Metagenomic mining and functional reconstitution of hexanoic acid biosynthetic enzymes from Baijiu pit bottom

Hexanoic acid is a key flavor compound in Baijiu and a valuable platform chemical, yet its efficient biosynthesis remains challenging. To overcome the carbon chain elongation bottleneck in the reverse β-oxidation (RBO) pathway in Saccharomyces cerevisiae, this study explored the metagenomic resource of a hexanoic acid-producing microbial community from the bottom of Jiang-flavor Baijiu pit. Three enriched hexanoic acid-producing consortia were obtained, all co-producing butanoic, hexanoic, and octanoic acids. Metagenomic analysis showed that while microbial compositions differed, the consortia shared similar core functional enzymes. Further correlation analysis identified 3-hydroxyacyl-CoA dehydrogenase (HBD) and acetate CoA-transferase (CAT) as key enzymes positively correlated with hexanoic acid synthesis. These enzymes were then expressed in an engineered RBO-pathway yeast strain. Expression of CAT-1 increased butanoic and hexanoic acid production by 25% and 34%, respectively, marking the first application of CAT in enhancing chain elongation in yeast. In summary, this study innovatively identified the key enzyme CAT from the microbiota of Jiang-flavor Baijiu pit bottom and achieved efficient hexanoic acid synthesis through heterologous expression. This work not only offers a strategy for mining high-performance enzymes but also provides theoretical support and technical references for the efficient biosynthesis of hexanoic acid.