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

摘要 在遗传上良好表征的微生物有机体中重建植物天然产物途径， 酿酒酵母是生产高价值药物的可持续和可扩展的方法 化合物.阿托西啶是数千种单萜吲哚生物碱（MIA）的通用前体， 如长春碱和喜树碱。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。在本提案中，我们将使用strictosidine 生物合成作为一个模型系统，探索使用新开发的酵母为基础的技术在加州大学洛杉矶分校， 斯坦福大学基因组技术中心（SGTC），用于在酵母中高滴度生产strictosidine。我们的实验室和 其他人已经表明，这种生物合成途径的关键部分受到相当大的干扰， 内源性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着损失。 我们的初步努力已经导致中间体荆芥拉酚的产品滴度增加，并建议更 针对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Tang实验室在天然产物生物合成方面的专业知识， 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，以及阐明迄今未知的MIA生物合成途径，涉及strictosidine。 我们将共同实现四个目标：1）使用高通量途径建设，以实现改进的基线 2）建立strictosidine和其他关键代谢物反应生长筛选 生物合成途径中间体; 3）采用新的基因组工程工具，快速创建，筛选和 从改进的基线开始，可以实现高水平的strictosidine生产的基因型酵母菌株 菌株;和4）复杂MIA的异源生产和下游途径探索，例如 长春碱和喜树碱。

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.