# Multi-gram enzymatic production of complex glycans by flow processes

> **NIH NIH R44** · ZYMTRONIX CATALYTIC SYSTEMS, INC. · 2020 · $354,457

## Abstract

Abstract: Zymtronix proposes to significantly improve production efficiency of glycans via new flow processes
based on chemoenzymatic catalysis to take significant steps towards broader commercial access. The first
glycans of interest are human milk oligosaccharides (HMOs), which have large commercial relevance in infant
nutrition, disease prevention and therapeutics. The primary hurdle towards the broader application of complex
synthetic glycans in research, preclinical and commercial applications is their affordability. The goal of the
proposed research is to lower the production costs of large and complex HMOs (>5 DP) by continuous flow
processes where each step of synthesis is conducted in modules. Zymtronix's technology enables (i) high
enzyme activity and stability ensuring the reuse of enzyme and (ii) enables the co-immobilization of multiple
enzymes to incorporate enzymatic recycling for sub-stoichiometric use of reagents to impart significant cost-
reductions. For this fast-track, we will produce large human milk oligosaccharides (HMOs), the third largest
component of breast milk that are particularly commercially relevant glycan models. While some simple
probiotic HMOs can be effectively produced via fermentation for infant formula (2'FL, 3 DP), complex and
branched HMOs are elusive and have been marginally produced at high cost via chemoenzymatic catalysis.
Zymtronix's technology solution will significantly improve production of glycans, starting with HMOs, while
imparting significant cost savings.
Fast-track Phase I specific aims are to immobilize 6 enzymes and to combine them in a defined sequence to
generate sialylated lacto-N-neopentaoses. (I) Aim 1: Immobilize 6 individual enzymes on sintered beads using
ZymTrap3D technology. 4 transferases and 2 activated-sugar-producing enzymes for in-situ synthesis of
GlcNAc-UDP, Gal-UDP and Neu5Ac-CMP, (II) Aim 2: Demonstrate sequential, modular synthesis of 1 mg of α-
2,3- and α-2,6-sialyl lacto-N-pentaoses on beads. (III) Aim 3: Produce 100 mg of α-2,3-sialyl lacto-N-
neopentaose using a flow cell.
Fast-track Phase II aims are to scale up production of three linear fucosyl- and sialyl-lacto-N-neopentaoses
from Aim1. (I) Aim 1: Development of HMO fucosylation and branching modules towards the 100 mg
production of 6 biantennary lacto-N-neohexaoses and heptaoses. The modularity of branching followed by
three possible `functionalization' modules, and a final elongation step allows the production of 6 biantennary
HMOs. (II) Aim 2: Development of flow cells with in situ reagent recycling for multi-gram scale HMO production.
Production and cost metrics will be assessed. (III) Aim 3: Scaling up engineering and production of 10 g and
then 100 g of fucosyl- and sialyl-lacto-N-neopentaoses. Work will include in-house enzyme production and
HPLC purification of HMOs.

## Key facts

- **NIH application ID:** 9909475
- **Project number:** 1R44GM136085-01
- **Recipient organization:** ZYMTRONIX CATALYTIC SYSTEMS, INC.
- **Principal Investigator:** Alexander Chris Hoepker
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $354,457
- **Award type:** 1
- **Project period:** 2020-04-03 → 2022-01-02

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9909475

## Citation

> US National Institutes of Health, RePORTER application 9909475, Multi-gram enzymatic production of complex glycans by flow processes (1R44GM136085-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9909475. Licensed CC0.

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