# In vitro and cellular tools for complex polysaccharide biosynthesis

> **NIH NIH R01** · UNIVERSITY OF NORTH CAROLINA CHARLOTTE · 2021 · $262,682

## Abstract

Bacterial surface polysaccharides play central roles in a wide range of biology, and could serve as
targets for novel anti-microbial agents, pathogen sensors, vaccine antigens or other important therapeutics. All
of these applications require robust methods to produce these materials that can be easily adapted from one
type of polysaccharide to another. One important way to go about doing this is to exploit the natural pathways
that are associated with the formation of these materials to build them either enzymatically or engineer a living
system to do it. Both of these options require more effective tools for the analysis of bacterial polysaccharide
biosynthesis and a better understanding of these natural pathways than is currently available. In this proposal
we aim to begin the development of new methods and tools for the production and analysis of complex
polysaccharide biosynthesis in vitro and in cells. We will start with the Escherichia coli exopolysaccharide
colanic acid (CA), which is thought to promote biofilm formation and help the organism survive in low pH
environments. We will first utilize a fluorescent bactoprenyl phosphate mimic to reconstitute the biosynthesis
of CA in vitro to elucidate the precise biochemical roles of all proteins involved. This system will be used to
produce standards that will then allow us to validate a new cellular probe developed to monitor CA
biosynthesis in cultured bacteria, and build tagged cellular polysaccharide precursors in mutant E. coli strains.
This CA biosynthesis system will then be replaced in E. coli with an operon from the mammalian symbiont
Bacteroides fragilis, which is responsible for the formation of capsular polysaccharide A (CPSA). CPSA is
thought to play important roles in the normal development of the mammalian immune system, and could be a
key therapeutic for autoinflammatory diseases. This operon replacement strategy in the CA biosynthesis locus
will provide a system for the production and excretion of this important biomolecule. The tools generated in this
proposal will allow for the optimization of the production of this material. The system developed could then be
applied to nearly any polysaccharide of this type, in nature, providing a robust genetically encoded factory for
polysaccharide production.

## Key facts

- **NIH application ID:** 10141249
- **Project number:** 5R01GM123251-05
- **Recipient organization:** UNIVERSITY OF NORTH CAROLINA CHARLOTTE
- **Principal Investigator:** JERRY M TROUTMAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $262,682
- **Award type:** 5
- **Project period:** 2017-05-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141249

## Citation

> US National Institutes of Health, RePORTER application 10141249, In vitro and cellular tools for complex polysaccharide biosynthesis (5R01GM123251-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10141249. Licensed CC0.

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