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

摘要 在基因良好表征的微生物中重建植物天然产物途径 生物体如酿酒酵母是一种可持续和可扩展的生产方法， 高价值药物化合物。单萜吲哚生物碱（MIA） 代表了具有显著生物活性的天然产物的多样性集合。MIA是 它们是不可或缺的药物成分，但也是昂贵的，并且难以从植物中分离 生产商在上一个资助周期中，我们成功地改造了可以产生 strictosidine，MIA的通用前体，滴度超过100 mg/L。在本提案中，我们 将设计strictosidine的下游步骤，以克服关键的代谢瓶颈， 开发基于酵母的新技术，用于工程化异源天然产物途径。 与Di Carlo实验室合作，我们将部署PicoShell支持的细胞分选， MIA途径的通量筛选。PicoShell技术允许基于微流体的、高 从液体培养物中生产单细胞包封。包封的酵母细胞可以生长 以单克隆的方式批量生产并产生感兴趣的化合物。PicoShell有效地 扩增来自单个酵母细胞的报告分子信号，并且可以基于 散射（生长速率）和荧光（滴度）。这样的工作流程使得酵母的合并 需要高通量筛选技术的途径工程，包括定向 进化和全基因组CRISPRi筛选。我们的初步工作表明， MIA途径中的荧光天然产物可以作为MIA途径的效率的报告基因。 在PicoShell使能的FACS分选期间的下游步骤。该合作提案将 利用Tang实验室在天然产物生物合成方面的专业知识， 迪卡罗实验室为酵母开发的工具。这将为完全重组铺平道路 重要的MIA在酵母中的高滴度，以及建立新的工具，酵母合成 生物学我们将共同努力实现三个目标：1）克服后一阶段的关键瓶颈， strictosidine步骤，特别是strictosidine葡萄糖苷酶（SGD）的低效率; 2）宿主 用CRISPR干扰和激活进行工程化，以增加strictosidine水平， 通过PicoShells实现的合理和全基因组筛选;以及3） 复杂的MIA伊波替尼和米曲宁，两种精神活性MIA，产生了显着的 作为阿片类药物成瘾的潜在治疗方法。

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. The family of monoterpene indole alkaloids (MIAs) represent a diverse collection of natural product with significant biological activities. MIAs are indispensable pharmaceutical ingredients, but are also expensive and difficult to isolate from plant producers. In the previous grant cycle, we successfully engineered yeast strains that can produce strictosidine, the universal precursor to MIAs, at titers exceeding 100 mg/L. In this proposal, we will engineer the downstream steps from strictosidine to overcome key metabolic bottlenecks, and develop new yeast based-technologies for engineering heterologous natural product pathways. In collaboration with the Di Carlo lab, we will deploy PicoShell enabled cell sorting to enable high throughput screening of MIA pathways. The PicoShell technology allows microfluidic-based, high throughput single-cell encapsulation from liquid culture. Encapsulated yeast cells can be grown in bulk in a monoclonal fashion and produce the compound of interest. PicoShell effectively amplifies reporter molecule signal from single yeast cells and can be sorted with FACS based on scatter (growth rate) and fluorescence (titer). Such workflow enables the merging of yeast pathway engineering with technologies that require high throughput screening, including directed evolution and genome wide CRISPRi screening. Our preliminary efforts have shown that a fluorescent natural product in the MIA pathway can serve as a reporter for the efficiency of the downstream steps during PicoShell enabled FACS sorting. This collaborative proposal will leverage Tang lab’s expertise in natural product biosynthesis with the new nanobiotechnology tools developed for yeast by the Di Carlo Lab. This will pave the way for complete reconstitution of important MIAs at high titers in yeast, as well as establishing new tools for yeast synthetic biology. Together we will address three aims: 1) overcoming key bottleneck step in post- strictosidine steps, specifically the low efficiency of strictosidine glucosidase (SGD); 2) host engineering with CRISPR interference and activation to increase strictosidine levels, using both rational and genome wide screening enabled by PicoShells; and 3) complete biosynthesis of complex MIAs ibogaine and mitragynine, two psychoactive MIAs that have generated significant interests as potential treatment for opioid addiction.