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

摘要 在基因特征良好的微生物中重建植物天然产物途径，如 酿酒酵母是一种可持续、可规模化生产高价值药物的方法。 化合物。马钱子苷是数千种单萜吲哚生物碱(MIA)的通用前体。 长春花碱和喜树碱。MIA是不可或缺的药物成分，但也很昂贵 由于生产困难和与植物生产者隔离。在这项提案中，我们将使用Strectosidine 生物合成作为一个模型系统，探索加州大学洛杉矶分校和加州大学洛杉矶分校新开发的酵母技术的使用 斯坦福大学基因组技术中心(SGTC)在酵母中高效生产斯特拉托西丁。我们的实验室和 其他研究表明，这种生物合成途径的关键部分与 内源酵母氧化还原活性酶，导致流向不可回收的分流产物的通量显著损失。 我们的初步努力提高了中间体奈派拉内醇的产品效价，并建议采用更多的 针对该途径中不同中间体的全球方法将导致重大改进。这 合作提案将利用唐实验室在天然产品生物合成方面的专业知识，通过新的合成 SGTC为酵母菌开发的生物工具。这将为完全重组重要的 酵母中的MIA，以及迄今未知的涉及Strectosidine的MIA生物合成途径的阐明。 我们将共同实现四个目标：1)使用高吞吐量通道建设以实现更好的基线 2)建立代谢产物响应型生长筛选法 生物合成途径中间体；3)使用新的基因组工程工具快速创建、筛选和 从改进的基线开始，对能够实现高水平生产的酵母菌株进行基因分型 菌株；4)复杂MIA的异源生产和下游途径探索，如 长春花碱和喜树碱，从Strictosidine开始。

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.