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 the CNs tetrahydrocannabinolic acid (THCA), and cannabidiolic acid (CBDA) are the highest abundance and therefore the most well studied, there are many other low abundant (rare) cannabinoids that are also made in plants (e.g. cannabigerolic acid (CBGA) and the “varins” cannabidivarinic acid (CBDVA) and tetrahydrocannabivarinic acid (THCVA). Plant production of CNs, particularly rare CNs, is problematic because of crop variability, 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 is developing an alternative, cell-free method to produce common and rare CNs (and other natural products) using enzymatic transformations. Our primary focus is production of the central CN precursors CBGA and cannabigerovarinic acid (CBGVA), because a variety of important CNs 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 among other indications. A key barrier to cell-based and cell-free production of CNs has been twofold. First, reliance on the native membrane protein to make CBGA, Geranyl:Olivetolate Transferase (GOT). Second, biosynthesis of CBGA requires synthesis of two complex and essential intermediates, olivetolic acid (OA) and geranyl pyrophosphate (GPP). In a major development, we were able to design a highly active, specific and water soluble GOT enzyme. With this soluble GOT enzyme, we designed a 7 enzyme system for the production of CBGA from low cost inputs, that we call SimplePath. Initial Phase I results yielded CBGA titers well over 12 g/L, exceeding cell-based methods by several orders of magnitude and suggesting SimplePath is suitable for commercial development. Our goal in this Phase II application is to expand our SimplePath approach and make necessary improvements to lower costs, improve titers, and broaden the number and type of products produced. At the end of Phase II we will perform techno-economic analysis on the optimized SimplePath system to guide commercialization efforts (and identify other weak points) that will be addressed in a Phase III or pilot scale project. Phase II work is necessary for establishing a consistent, highly pure supply of a range of CNs to be used as therapeutics or nutraceuticals at costs that can compete with or surpass both microbial and plant derived CN production.