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

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2023 · $362,777

## Abstract

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.

## Key facts

- **NIH application ID:** 10735587
- **Project number:** 2R01AT010001-06
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Dino Di Carlo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $362,777
- **Award type:** 2
- **Project period:** 2018-07-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10735587

## Citation

> US National Institutes of Health, RePORTER application 10735587, Engineering Yeast towards High Titer Production of Monoterpene Indole Alkaloid Natural Products (2R01AT010001-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10735587. Licensed CC0.

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