# Engineering bacteria for production and delivery of the halogenated prodrug lead L-4-chlorokynurenine

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $76,756

## Abstract

PROJECT SUMMARY/ABSTRACT
 Studies of the human microbiome have demonstrated that bacteria play a once overlooked, but important
role in health and disease. In turn, therapeutic interventions that attempt to reprogram the microbiome to promote
health or treat disease have garnered increased attention. The utility of using live bacteria as medicine was once
limited by their native behavior. Modern synthetic biology tools, however, make it possible to genetically encode
functions into microorganisms, expanding their potential roles as therapeutics. One such burgeoning application
is to engineer microbes to function as synthetic factories within the gut, enabling the delivery of medicinal
compounds from within. Live bacterial therapeutics programmed to deliver metabolic enzymes, for example,
have been efficacious towards rescuing inborn errors of metabolism in mouse models. To date, the delivery of
therapeutic compounds by bacteria in vivo has been limited to ribosomally-synthesized active agents (i.e.
enzymes, proteins, peptidic hormones). Despite the known biosynthetic capability of microbes to make a
dazzling array of small molecule natural products, there are no existing studies that have harnessed this
prowess towards the development of a microbiome-based therapeutic. In this proposal, I aim to engineer
the probiotic Escherichia coli Nissle (EcN) to produce the neuropharmaceutical prodrug candidate L-4-
chlorokynurenine (4CK) and then test its ability to function inside a mouse model. In doing so, I will explore the
capacity of probiotics to synthesize non-native metabolites inside an animal host, which could give rise to a new
mechanism for the sustained delivery of small molecule drugs.
 In aim 1, I will genetically program EcN to synthesize 4CK and optimize production. This will require
cloning and refactoring a heterologous 4CK biosynthetic pathway as well as manipulating the endogenous
metabolism of the EcN chassis. In aim 2, I will use a growth-coupled selection system in Pseudomonas putida
to evolve the biosynthetic enzymes. Improved enzyme variants will be re-engineered into EcN. In aim 3, the
optimized strain will be administered to mice and systemic distribution of 4CK evaluated. This will include
assessing production in the gut, absorption into the bloodstream and transport to the brain. This proposal is
designed to provide a multidisciplinary training opportunity combining my interests in natural products,
bioengineering, microbiome science, and biomedical research and is strongly supported by a diverse team of
advisors who are experts in these fields.
.

## Key facts

- **NIH application ID:** 10775705
- **Project number:** 5F32GM149120-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Leah Bushin
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $76,756
- **Award type:** 5
- **Project period:** 2023-03-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10775705

## Citation

> US National Institutes of Health, RePORTER application 10775705, Engineering bacteria for production and delivery of the halogenated prodrug lead L-4-chlorokynurenine (5F32GM149120-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10775705. Licensed CC0.

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