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: 10735587
• 负责人: Dino Di Carlo
• 金额: $ 36.28万
• 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 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735587
• 关键词: Achievement; Anabolism; Behavior; Biological; Biological Models; CRISPR interference; CRISPR-mediated transcriptional activation; Cell Separation; Cells; Chemicals; Chemistry; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collection; Complex; Data; Derivation procedure; Directed Molecular Evolution; Disease; Encapsulated; Engineering; Enzymatic Biochemistry; Enzymes; Evaluation; Family; Fluorescence; Genes; Genetic Screening; Genome; Grant; Growth; Hydrogels; Ibogaine; Indole Alkaloids; Investigation; Letters; Liquid substance; Maintenance; Metabolic; Metabolic Biotransformation; Methods; Microfluidic Microchips; Microfluidics; Monoterpenes; Mutagenesis; Natural Products; Opiate Addiction; Opioid; Organism; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phytochemical; Plant alkaloid; Plants; Positioning Attribute; Process; Production; Psychotropic Drugs; Reporter; Reporting; Research Project Grants; Saccharomyces cerevisiae; Signaling Molecule; Sorting; Source; System; Technology; Therapeutic; Vinblastine; Yeasts; addiction; genome wide screen; genome-wide; glucosidase; healthy lifestyle; high throughput screening; improved; interest; microbial; microbial host; mutant; nanobiotechnology; novel strategies; particle; pharmacologic; plant metabolites; promoter; prophylactic; reconstitution; reconstruction; screening; small molecule; strictosidine; success; synthetic biology; tool

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.

摘要 在基因特征良好的微生物中重建植物天然产物途径 像酿酒酵母这样的生物是一种可持续和可扩展的生产方法 高价值的药物化合物。单萜吲哚生物碱(MIA)家族 代表具有重大生物活性的各种天然产品的集合。三军情报局 不可缺少的药物成分，但也很昂贵，很难从植物中分离出来 制片人。在之前的资助周期中，我们成功地培育出了能够产生 作为MIA的普遍前体，其滴度超过100 mg/L。在这项建议中，我们 将设计从Strectosidine开始的下游步骤，以克服关键的代谢瓶颈，以及 开发基于酵母的工程异源天然产物途径的新技术。 在与Di Carlo实验室的合作下，我们将部署启用PicoShell的细胞分类，以实现高 MIA通路的吞吐量筛选。PicoShell技术允许基于微流控的、高 从液体培养中获得单细胞包裹量。微囊化酵母细胞可以培养 以单克隆化的方式散装生产感兴趣的化合物。PicoShell有效 从单个酵母细胞中放大报告分子信号，并可根据以下条件用FACS进行分选 散射(生长率)和荧光(滴度)。这样的工作流程使酵母的合并成为可能 使用需要高通量筛选的技术的途径工程，包括定向筛选 进化与全基因组CRISPRi筛选。我们的初步努力表明， MIA途径中的荧光天然产物可以作为MIA途径效率的报告 PicoShell过程中的下游步骤启用了FACS分类。这份协作性提案将 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纳米生物技术 迪卡洛实验室为酵母菌开发的工具。这将为彻底重建铺平道路。 酵母中重要的高滴度MIA，以及建立酵母合成的新工具 生物学。我们将共同解决三个目标：1)克服后 限制性步骤，特别是限制性葡萄糖苷酶(SGD)的低效率；2)宿主 具有CRISPR干扰和激活的工程，以提高Strettosidine水平，使用两者 由PicoShells实现的Rational和全基因组筛选；以及3)完整的生物合成 复杂的MIA伊波加因和米拉吉宁，这两种精神活性MIA已经产生了显著的 兴趣是阿片成瘾的潜在治疗方法。