into selected LAB strains and analyze the fermentation products. The thl gene from Clostridium beijerinckii P260 was amplified by genomic PCR using gene specific primers designed from the published genome sequences of C. beijerinckii NCIMB 8025. The 1.2 kb thl gene was cloned into the pETBlue vector and overexpressed in Escherichia coli Tuner (DE3) pLacI cells. Functional enzyme activity was detected spectrophotometrically by measuring the decrease in absorbance at 303nm, which reflects the change in acetoacetyl-CoA concentrations. The thl gene was subsequently introduced into Lactococcus lactis and Lactobacillus buchneri strains, and GC analysis indicated about 28 mg/L and 66 mg/L of butanol was produced in the recombinant strains respectively. This study reports the first step toward developing a butanolgenic LAB through introduction of the butanol pathway into butanol tolerant strains of LAB.

Last Modified: 9/10/2014
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