Engineering Yeast towards High Titer Production of Monoterpene Indole Alkaloid Natural Products

工程酵母用于高滴度生产单萜吲哚生物碱天然产物

• 批准号: 10427220
• 负责人: Yi Tang
• 金额: $ 38.68万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427220
• 关键词: Address; Anabolism; Biological; Biological Models; CRISPR interference; CRISPR-mediated transcriptional activation; Camptothecin; Catharanthus roseus; Chemistry; Cloning; Collaborations; Collection; Complex; Defect; Disease; Drug Metabolic Detoxication; Drug usage; Engineering; Enzymatic Biochemistry; Enzymes; Genes; Genetic Transcription; Genome; Genome engineering; Genotype; Goals; Growth; Hodgkin Disease; Indole Alkaloids; Investigation; Letters; Libraries; Maintenance; Malignant Neoplasms; Metabolic; Metabolic Biotransformation; Metabolism; Methods; Monoterpenes; Morphine; Mutation; Natural Products; Organism; Oxidation-Reduction; Partner in relationship; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Plant alkaloid; Plants; Procedures; Production; Reporter; Reporting; Saccharomyces cerevisiae; Shunt Device; Solubility; System; Technology; Therapeutic; Toxic effect; Transcriptional Regulation; Variant; Vinblastine; Work; Yeasts; base; comparative; cost; design; fitness; healthy lifestyle; improved; leukemia treatment; liquid chromatography mass spectrometry; microbial; novel strategies; overexpression; pathway tools; promoter; prophylactic; reconstitution; reconstruction; response; screening; small molecule; strictosidine; synthetic biology; tool; transcriptomics

ABSTRACT Reconstruction of plant natural product pathways in genetically well-characterized microbial organisms such as Saccharomyces cerevisiae is a sustainable and scalable method of producing high value pharmaceutical compounds. Strictosidine is the universal precursor to thousands of monoterpene indole alkaloids (MIAs) such as vinblastine and camptothecin. MIAs are indispensable pharmaceutical ingredients, but are also expensive due to difficulties in production and isolation from plant producers. In this proposal, we will use strictosidine biosynthesis as a model system to explore the use of newly developed yeast-based technologies at UCLA and Stanford Genome Technology Center (SGTC) for high-titer production of strictosidine in yeast. Our labs and others have shown that critical parts of this biosynthetic pathway are subject to considerable crosstalk with the endogenous yeast redox active enzymes, resulting in significant loss of flux toward irrecoverable shunt products. Our preliminary efforts have led to increase in product titer of the intermediate nepetalactol, and suggest a more global approach aimed at the different intermediates in the pathway will lead to significant improvements. This collaborative proposal will leverage the Tang labs expertise in natural product biosynthesis with the new synthetic biological tools developed for yeast by SGTC. This will pave the way for complete reconstitution of important MIAs in yeast, as well as elucidation of hitherto unknown MIA biosynthetic pathways involving strictosidine. Together we will address four aims: 1) Use high-throughput pathway construction to achieve improved baseline production of strictosidine; 2) establish metabolite-responsive growth screenings for strictosidine and other key biosynthetic pathway intermediates; 3) employ new genome-engineered tools to rapidly create, screen and genotype yeast strains that can achieve high level of strictosidine production starting from the improved baseline strain; and 4) heterologous production and downstream pathway exploration of complex MIAs, such as vinblastine and camptothecin, starting from strictosidine.

抽象的 在遗传特征良好的微生物中重建植物天然产物途径，例如 酿酒酵母是一种可持续且可扩展的高价值药物生产方法 化合物。马球糖苷是数千种单萜吲哚生物碱 (MIA) 的通用前体，例如 如长春花碱和喜树碱。 MIA是不可或缺的药物成分，但价格昂贵 由于生产困难以及与植物生产者的隔离。在本提案中，我们将使用胡豆素 生物合成作为模型系统，探索加州大学洛杉矶分校新开发的基于酵母的技术的使用 斯坦福基因组技术中心 (SGTC) 用于在酵母中高滴度生产胡胡豆素。我们的实验室和 其他人已经表明，该生物合成途径的关键部分受到与 内源性酵母氧化还原活性酶，导致流向不可恢复的分流产物的通量显着损失。 我们的初步努力提高了中间体荆芥内酯的产物滴度，并提出了更多的建议 针对途径中不同中间体的全球方法将带来重大改进。这 合作提案将利用唐实验室在天然产物生物合成方面的专业知识和新的合成方法 SGTC 为酵母开发的生物工具。这将为全面重建重要的 酵母中的 MIA，以及迄今为止未知的涉及胡豆苷的 MIA 生物合成途径的阐明。 我们将共同实现四个目标：1）利用高通量途径构建来实现改进的基线 胡胡苷的生产； 2) 建立胡胡苷和其他关键代谢物响应性生长筛选 生物合成途径中间体； 3）采用新的基因组工程工具来快速创建、筛选和 从改进的基线开始，可以实现高水平胡豆苷生产的基因型酵母菌株 拉紧; 4）复杂MIA的异源生产和下游途径探索，例如 长春花碱和喜树碱，以胡胡豆苷为原料。