SimplePath II: A Cell-free Enzymatic Platform for Cannabinoid Biosynthesis

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Project Summary Cannabinoids (CNs) are bioactive natural products with many current and potential theoretical therapeutic uses that are generally extracted from natural plant sources. While a few CNs are found at high abundance in plants and therefore the most well studied, there are many other rare CNs that are also made in plants (e.g. the “varins” cannabidivarin, CBDV, and tetrahydrocannabivarin, THCVA). Plant production of CNs, particularly rare CNs and the acid forms of all CNs, is problematic because of crop variability, low abundance, purification challenges, and environmental concerns. Consequently, there is considerable interest in producing both common and rare CNs by metabolic engineering of microbes. Microbial production of CNs also faces daunting challenges, however, and published titers so far are several orders of magnitude below cost competitive levels (8 mg/L). Invizyne Technologies has developed an alternative cell-free method to produce common and rare CNs (and other natural products) using enzymes. Our primary focus is production of the central CN precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA), because a variety of important cannabinoids can be produced from CBGA/CBGVA in single enzymatic steps. Moreover, CBGA itself is bioactive and shows promise for treatment of glaucoma, inflammatory bowel disease, and Huntington’s disease. A key barrier to non-plant based production of CNs has been reliance on the native enzyme that makes CBGA/CBGVA, Geranyl:Olivetolate Transferase (GOT), which is a membrane protein. In a major development, we were able to design a highly active, specific, and water soluble GOT enzyme. With this GOT enzyme in hand, we designed and implemented an enzyme system for the production of CBGA/CBGVA, that we call SimplePath, that far exceeds cell-based production parameters by many orders of magnitude. Not only is SimplePath tractable for commercial development, but it can already be used to make CNs at titers and scale large enough to provide material for preclinical testing. Additionally, SimplePath can be used to make a variety of other CN analogs beyond CBGA/CBGVA including new to nature molecules with potential to treat a number of indications. Our goal in this Administrative Supplement to our Phase II award is to reduce costs and logistical burdens for the SimplePath platform while promoting diversity and enhancing leadership opportunities for the Administrative Candidate. As part of this effort, we will streamline logistics by setting benchmarks for the quality of enzymes produced as well as implementing co-expression of enzymes. At the end of the Administrative Supplement, we will perform techno-economic analysis on the effect of co-expression and enzyme quality on the costs of the SimplePath system to guide pilot scale commercialization efforts and identify additional weak points that will require focus. We also anticipate that our Administrative Supplement candidate will benefit significantly from leadership and mentoring activities which will further support efforts at Invizyne to promote diversity.