# Economical Modular One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides

> **NIH NIH R21** · UNIVERSITY OF GEORGIA · 2024 · $188,750

## Abstract

Project Summary
Glycans play essential roles in virtually all aspects of biology. Thus, they have broad applications, including
supplementation of necessary human milk oligosaccharides (HMOs) to infant formula as therapeutics to prevent
infection by multiple pathogens, maintain proper microbiota, prevent allergies, and treat necrotizing enterocolitis
in infants. However, present synthesis methods for the more complex HMOs fail in affordable scalability that
hinders their development as glycan therapeutics. Although current one-pot multienzyme (OPME) systems for
HMO synthesis have streamlined numerous synthetic reaction steps, little has been done on optimization and
scalability. We hypothesize that a modular OPME system for glycan synthesis can be optimized and integrated
with inexpensive polyphosphate-based energy regeneration to decrease cost and significantly increase yield of
desirable glycans. In Aim 1, we will establish reference OPME reactions for single sugar transfer steps that start
from simple building blocks of monosaccharides and glycan acceptors as input, and provide enzymes for high-
energy sugar donor synthesis, monosaccharide transfer and energy regeneration. These studies will develop
scalable modules for optimization of chemical input (donor and acceptor building blocks, catalytic quantities of
nucleotides, divalent cations, controlled pH, and enzyme catalysts) to establish cross-platform compatible
reaction conditions. In Aim 2, we will establish a scalable, coupled polyphosphate energy regeneration system
for OPME glycan synthesis. Optimization of polyphosphate as an energy source will require control of
polyphosphate and divalent cation concentrations, neutralization of pH changes, and integration of enzymes that
generate ATP (RpPPK2-3) and distribute high-energy phosphate equivalents to other nucleotide forms (NDK).
Our goals are to create a universal energy source that integrates the continual synthesis of UDP-, GDP-, CMP-,
and ADP-sugar donors in large-scale OPME reactions. In Aim 3, we will generate proof-of-concept scalable
OPME synthesis of model HMO targets of biological interest for optimization. The approach will combine
synthetic modules for sugar donor synthesis and glycan extension with optimized energy regeneration and
determine conditions for cross-platform compatibility for all enzymatic steps in the energy-coupled OPME
(ecOPME) platform. The HMO targets also provide opportunities to test combined ecOPME reactions as well as
sequential ecOPME sugar additions where enzyme competition would yield undesired products. The goals are
to integrate multiple enzymatic transfer steps through the selective use of glycosyltransferase acceptor specificity
coupled with energy regeneration to result in a proof-of-concept modular platform for efficient, flexible, and
scalable synthesis of target therapeutic HMOs starting from simple monosaccharide building blocks.

## Key facts

- **NIH application ID:** 10834878
- **Project number:** 5R21HD110982-02
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** KELLEY W. MOREMEN
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $188,750
- **Award type:** 5
- **Project period:** 2023-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10834878

## Citation

> US National Institutes of Health, RePORTER application 10834878, Economical Modular One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides (5R21HD110982-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10834878. Licensed CC0.

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