Harnessing endogenous EcHpaBC for P450-Independent dihydroquercetin biosynthesis

Dihydroquercetin (DHQ), a natural flavonoid with notable pharmacological properties, has faced microbial production challenges due to the poor functional expression of heterologous P450 enzymes in prokaryotic hosts. Here, we reported a P450-independent biosynthetic pathway for DHQ in Escherichia coli using naringenin as the precursor and the endogenous flavin monooxygenase EcHpaBC from E. coli as the core hydroxylase. Screening identified a flavonoid 3-hydroxylase (CsF3H) derived from the tea plant Camellia sinensis , which efficiently converted naringenin to dihydrokaempferol (DHK). Subsequently, EcHpaBC hydroxylated DHK at the 3′ position to yield DHQ, achieving an initial titer of 7.11 ± 0.45 mg/L. Semi-rational engineering of the rate-limiting EcHpaB subunit generated the optimal variant N460M, which increased the DHQ titer by 117.3%. Systematic optimization strategies—including plasmid copy number tuning, cofactor engineering, and fermentation condition optimization—ultimately enabled the engineered strain to achieve a final DHQ titer of 79.46 ± 1.42 mg/L, representing an 11-fold improvement over the unoptimized strain, corresponding to a molar yield of 0.261 mol mol −1 . To our knowledge, this titer represents the highest DHQ production achieved in E. coli through a non-P450 hydroxylase pathway. This pathway offered advantages in genetic simplicity, operational stability, and lower fermentation cost, thereby providing a scalable prokaryotic platform for flavonoid production that bypassed the redox partner requirement and folding limitations commonly associated with P450 enzymes.