On Oct 8, 2021, Hu et al. from State Key Laboratory of Food Science and Technology, Jiangnan University, published a paper in the Biotechnology for Biofuels, titled "Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin". The core of their work is the development of an engineered Escherichia coli Cell Factory capable of synthesizing lacto-N-triose II (LNT II), a crucial trisaccharide building block. The innovative foundation of this project lies in leveraging N-acetylglucosamine (GlcNAc), a readily available monomer derived from abundant chitin biomass, as the primary feedstock. The main finding is a remarkable demonstration of metabolic engineering prowess, achieving an impressive final LNT II titer of 15.8 g/L through rigorous pathway design, flux balancing, and fermentation optimization.

Overview

The importance of complex carbohydrates, particularly Human Milk Oligosaccharides (HMOs), cannot be overstated. These structurally diverse glycans are not metabolized directly by the infant but act as powerful prebiotics, shaping the gut microbiome and offering profound benefits to immune and cognitive development. Among these, lacto-N-neotetraose (LNnT) and its precursor, LNT II, are vital core structures.

The challenge for industrial applications, however, lies in production. Traditional chemical synthesis is often complex, expensive, and environmentally intensive, while pure enzymatic synthesis faces issues related to cofactor regeneration and reaction equilibrium shifts (hydrolysis activity). Synthetic Glycobiology seeks to overcome these limitations through Microbial Cell Factories. By engineering microorganisms, we harness the efficiency of cellular metabolism to perform complex, multi-step glycosylation reactions.

Furthermore, this research is strategically positioned at the intersection of synthetic biology and the bio-economy by focusing on sustainable feedstock utilization. Chitin, the second most abundant natural polymer after cellulose, is a vast, underutilized biomass source, mainly sourced from crustacean shells and fungal cell walls. GlcNAc, the monomer of chitin, is an ideal, renewable starting material. Therefore, the successful integration of a GlcNAc-to-LNT II pathway represents a powerful, green alternative to using expensive or non-renewable carbon sources, aligning the production of high-value glycans with principles of resource sustainability.

Research Results

• Initial Pathway Design: Heterologous Glycosyltransferase Introduction

The first critical step in constructing the LNT II cell factory was identifying and integrating the key glycosyltransferase. LNT II is synthesized by transferring GlcNAc residue from UDP-GlcNAc to β-lactose. This reaction is catalyzed by a β-1,3-acetylglucosaminyltransferase.

The researchers introduced the heterologous gene NmLgtA from Neisseria meningitides, which encodes the required β-1,3-acetylglucosaminyltransferase, into the E. coli host strain. This was the initial proof-of-concept, establishing the minimal biosynthesis route. The wild-type E. coli cannot synthesize LNT II, but it naturally produces the essential precursor, UDP-GlcNAc, from fructose-6-phosphate (Fru-6-P). By feeding the host GlcNAc as the sole carbon source and β-lactose as the acceptor, the engineered strain was able to initiate LNT II production, albeit at a modest starting titer of 0.12 g/L. This demonstrated the pathway feasibility but highlighted severe metabolic bottlenecks.

Fig.1 Construction the biosynthesis pathway for LNT II production using GlcNAc substrate in E. coli. (Hu, et al., 2021)

• Flux Balancing and Precursor Supply Enhancement: Overcoming Bottlenecks

The low initial titer indicated a clear metabolic bottleneck, primarily centered around the supply of the nucleotide sugar precursor, UDP-GlcNAc. In the engineered pathway, GlcNAc must first be converted efficiently into UDP-GlcNAc before it can be used by the NmLgtA enzyme.

To boost the metabolic flux towards the LNT II synthesis pathway, the team focused on strengthening the enzymatic reactions responsible for UDP-GlcNAc synthesis. This involved the combinatorial overexpression of two key endogenous genes: GlmU and NagA. The simultaneous overexpression of these two enzymes along with NmLgtA proved to be a critical innovation. The titer rapidly increased from 0.12 g/L to 2.44 g/L, a 20-fold increase. This result vividly illustrates the principle of metabolic flux control in synthetic glycobiology—where engineering not just the pathway, but the flux of the precursor molecules, is necessary for high-yield production.

• Product Stability and Final Titer Optimization: Blocking Side Reactions

The researchers systematically targeted genes in E. coli that could interfere with the synthesis or stability of the LNT II building blocks:

• lacZ Knockout: The native lacZ gene encodes β-galactosidase, an enzyme that hydrolyzes the lactose acceptor molecule. By knocking out this gene, the team ensured that the expensive β-lactose substrate remained available for the NmLgtA reaction, preventing wasteful consumption.
• nanE Knockout: The nanE gene encodes an N-acetylglucosamine-6-phosphate 2-epimerase, which could potentially divert GlcNAc-6-phosphate away from the desired UDP-GlcNAc synthesis route. Blocking this gene further tightened metabolic control over the GlcNAc feedstock.

These genetic modifications, coupled with optimized fed-batch fermentation and a fine-tuned GlcNAc and β-lactose feeding strategy, culminated in the breakthrough. The final engineered strain produced LNT II at an unprecedented titer of 15.8 g/L after 76 hours of fermentation. This is a nearly 132-fold improvement over the initial construct and represents a truly industrial-scale concentration for such a complex glycan precursor.

Fig.2 Effects of inactivating lacZ and nanE on DCW, LNT II titer, residue concentration of GlcNAc, and lactose. (Hu, et al., 2021)

Conclusion

The success of engineering E. coli to produce LNT II at a titer exceeding 15 g/L is more than just a numbers game; it is a profound methodological triumph for synthetic glycobiology. The work establishes a highly efficient, closed-loop biosynthesis platform that leverages renewable chitin biomass, effectively transforming a waste product into a high-value nutraceutical precursor.

This research demonstrates that complex glycosylation pathways are reliably installed and rigorously optimized in microbial hosts by applying a systematic approach to enzyme selection, precursor flux balancing, and competitive pathway elimination. The resulting platform has immediate, innovative applications in infant nutrition and functional food development, providing a scalable and cost-effective source of LNT II for subsequent enzymatic conversion to LNnT.