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.

项目摘要 大麻素（CNS）是生物活性天然产物，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提取的。而CNS四氢大麻酚酸（THCA）和 大麻二醇酸（CBDA）是最高的丰度，因此是研究最合理的，还有许多其他 植物（例如大麻酸（CBGA）和“ varis”）也制成的低丰富（稀有）大麻素 大麻丙酸（CBDVA）和四氢大麻酶酸（THCVA）。 CNS的植物生产， 特别是罕见的中枢神经系统，由于作物变异性，纯化挑战和环境而存在问题 关注。因此，有很大的兴趣通过代谢产生常见和稀有的中枢神经系统 微生物的工程。 CNS的微生物生产也面临着艰巨的挑战，并发表了 到目前为止，滴度较低的几个数量级低于竞争水平（8 mg/L）。 Invizyne Technologies正在开发一种替代的，无细胞的方法来生成常见和稀有的CNS（和 其他天然产物）使用酶转化。我们的主要重点是中央CN的生产 前体CBGA和大麻丙烯酸（CBGVA），因为可以产生各种重要的CNS 从单个酶促步骤中的CBGA/CBGVA。而且，CBGA本身具有生物活性，并显示出希望 青光眼，炎症性肠病和亨廷顿氏病的治疗。 CNS的基于细胞和无细胞生产的关键障碍是双重的。首先，对本地的责任 膜蛋白使CBGA，香精：橄榄酸酯转移酶（GOT）。其次，CBGA的生物合成 需要合成两个复合物和必需的中间体：橄榄油酸（OA）和焦磷酸香豆基 （GPP）。在重大发展中，我们能够设计一种高度活跃，特异性和水实心获得酶。 通过这种可溶性酶，我们设计了一个7酶系统，用于从低成本生产CBGA 输入，我们称为简单路径。初始阶段I结果产生的CBGA滴度远超过12 g/L，超过基于细胞的滴度 通过几个数量级的方法，建议简单路径适合商业开发。 我们在这二阶段应用中的目标是扩展我们的简单路径方法并进行必要的改进 为了降低成本，提高滴度并扩大生产的产品的数量和类型。在第二阶段结束时 我们将对优化的简单路径系统进行技术经济分析，以指导商业化 努力（并确定其他弱点）将在第三阶段或试点量表项目中解决。第二阶段的工作 对于建立一致，高度纯净的CNS供应的必要条件是必要的，以用作治疗或 营养物的成本可以与微生物和植物衍生的CN生产竞争或超过竞争。