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.

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 酿酒酵母等生物体是一种可持续且可扩展的生产方法 高价值的药物化合物。单萜吲哚生物碱 (MIA) 家族 代表具有重要生物活性的多种天然产物。 MIA 是 不可或缺的药物成分，但价格昂贵且难以从植物中分离 生产者。在上一个资助周期中，我们成功地设计了可以生产的酵母菌株 胡豆素是 MIA 的通用前体，滴度超过 100 mg/L。在这个提案中，我们 将设计胡豆素的下游步骤以克服关键的代谢瓶颈，以及 开发基于酵母的新技术来设计异源天然产物途径。 我们将与 Di Carlo 实验室合作，部署支持 PicoShell 的细胞分选，以实现高 MIA 途径的通量筛选。 PicoShell 技术允许基于微流体的高 液体培养物中单细胞封装的吞吐量。封装的酵母细胞可以生长 以单克隆方式批量生产并产生感兴趣的化合物。 PicoShell 有效 放大来自单个酵母细胞的报告分子信号，并可以基于 FACS 进行分选 散射（生长率）和荧光（滴度）。这样的工作流程使得酵母能够合并 使用需要高通量筛选的技术进行通路工程，包括定向 进化和全基因组 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报告效率 PicoShell 启用 FACS 排序期间的下游步骤。该合作提案将 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纳米生物技术 Di Carlo 实验室为酵母开发的工具。这将为彻底重建铺平道路 酵母中重要的 MIA 的高滴度，以及建立酵母合成的新工具 生物学。我们将共同实现三个目标：1）克服后期的关键瓶颈步骤 胡豆素步骤，特别是胡豆素葡萄糖苷酶（SGD）效率低； 2）主机 通过 CRISPR 干扰和激活进行工程设计，以提高胡豆素水平，同时使用 PicoShells 实现合理的全基因组筛选； 3) 完整的生物合成 复杂的 MIA 伊博加因和帽柱木碱，这两种精神活性 MIA 已产生显着的 兴趣作为阿片类药物成瘾的潜在治疗方法。