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。而中枢神经系统四氢大麻酚酸(THCA)，以及 大麻二酚酸(CBDA)是含量最高的，因此是研究最充分的，还有许多其他 低含量(稀有)的大麻类化合物，也存在于植物中(例如大麻酚酸(CBGA)和“Varins”) 大麻二芳酸(CBDVA)和四氢大麻呋喃二酸(THCVA)。CNS的工厂生产， 尤其是罕见的CNS，由于作物的变异性、提纯挑战和环境 担忧。因此，人们对通过代谢产生常见和罕见的CNS有相当大的兴趣 微生物工程学。然而，中枢神经系统的微生物生产也面临着艰巨的挑战，并发表了 到目前为止，效价比成本竞争水平(8毫克/L)低几个数量级。 Invizyne Technologies正在开发一种替代的、无细胞的方法来生产常见和稀有的CNS(和 其他天然产物)使用酶转化。我们的主要关注点是制作中央CN 前体CBGA和大麻香草酸(CBGVA)，因为可以产生各种重要的CNS 从CBGA/CBGVA中分离得到单酶解产物。此外，CBGA本身具有生物活性，并显示出前景 治疗青光眼、炎症性肠病和亨廷顿病等适应症。 基于细胞和无细胞生产CNS的一个关键障碍是双重的。第一，对本地人的依赖 生成CBGA的膜蛋白，香叶醇：橄榄酸转移酶(GOT)。CBGA的生物合成 需要合成两个复杂和重要的中间体，橄榄酸(OA)和香叶基焦磷酸 (GPP)。在一项重大开发中，我们能够设计出一种高活性、特异性和水溶性的GOT酶。 以该可溶GOT酶为原料，设计了一种低成本生产CBGA的7酶体系。 输入，我们称之为SimplePath。第一阶段的初步结果显示，CBGA效价远高于12g/L，超过了基于细胞的 方法对SimplePath进行几个数量级的测试，表明SimplePath适合于商业开发。 我们在此第二阶段应用程序中的目标是扩展SimplePath方法并进行必要的改进 降低成本，提高效价，拓宽生产产品的数量和类型。在第二阶段结束时 我们将对优化的SimplePath系统进行技术经济分析，以指导商业化 将在第三阶段或试点规模项目中解决的努力(并确定其他弱点)。第二阶段工程 对于建立一系列用作治疗或治疗的一致的、高纯度的中枢神经系统是必要的 营养食品的成本可以与微生物和植物来源的CN产品竞争或超过。

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.