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

项目摘要 大麻素是一种具有生物活性的天然产物，具有许多当前和潜在的理论治疗用途 通常从天然植物中提取。虽然在植物中发现了高丰度的氯化萘， 此外，还有许多其他稀有氯化萘也可在植物中产生（如“varins”）， 大麻二酚，CBDV和四氢大麻二酚，THCVA）。植物生产氯化萘，特别是稀有氯化萘， 所有氯化萘的酸形式都存在问题，因为作物变异性大、丰度低、纯化困难， 环境问题。因此，生产常见氯化萘和稀有氯化萘的兴趣很大 通过微生物的代谢工程。然而，微生物生产氯化萘也面临着严峻的挑战， 并且迄今为止公布的滴度比成本竞争水平（8 mg/L）低几个数量级。 Invizyne Technologies开发了一种替代的无细胞方法来生产常见和稀有氯化萘（以及 其他天然产物）。我们的主要重点是生产中央CN前体 大麻萜酚酸（CBGA）和大麻萜伐林酸（CBGVA），因为多种重要的大麻素 可以在单一酶促步骤中由CBGA/CBGVA产生。此外，CBGA本身具有生物活性， 有望用于治疗青光眼、炎症性肠病和亨廷顿病。 氯化萘非植物生产的一个主要障碍是依赖于生成氯化萘的天然酶， CBGA/CBGVA，Geranyl：Olivetolate Transferase（GOT），一种膜蛋白。在一个重大的发展， 我们能够设计一种高活性、特异性和水溶性的GOT酶。有了这种GOT酶， 我们设计并实现了一种用于生产CBGA/CBGVA的酶系统，我们称之为SimplePath， 这远远超过基于细胞的生产参数许多数量级。SimplePath不仅 该技术易于商业开发，但已经可以用于生产氯化萘，其滴度和规模都足够大 为临床前试验提供材料。此外，SimplePath还可以用于创建各种其他CN CBGA/CBGVA以外的类似物，包括具有治疗多种适应症潜力的新分子。 我们在第二阶段合同的行政补充中的目标是减少成本和后勤负担， SimplePath平台，同时促进多样性和加强行政领导机会， 候选人作为这项工作的一部分，我们将通过设定酶质量的基准来简化物流 并实现酶的共表达。在行政补充的最后，我们 将对共表达和酶质量对成本的影响进行技术经济分析， SimplePath系统，用于指导试点规模的商业化工作，并确定其他弱点， 需要专注我们还预计，我们的行政补充候选人将大大受益于 这将进一步支持Invizyne促进多样性的努力。

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.