Enhancing Cannabichromenic Acid Biosynthesis in Saccharomyces cerevisiae

Cannabichromene (CBC), a valuable but extremely low-abundance component of cannabinoids in Cannabis sativa L., is known for its ability to promote neurogenesis. The scarcity of CBC in natural C. sativa is primarily attributed to the inefficiency of the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4 phosphate (DOXP/MEP) and fatty acid metabolism pathways, along with the limited competitive advantage of cannabichromenic acid synthetase (CBCAS) compared to other cannabinoid synthases. In this study, we constructed capable of biosynthesizing cannabichromenic acid (CBCA) from glucose and olivetolic acid. First, we enhanced the supply of the precursor isopentenyl diphosphate/dimethylallyl diphosphate by introducing a two-step isopentenol utilization pathway (IUP). Additionally, we increased the CBCA titer by co-overexpressing endoplasmic reticulum auxiliary protein genes. Moreover, we improved the selectivity and catalytic activity of CBCAS through rational design. By localizing the IUP to peroxisomes, geranylgeranyl pyrophosphate and CBCA titers were further increased by 1.6-fold and 28%, respectively. Notably, the yeast strain synthesized CBCA at a rate 25.8% higher than that of C. sativa. Our findings suggest that microbial synthesis offers a promising alternative to traditional C. sativa for sustainable CBCA production.