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

SimplePath II：大麻素生物合成的无细胞酶平台

• 批准号: 10698019
• 负责人: Tyler P Korman
• 金额: $ 63.12万
• 依托单位: INVIZYNE TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10698019
• 关键词: Acids; Address; Anabolism; Animals; Anticonvulsants; Cannabinoids; Cell-Free System; Cells; Complex; Crowding; Development; Diphosphates; Economics; Engineering; Enzymes; Epilepsy; Evaluation; Face; Frustration; Glaucoma; Goals; Government; Huntington Disease; Industrialization; Inflammatory Bowel Diseases; L Cells; Licensing; Marketing; Medical; Membrane Proteins; Methods; Microbe; Molecular Target; Natural Extract; Natural Products; Natural Source; Nutraceutical; Pathway interactions; Persons; Phase; Plant Sources; Plants; Preclinical Testing; Production; Productivity; Publishing; Recycling; Reporting; Research; Route; Small Business Innovation Research Grant; System; Technology; Testing; Therapeutic; Therapeutic Uses; Transferase; Universities; Water; Work; analog; bioactive natural products; cannabigerolic acid; cannabinoid treatment; commercialization; cost; cost effective; design; economic evaluation; improved; innovation; interest; manufacture; manufacturing facility; metabolic engineering; microbial; novel; preclinical trial; prenyl; prenylation; therapeutic evaluation

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.

项目概要 大麻素（CN）是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 通常是从天然植物来源中提取的。而 CNs 四氢大麻酚酸 (THCA) 和 大麻二酚酸 (CBDA) 的丰度最高，因此研究最深入，还有许多其他物质 植物中产生的低丰度（稀有）大麻素（例如大麻二酚酸 (CBGA) 和“varins” 大麻二维酸（CBDVA）和四氢大麻二维酸（THCVA）。氯化萘的工厂生产， 特别是稀有的氯化萘，由于作物变异、纯化挑战和环境问题而存在问题 的担忧。因此，人们对通过代谢生产常见和稀有氯化萘产生了很大的兴趣。 微生物工程。然而，微生物生产氯化萘也面临着严峻的挑战，并且已发表 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的 CN（以及 其他天然产物）使用酶促转化。我们的主要重点是中央 CN 的生产 前体 CBGA 和大麻二酚酸 (CBGVA)，因为可以产生多种重要的 CN 通过单一酶促步骤从 CBGA/CBGVA 中获得。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等适应症。 基于细胞和无细胞生产氯化萘的主要障碍是双重的。第一，对本土的依赖 用于制造 CBGA、香叶基：橄榄叶酸转移酶 (GOT) 的膜蛋白。二、CBGA的生物合成 需要合成两种复杂且必需的中间体：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。 利用这种可溶性 GOT 酶，我们设计了 7 种酶系统，用于低成本生产 CBGA 输入，我们称之为 SimplePath。初始 I 期结果产生的 CBGA 滴度远远超过 12 g/L，超过了基于细胞的滴度 方法提高了几个数量级，表明 SimplePath 适合商业开发。 我们在此第二阶段应用程序中的目标是扩展我们的 SimplePath 方法并进行必要的改进 降低成本、提高效价并扩大生产产品的数量和类型。第二阶段结束时 我们将对优化后的SimplePath系统进行技术经济分析，指导商业化 将在第三阶段或试点项目中解决的努力（并确定其他薄弱环节）。二期工作 对于建立一系列用于治疗或治疗的氯化萘的一致、高纯度供应是必要的 营养保健品的成本可以与微生物和植物衍生的 CN 生产相竞争或超过。