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

项目摘要 大麻素(CNS)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 通常从天然植物来源中提取。而在植物中发现的少量CNS含量很高 因此，研究最充分的是，还有许多其他稀有的中枢神经系统也是在植物中制造的(例如“Varins” 大麻地黄碱(CBDV)和四氢大麻碱(THCVA)。工厂生产CNS，特别是稀有的CNS和 所有CNS的酸形式，是有问题的，因为作物的变异性，低丰度，纯化挑战，以及 环境问题。因此，人们对生产常见和稀有的CNS有相当大的兴趣 通过微生物的新陈代谢工程。然而，中枢神经系统的微生物生产也面临着艰巨的挑战， 到目前为止，公布的滴度比成本竞争水平(8毫克/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罕见的CNS(和 其他天然产品)使用酶。我们的主要关注点是生产中央CN前体 大麻酸(CBGA)和大麻酸(CBGVA)，因为多种重要的大麻素 可以由CBGA/CBGVA在单一的酶促步骤中生产。此外，CBGA本身具有生物活性，并显示出 有望治疗青光眼、炎症性肠病和亨廷顿病。 非植物性生产CNS的一个关键障碍是依赖于使 CBGA/CBGVA，Geranyl：橄榄酸酯转移酶(GOT)，是一种膜蛋白。在一个重大的发展中， 我们能够设计出一种活性高、特异性强、可溶于水的GOT酶。有了这种酵素， 我们设计并实现了一个用于生产CBGA/CBGVA的酶系统，我们称之为SimplePath， 这远远超过了以电池为基础的生产参数许多个数量级。不仅SimplePath 易于商业开发，但已经可以用于在滴度和规模上制造足够大的CNS 为临床前试验提供材料。此外，SimplePath还可以用来制作各种其他CN CBGA/CBGVA以外的类似物，包括新的天然分子，具有治疗许多适应症的潜力。 我们在第二阶段奖励的行政副刊中的目标是降低成本和后勤负担 SimplePath平台，同时促进多样性并增强行政部门的领导机会 候选人。作为这项努力的一部分，我们将通过为酶的质量设定基准来简化物流 生产和实施酶的共表达。在行政副刊末尾，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，以指导试点规模的商业化工作，并确定将 需要集中精力。我们还预计，我们的行政副刊候选人将从 领导和指导活动，这将进一步支持因维辛促进多样性的努力。

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.