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。而氯化萘四氢大麻酚酸（THCA）和 大麻二酚酸（CBDA）是丰度最高的，因此是研究最充分的，还有许多其他 也在植物中产生的低丰度（稀有）大麻素（例如大麻萜酚酸（CBGA）和“varins” 大麻二羧酸（CBDVA）和四氢大麻二羧酸（THCVA）。植物生产氯化萘， 由于作物的变异性、提纯的挑战和环境因素， 性问题因此，人们对通过代谢产生常见和罕见氯化萘的兴趣很大。 微生物工程然而，微生物生产氯化萘也面临着严峻的挑战， 到目前为止，滴度比成本竞争水平（8 mg/L）低几个数量级。 Invizyne Technologies正在开发一种替代的无细胞方法来生产常见和稀有氯化萘（以及 其它天然产物）。我们的主要重点是生产中央CN 前体CBGA和CBGVA，因为可以产生多种重要的氯化萘 从CBGA/CBGVA在单一的酶促步骤。此外，CBGA本身是生物活性的，并显示出对 治疗青光眼、炎症性肠病和亨廷顿氏病以及其他适应症。 细胞和无细胞生产氯化萘的主要障碍有两个。第一，对本土的依赖 膜蛋白以制备CBGA，香叶基：橄榄醇酸转移酶（GOT）。第二，CBGA的生物合成 需要合成两个复杂的和必需的中间体，橄榄油酸（OA）和焦磷酸香叶酯 （GPP）。在一个重大的发展，我们能够设计一个高活性，特异性和水溶性GOT酶。 利用这种可溶性GOT酶，我们设计了一个7酶系统，用于低成本生产CBGA 输入，我们称之为SimplePath。最初的第一阶段结果产生的CBGA滴度远高于12 g/L，超过了基于细胞的滴度 方法进行了几个数量级的改进，并表明SimplePath适合商业开发。 我们在第二阶段应用中的目标是扩展我们的SimplePath方法并进行必要的改进 以降低成本，提高效价，并扩大生产的产品数量和类型。在阶段II结束时 我们将对优化的SimplePath系统进行技术经济分析，以指导商业化 在第三阶段或试点规模项目中将解决的工作（并确定其他弱点）。二期工程 是确保一系列氯化萘持续、高纯度供应的必要条件，这些氯化萘可用于治疗或 这些营养保健品的成本可以与微生物和植物来源的氯化萘生产竞争或超过微生物和植物来源的氯化萘生产。

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.