17-45: Substrate specificity to carbon elongation of a thiolase from Megasphaera species

A thiolase is the enzyme coupling two acyl CoA molecules (acetyl CoA, propionyl CoA, and butyryl CoA, etc.) and thus is related to the carbon elongation for the synthesis of fatty acids. In this study, the enzymatic activity and substrate specificity of a thioase annotated from the genome of Megasphara strain producing pentanoic acid and hexanoic acid was investigated. The thiolase is thought to be a key enzyme related to the carbon elongation for the production of carboxylic acids, such as pentanoic acid and hexanoic acid. First of all, codons of the thiolase were optimized. And then, the thioase was expressed to Escherichia coli BL21 (DE3) and purified by Ni-NTA column. The expressed and purified thiolase was analyzed using SDS page and Bradford protein assay. The basic activity of the thiolase was confirmed by colorimetric reaction using acetoacetyl CoA as a reverse reaction. In order to evaluate the substrate specificity of the purified thiolase, various CoAs (acetyl CoA, propionyl CoA, and Butyryl CoA) were used for the condensation reaction by the thiolase and the metabolites were analyzed by HPLC/DAD and HPLC/MS/MS. The substrate specificity of each thiolase from Escherichia coli, Clostridium tyrobutyricum, and Ralstonia eutroph, respectively, was compared with the thiolase from Megasphaera sp. The thiolase from Megasphaera sp. showed broader substrate specificity and better activity for butyl CoA.