Evaluation of acyl-CoA transferases isolated from Megasphaera species for bio-fuel production

An acyl-CoA transferase (ACT) is the enzyme, which transfers a CoA molecule of acetyl-CoA into fatty acid anion and produces acetate and acyl-CoA. Several ACTs participate the various CoA dependent pathways such as butanoate, propionate, and benzoate metabolism. In order to produce the medium-length carbon elongated products, the CoA dependent fatty acid synthesis is an important metabolic step. Thus 8 ACTs of Megasphaera sp. isolated from cow rumen were selected using genome analysis tool, CL genomics. The activities of 8 ACTs were evaluated using Escherichia coli through converting acyl-CoAs into the corresponding alcohols by an alcohol dehydrogenase. The ACTs were inserted into pGS 21a (expression vector) containing an alcohol dehydrogenase gene and expressed in gene modified Escherichia coli BL21 (DE3). After IPTG was added for induction of ACTs, fatty acids such as acetic acid, propionic acid, butyric acid, pentanoic acid, and hexanoic acid were added into medium (terrific broth) as same concentration. The strains were cultivated at 37 °C for 72 hours and the cultivation broth was sampled by 24 hour for the product analysis. The fermentative products such as alcohols and fatty acids were measured by gas chromatography equipped with flame ionized detector. The E. coli strain containing ACTs converted added fatty acids into the corresponding alcohols. The amount of consumed fatty acids and produced alcohols were compared, respectively, for the evaluation of the ACTs.