Biosynthesis of cannabinoid precursor olivetolic acid by overcoming rate-limiting steps in genetically engineered Yarrowia lipolytica

Natural products acting on our central nervous systems are in utmost demand to fight against pain and mental disorders. Cannabinoids (CBDs) are proven neuroactive agents to treat anxiety, depression, chronic pain diseases, seizure, strokes and neurological disorders. The scarcity of the hemp-sourced CBD products and the prohibitive manufacturing cost limit the wide application of CBDs. Yeast metabolic engineering offers the flexibility to meet the ever-increasing market demand. In this work, we took a retrosynthetic approach and sequentially identified the rate-limiting steps to improve the biosynthesis of the CBD precursor olivetolic acid (OLA) in Yarrowia lipolytica. We debottlenecked the critical enzymatic steps to overcome the supply of hexanoyl-CoA, malonyl-CoA, acetyl-CoA, NADPH and ATPs to redirect carbon flux toward OLA. Implementation of these strategies led to an 83-fold increase in OLA titer in shaking flask experiment. This work may serve as a baseline for engineering CBD biosynthesis in oleaginous yeast species.