Lacto-N-neotetraose (LNnT) is expected to be added to infant food as a nutritional fortifier. Exploring its production methods of it and its derivatives has become a major research hotspot. CD BioGlyco presents multiple strategies to achieve efficient production of LNnT.

LNnT Is One of the Important Core Structures of HMOs

Human milk oligosaccharides (HMOs) can be divided into three types of oligosaccharides, which are neutral oligosaccharides, fucosylated oligosaccharides, and sialylated oligosaccharides. As one of the important core structures of HMOs, LNnT is the simplest type II sugar, consisting of galactose (Gal), N-acetylglucosamine (GlcNAc), and glucose (Glc), the structure is shown in Figure 1. It can be synthesized into various derivatives through sialylation and fucosylation, including lacto-N-fucopentaose, lacto-N-neofucopentaose, lacto-N-neodifuco-hexaose, and sialyllacto-N-tetraose. The derivatives have unique effects on the human body, which have been confirmed by many studies. In short, LNnT and its derivatives have great application potential and commercial value in the food and pharmaceutical industries. The exploration of related production methods has become a major research hotspot.

Physiological Functions of LNnT

• Prebiotic function

LNnT has a prebiotic effect and can promote the growth and metabolic activities of bifidobacteria in the intestine, which can significantly increase the number and population abundance of the strains.

• Immune regulation function

For example, LNnT and its derivatives can stimulate type II immunity by increasing the number of Grl cells and antigen-presenting cells.

• Anti-pathogen and toxin function

LNnT and its sialylated derivatives can block the adhesion of Streptococcus pneumonia to intestinal epithelial cells, thus inhibiting the occurrence of pneumonia.

Key Technologies

• Enzymatic Synthesis: Precision at the Molecular Level

We employ highly specific glycosyltransferases, precisely control the sequence and linkage of the sugar units, ensuring the final product is identical to the LNnT found in human milk.

• Metabolic Engineering: A Whole-Cell Biocatalyst

To achieve scalable and cost-effective production, we integrate enzymatic synthesis with state-of-the-art metabolic engineering. By engineering these microbial hosts, we optimize their metabolic pathways to not only produce the necessary enzymes for synthesis but also to generate the required sugar precursors.

Innovating HMO Production: CD BioGlyco's Solution for LNnT Synthesis

CD BioGlyco provides a variety of Methods to produce LNnT and its derivatives. Due to the specificity of the enzymatic reaction, the latter two synthetic methods are the most efficient and promising, and progress has been especially great in developing and improving these two methods through the continuous efforts of our researchers.

Our production strategy includes but is not limited to the following:

• Enzymatic synthesis
  • The common precursor of LNnT, lacto-N-triose II (LNT II), is first synthesized by the chemical enzymatic method or a one-pot multi-enzyme system. And then the conversion of LNnT is realized by linking galactose to the acceptor substrate through β-1, 4 bonds.
  • After the synthesis of LNnT, an effective donor is selected to react with lactose, and the sugar synthase is immobilized in a fixed bed filled with agarose beads to form a continuous synthesis of LNT II.
• Metabolic Engineering
  • The key transferases of the metabolic pathway, such as glucosamine transferase, are injected into the derived Escherichia coli cells with the plasmid as a carrier for cultivation and production.
  • Through chromosomal integration, a plasmid-free engineered Escherichia coli strain is constructed and fed-batch culture to synthesize LNT II with glycerol or galactose as the main carbon source.

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As a recognized expert in the production of LNnT, CD BioGlyco has developed efficient and high-quality synthetic strategies. If you have any needs in this regard, please feel free to contact us.