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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

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 Hughes, Stephen
item Qureshi, Nasib
item Rich, Joseph

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2010
Publication Date: July 1, 2010
Citation: Liu, S., Bischoff, K.M., Hughes, S.R., Qureshi, N., Rich, J.O. 2010. Functional expression of the Thiolase gene THL from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri [abstract]. Genetics of Industrial Microorganisms. p. 36.

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: 9/2/2014
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