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

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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.