Functional expression of the thiolase gene thl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri.
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 two 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 303 nm, 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 the introduction of the butanol pathway into butanol-tolerant strains of LAB.
Liu
Siqing
S
Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture(3), 1815 N. University St., Peoria, IL 61604, USA. Siqing.liu@ars.usda.gov
Bischoff
Kenneth M
KM
Qureshi
Nasib
N
Hughes
Steven R
SR
Rich
Joseph O
JO
D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D046970:Clostridium beijerinckii / Q000201:enzymology*; D017124:Conserved Sequence / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007778:Lactobacillus / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity
D001426:Bacterial Proteins; D000440:Butanols; D011994:Recombinant Proteins; D000101:Acetyl-CoA C-Acetyltransferase