Improvement of cannabichromenic acid synthase activity and construction of recombinant yeast strain

Cannabichromenic acid synthase (CBCAS) catalyzes a pivotal step in the biosynthesis of cannabichromene (CBC), and its activity level directly affects the production of CBC. In this study, a combined approach involving molecular docking and site-directed mutagenesis was employed to engineer the enzyme, with the goal of generating CBCAS variants that exhibit increased catalytic activity. The CBCAS gene was cloned from a cannabis cultivar with a high CBC content. The three-dimensional structure of the CBCAS protein was predicted using AlphaFold2. Molecular docking between the enzyme and its substrate, cannabigerolic acid (CBGA), was performed using Discovery Studio software. Mutation sites predicted to increase substrate binding affinity were identified, and the resulting recombinant plasmids were constructed and transformed into Pichia pastoris (P. pastoris) to generate engineered strains with increased CBCAS activity. Molecular docking analysis revealed that the CBCAS^Q106Y+E534R and CBCAS^Q124L+E534K double mutants exhibited increased noncovalent interactions and increased hydrophobic contact with the substrate, which likely accounted for their strengthened binding affinity for CBGA. Enzyme activity analysis revealed that compared with those of the wild type (CBCAS^WT), the enzymatic activities of CBCAS^Q106Y+E534R and CBCAS^Q124L+E534K were both significantly elevated (P < 0.05). These mutants catalyzed the conversion of CBGA to yield 93.3 ng/mL and 91.4 ng/mL CBC, representing increases of 37.5% and 34.7%, respectively, relative to CBCAS^WT. Heterologous expression of cannabis CBCAS was successfully achieved in P. pastoris, and mutant enzymes with increased catalytic activity were obtained, laying a foundation for the industrial production and application of recombinant CBCAS.