Integrated cascade biocatalysis of cannabigerolic acid through cyclodextrin-mediated stabilization and prenyltransferase engineering

Cannabigerolic acid (CBGA) is a key precursor for therapeutic cannabinoids, but its sustainable production is limited by enzymatic instability and costly cofactors. Here, integrated bioprocess enabled efficient CBGA biosynthesis. Cyclodextrin-mediated stabilization enhanced in vitro CBGA yield by 7-fold to 4 mM by reducing enzymatic degradation. A lycopene-based biosensor identified rate-limiting kinases, doubling geranyl pyrophosphate supply. Combinatorial engineering of NphB, based on prior beneficial mutations yielded a variant with 8.8-fold lower K_m and 1.5-fold higher k_cat. Computational modeling clarified the mechanism. Incorporating adenosine triphosphate (ATP) regeneration system enabled 5 mM CBGA production in vitro with 0.64 mM ATP supplied, reducing cofactor demand. In vivo validation in E. coli achieved 16 µM CBGA production, demonstrating proof-of-concept for microbial production. This work demonstrates a scalable, cost-effective platform for cannabinoid precursor biosynthesis, highlighting that bringing together stabilization, pathway, enzyme, and cofactor engineering is crucial for sustainable industrial production.