UCLA

Cannabinoids are a large class of bioactive natural products originally derived from the Cannabis sativa plant that regulate the cannabinoid receptors CB1 and CB2 of the human endocannabinoid system . Cannabinoid based medicines (CBMs) have shown promise as pharmacological agents, acting as antidepressants, analgesics, anticonvulsants, and antiemetics . Cannabinoids have also shown to demonstrate preliminary beneficial therapeutic effects in the treatment of cancer cells . In recent years, there has been significant interest from the synthetic biology community to produce these cannabinoids using microbial and cell-free strategies because of (i) the flexibilities in engineering the pathway to access rare or unnatural cannabinoids, (ii) the challenges associated with chemical synthesis, and (iii) the inconsistent and relatively low production of cannabinoids from plants with microbial production of naturally occurring and new cannabinoids becoming a growing, disruptive technology to the ~$10 billion global cannabis industry. However, the microbial synthesis of cannabinoids and related molecules requires access to the intermediate olivetolic acid (OA), a major bottleneck in the pathway due to its low production. Whereas plant enzymes have been expressed in E.coli and yeast biosynthesis, moderate yields and shunt product formation are major hurdles. Here in, I describe the elucidation of a non-plant biosynthetic pathway using genome mining consisting of fungal tandem polyketide synthases that produces olivetolic acid as well as the related octanoyl-primed derivative sphaerophorolcarboxylic acid (SA) in high titers using the model fungal organism Aspergillus nidulans. When compared with the plant pathway production of olivetolic acid, this biosynthetic pathway provides increased production, diversity, and selectivity. This platform can be groundbreaking for the production of cannabinoid-based medicines (CBMs). This platform has potential to not only produce common cannabinoids like Δ9-tetrahydrocannabionol ( Δ9-THC) and cannabidiol (CBD), but also rare and potentially more potent cannabinoids such as tetrahydrocannabiphorol (THCP), the heptyl alkyl chain version of Δ9-THC, due to the diversity of analog products produced. Here in, I also describe and detail the methods and results in increasing the titer of this production platform, the expansion of the diversity of products produced through the platform, and the attempt to go downstream to the final elaborated cannabinoid molecules in order to develop a de novo strain that produces a wide variety of cannabinoids. Therefore, in this work, I describe the development of an olivetolic acid platform that represents a new strategy to produce cannabinoid precursors in microbes without relying on plant enzymes, leading to higher titers and a flexible engineering pathway to access rare or unnatural cannabinoids.