Co-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, produces 2184.6 ± 111.8 mg/L HMV, comprising 82.2% of the total 2-hydroxy acids produced. Compared to mono-culture using glucose as a single substrate, this consortium exhibits less catabolic interference and enhanced HMV biosynthesis efficiency. This study shows that substrate utilization and pathway division of labor in synthetic consortium convert mixed sugars into high-value chemicals, boosting titer and robustness for scalable green production.