Hexanoic acid has been highlighted as an industrial chemical and potential biofuel precursor due to its various applications. Hexanoic acid can be synthesized through the reverse β-oxidation (r-BOX) pathway. The r-BOX pathway is of particular interest due to its redox-mediated chain elongation mechanism consisting of iteration, elongation, and termination steps. In this pathway, a short-chain carboxylate (e.g. butyrate, C4) is elongated by two-carbon units donated by electron donors, yielding hexanoic acid (HA, also known as caproic acid, C6). This study aimed to introduce an optimized r-BOX pathway into Clostridium tyrobutyricum (C. tyro) to enable the production of hexanoic acid. Genes involved in the r-BOX pathway were introduced into C. tyro, either individually or in combination, to identify the rate-limiting steps in the pathway. As a result, C. tyro strains overexpressing thiolase (thl_ck) and 3-hydroxybutyryl-CoA dehydrogenase (hbd_ck) from Clostridium kluyveri concurrently produced 1.29 g/L of hexanoic acid. In addition, several genes involved in the termination step, including CoA-transferase (cat) and thioesterase (tesA) known to play key roles in converting CoA-derivatives into their corresponding free acids, were also expressed to evaluate their effects on HA biosynthesis. These mutants were studied for their fermentation kinetics in serum bottles and bioreactors to evaluate their techno-economic feasibility.
