Work

Engineering the substrate-binding pocket of olivetol synthase to suppress derailment byproducts in olivetolic acid biosynthesis in Escherichia coli

Year: 2026

Type: article

Abstract: Phytocannabinoids are high-value meroterpenoids with significant therapeutic potential; however, their microbial production is currently hindered by the low efficiency and poor selectivity of key biosynthetic enzymes. Olivetol synthase (OLS), a type III polyketide synthase (PKS), catalyzes the formation of a linear tetraketide-CoA intermediate, which is subsequently cyclized by olivetolic acid cyclase (OAC) to generate olivetolic acid (OA)—the universal precursor for cannabinoids. Nevertheless, heterologous expression of OLS in Escherichia coli often leads to the premature release of triketide intermediates, resulting in the predominant accumulation of the derailment byproduct, pentyl diacetic acid lactone (PDAL). In this study, we employed a structure-guided enzyme engineering strategy to enhance the catalytic performance and product selectivity of OLS. Through comparative structural analysis with the highly efficient homolog OLS4, combined with in silico cavity volume assessment, hotspot prediction, and molecular docking, we identified key residues within the active-site pocket that govern intermediate stabilization. Notably, our optimized OLS variant, M187V/S332A, achieved a 3.9-fold increase in OA productivity (1.6 mg/L) compared to wild-type OLS (0.4 mg/L), while simultaneously reducing PDAL byproduct formation by 37.0%. These results suggest that precise modulation of the catalytic pocket architecture can effectively redirect metabolic flux away from derailment pathways and toward the desired product. Overall, this study provides a structural and mechanistic framework for engineering OLS and potentially other type III PKS enzymes, establishing an efficient platform for the microbial biosynthesis of cannabinoid precursors.

Source: Journal of Biological Engineering

DOI: 10.1186/s13036-026-00760-8