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In this study, the marine bacterium <jats:italic>Vibrio diabolicus<\/jats:italic>, characterized by its rapid growth and strong ability to utilize starch, was systematically engineered for efficient conversion of starch into acetoin. A suicide plasmid\u2010mediated 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\u2010butanediol dehydrogenase gene and polyhydroxyalkanoate synthase gene were deleted, thereby eliminating carbon flux into competing pathways for 2,3\u2010butanediol and poly\u20103\u2010hydroxybutyrate biosynthesis. Subsequently, multiple copies of the <jats:italic>budAB<\/jats:italic> operon were integrated into the chromosome to strengthen the acetoin biosynthetic route. The final engineered strain produced 13.21\u00a0g\/L of acetoin within 12\u00a0h of shake flask cultivation, reflecting a significant enhancement in production efficiency. This study presents the first successful case of metabolic engineering in <jats:italic>V. diabolicus<\/jats:italic> for direct and efficient production of acetoin from starch, highlighting its significant potential for industrial\u2010scale bioproduction.<\/jats:p>","DOI":"10.1002\/biot.70094","type":"journal-article","created":{"date-parts":[[2025,8,11]],"date-time":"2025-08-11T05:56:05Z","timestamp":1754891765000},"source":"Crossref","is-referenced-by-count":1,"title":["Chromosomal Integration of <i>budAB<\/i> Operons and Pathway Rewiring Enhance Acetoin Production From Starch in <i>Vibrio diabolicus<\/i>"],"prefix":"10.1002","volume":"20","author":[{"given":"Yuan","family":"He","sequence":"first","affiliation":[{"name":"State Key Laboratory of Green Biomanufacturing, National Energy R&amp;D Center for Biorefinery, and Beijing Key Laboratory of Green Chemicals Biomanufacturing Beijing University of Chemical Technology  Beijing People's Republic of 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