Metabolic engineering of Clostridium tyrobutyricum for hexanoic acid and hexanol production

n-Hexanoic acid and n-hexanol, a six-carbon linear carboxylic acid and its corresponding alcohol, are important building blocks for pharmaceuticals, cosmetics, fuels, and food additives. Microbial biosynthesis offers a sustainable alternative to petroleum-based production, yet yields are often constrained by product toxicity and limited pathway efficiency. In this study, we engineered Clostridium tyrobutyricum, a strong butyrate producer previously optimized for high-titer butanol production, for efficient C6 acid and alcohol formation through a CoA-dependent reverse β-oxidation pathway. Key enzymes from multiple microorganisms were systematically assessed for their ability to extend the carbon chain from butyryl-CoA. Among them, thiolase from Ruminococcaceae bacterium CPB6 (Thl_CPB6) was identified as the essential catalyst driving condensation of acetyl-CoA and butyryl-CoA to form C6-acyl-CoA intermediates. Co-expression of Thl_CPB6 with native 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), butyryl-CoA dehydrogenase (Bcd), and butyryl-CoA:acetate CoA-transferase (Cat1) enabled de novo hexanoate synthesis, while replacing Cat1 with the CPB6 homolog further enhanced hexanoic acid titers to 4.4 g/L, which, to our knowledge, is the highest reported in an engineered recombinant strain. Furthermore, introducing Thl_CPB6 into a butanol-producing engineered C. tyrobutyricum strain led to the formation of up to 91 mg/L hexanol. Collectively, these results establish C. tyrobutyricum as a robust microbial chassis for renewable production of medium-chain acids and alcohols via CoA-dependent reverse β-oxidation, paving the way toward sustainable biosynthesis of value-added chemicals from lignocellulosic bioresources.