Engineering of prenyltransferase NphB for improving the regioselective olivetolic acid and geranyl pyrophosphate

Abstract

The increasing global demand for cannabinoid-derived drugs, particularly from Cannabis sativa., necessitates efficient enzymatic synthesis methods. Cannabigerolic acid (CBGA), a crucial precursor for various cannabinoids, holds significant importance. In this context, the prenyltransferase NphB, derived from Streptomyces sp., is a focal point in cannabinoid biosynthesis. NphB is a soluble aromatic prenyltransferase from Streptomyces sp. which has been shown to be able to prenylate diverse aromatic substrates including olivetolic acid (OA) to form CBGA. However, during CBGA production by NphB, unwanted side product has been also produced, consequently reducing yield of CBGA. To enhance specificity and catalytic efficiency, I employed the computational enzyme design tool FuncLib. Through the analysis, critical residues influencing regioselectivity and activity were identified. The mutants including the residues produced CBGA over 20.4-fold increase than wild-type without side product. Our NphB mutants hold promise for producing valuable cannabinoids with improved regioselectivity and catalytic activity, thereby advancing cannabinoid-based therapeutics.