biostudies-literatureLiu YFundamental Research Funds for the Central Universitiesthe National Natural Science Foundation of ChinaBeijing Natural Science Foundation117https://www.ebi.ac.uk/biostudies/studies/S-EPMC12645685biostudies-literatureUnknown18(1)2-Hydroxy-3-methylvalerate (HMV), a kind of bioactive aliphatic 2-hydroxy acids, is a building block and a key intermediate for pharmaceuticals, including beauvericin, yet its microbial synthesis remains largely unexplored. Here, we present a synthetic biology strategy for high-titer production of HMV that couples mixed-sugar utilization with a division-of-labor microbial consortium. Co-feeding glucose and xylose synchronized substrate uptake with product formation, eliminating intermediate overflow and rerouting carbon from by-products into efficient HMV biosynthesis. To reduce the metabolic burden of the host, the HMV pathway is divided into two engineered strains: one optimized for glucose-to-intermediate conversion, the other for xylose-to-HMV completion. The best consortium, KMV-G-X, Biotechnology for biofuels and bioproductsCo-utilization of glucose and xylose in synthetic Escherichia coli consortia for efficient 2-hydroxy-3-methylvalerate biosynthesis.PMC12645685buctrc202341, JD2537225201722578021, 22308018Liu YWang SSu HfalseCo-utilization of glucose and xylose in synthetic Escherichia coli consortia for efficient 2-hydroxy-3-methylvalerate biosynthesis.2-Hydroxy-3-methylvalerate (HMV), a kind of bioactive aliphatic 2-hydroxy acids, is a building block and a key intermediate for pharmaceuticals, including beauvericin, yet its microbial synthesis remains largely unexplored. Here, we present a synthetic biology strategy for high-titer production of HMV that couples mixed-sugar utilization with a division-of-labor microbial consortium. Co-feeding glucose and xylose synchronized substrate uptake with product formation, eliminating intermediate overflow and rerouting carbon from by-products into efficient HMV biosynthesis. To reduce the metabolic burden of the host, the HMV pathway is divided into two engineered strains: one optimized for glucose-to-intermediate conversion, the other for xylose-to-HMV completion. The best consortium, KMV-G-X, 2025-01-01T00:00:00Z2025 Nov2026-06-05T19:12:46.863Z2026-05-20T03:14:49.066ZS-EPMC126456854129187410.1186/s13068-025-02716-4