Establishing a coumarin production platform by protein and metabolic engineering

Coumarins are a vast family of natural products with diverse biological activities. Cinnamyl-CoA ortho-hydroxylases (CCHs) catalyze the gateway and rate-limiting steps in coumarin biosynthesis. However, engineering CCHs is challenging due to the large size of the substrates and the vague structure-activity relationships. Herein, directed evolution and structure-guided engineering are performed to engineer a CCH (AtF6'H from Arabidopsis thaliana) using a fluorescence-based screening method, yielding the transplantable surface mutations and substrate-specific pocket mutations with improved activity. Structural analysis and molecular dynamics simulations elucidate the conformational changes that lead to increased catalytic efficiency. Applying appropriate variants with the optimized upstream biosynthetic pathways improves the titer of three simple coumarins by 5-22-fold. Further introducing glycosylation modules results in the production of four coumarin glucosides, among which the titer of aesculin increased by 15.7-fold and reached 3 g/L in scale-up fermentation. This work unleashed the potential of CCHs and established an Escherichia coli platform for coumarins production.