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