De novo biosynthesis of cannabinoids and their analogs in Yarrowia lipolytica

<i>Cannabis sativa</i> has long been a cornerstone of both medicinal and cultural practices, with its therapeutic use spanning over 2700 years. Central to its therapeutic effects are cannabinoids, which interact with the endocannabinoid system to influence various physiological processes such as anxiety, pain, and inflammation. Despite its benefits, cannabinoid production faces challenges and scarcity from plant extraction. This work leverages <i>Yarrowia lipolytica</i> as a platform for cannabinoid biosynthesis. By optimizing the precursor supply, engineering biomolecular condensate-like dual prenyltransferase expression and expanding endogenous metabolism with a noncanonical polyketide synthase, we achieved the <i>de novo</i> biosynthesis of various cannabinoids and their analogs. Our engineered <i>Y. lipolytica</i> produced ∼3.5 ​mg/L cannabigerolic acid, 18.8 ​mg/L orsellinic acid, and 0.5 mg/L cannabigerorcinic acid. Additionally, the CBGA titer reached 15.7 ​mg/L with olivetolic acid supplementation. This work demonstrates the versatility of <i>Y. lipolytica</i> as a promising host for the production of cannabinoids and their analogs, which opens avenues for further research and medicinal applications.