LAUSR

The microbial production of medium‐chain carboxylates has attracted considerable interest owing to their potential applications in biofuels and specialty chemicals; however, the underlying biosynthetic mechanisms remain incompletely understood. The present study evaluates the medium‐chain carboxylate‐producing microbe Megaspahera hexanoica using genomic analysis, transcriptome analysis, and metabolic engineering. Additionally, the n‐caproate synthesis pathway of M. hexanoica is characterized with fructose as an electron donor, and the substrate specificity of the respective proteins is evaluated by constructing an n‐caproate biosynthetic pathway in Escherichia coli. Among all r‐BOX or RBO genes, thl_1583, which encodes β‐ketothiolase (MhTHL), is identified as the most critical enzyme for the carbon chain elongation mechanism in M. hexanoica. Therefore, MhTHL is compared with other well‐studied β‐ketothiolases (CkTHL from Clostridium kluyveri, ReBktB from Ralstonia eutropha (Cupriavidus necator), EcAtoB from E. coli, and CaTHL from C. acetobutylicum). MhTHL is found to exhibit the highest n‐caproate production, as evidenced by the protein crystal structure of MhTHL. Structural comparisons with other thiolases show that MhTHL has a larger substrate‐binding pocket than ReBktB. Thiolase mutants generated by site‐directed mutagenesis reveal that two residues (Leu87 and Val351) are essential for determining the size of the substrate‐binding pocket.