Chromosomal Integration of budAB Operons and Pathway Rewiring Enhance Acetoin Production From Starch in Vibrio diabolicus

ABSTRACT Acetoin is a key platform chemical with diverse industrial applications. In this study, the marine bacterium Vibrio diabolicus , characterized by its rapid growth and strong ability to utilize starch, was systematically engineered for efficient conversion of starch into acetoin. A suicide plasmid‐mediated homologous recombination system was first developed to investigate the roles of four endogenous amylase genes. Based on transcriptomic analysis, two strong constitutively active endogenous promoters were identified and functionally validated to enhance gene expression. To increase acetoin production, the 2,3‐butanediol dehydrogenase gene and polyhydroxyalkanoate synthase gene were deleted, thereby eliminating carbon flux into competing pathways for 2,3‐butanediol and poly‐3‐hydroxybutyrate biosynthesis. Subsequently, multiple copies of the budAB operon were integrated into the chromosome to strengthen the acetoin biosynthetic route. The final engineered strain produced 13.21 g/L of acetoin within 12 h of shake flask cultivation, reflecting a significant enhancement in production efficiency. This study presents the first successful case of metabolic engineering in V. diabolicus for direct and efficient production of acetoin from starch, highlighting its significant potential for industrial‐scale bioproduction.