Through competent assistance by our researchers and the fastest delivery of high-quality lacto-N-tetraose (LNT) products at fair prices, CD BioGlyco has become a “preferred supplier” of many clients. We have developed many effective strategies to deal with the uncertainty of metabolic patterns in model microorganisms.

Relationship Between Human Milk and LNT

Human milk oligosaccharides (HMOs), the third largest molecular constituent in breast milk, have received considerable attention for their importance to infant health, much of which stems from the physiological factor that they can promote the development of a healthy microbiome. LNT is a core structure shared by long-chain HMOs and many glycolipids, its content is 8 times that of Lacto-N-neotetraose.

Fig.1 The chemical structure of LNT. (Deng, et al., 2021)

Safety of LNT in Food Applications

More accurately mimicking the composition of human breast milk is certainly a production priority in commercial infant formula, which requires the addition of naturally occurring structural products identical to the milk oligosaccharides produced by humans. One study specifically evaluated the safety of LNT, as part of an in vitro chronic subchronic gavage toxicity study and genotoxicity testing. Study data showed no compound-related adverse effects. This supports the safe use of LNT in infant formula and the corresponding composition ratio.

LNT Production Service at CD BioGlyco

Due to the development of metabolic engineering and synthetic biology strategies, the microbial production of LNT provided by us has developed rapidly. Our researchers have accumulated mature production technologies and are constantly improving and innovating, including but not limited to the following:

• Synthesis by chemoenzymatic methods
• First synthesizing lacto-N-triose by chemical method. Then, using it as an acceptor, a molecule of galactose is introduced into the lacto-N-triose acceptor through a one-pot two-enzyme reaction to synthesize LNT.
• Lactate-N-disaccharide derivative is prepared by the chemoenzymatic method as donor substrate, and LNT was synthesized by glycosylation of wild-type and glycosidase variants from Bifidobacterium bifidum.
• Biological production methods
• Construct engineered Escherichia coli.

The transferases of exogenous strains and derived Escherichia coli are simultaneously introduced into other derived Escherichia coli strains to construct engineered Escherichia coli.

• The pathway is optimized through genome combination, fine-tuning, overexpression, and other strategies.
• Scale up production using shake flask fermentation and fed-batch.

Biological Strategies to Optimize Production at CD BioGlyco

• Improve intracellular lactose supply

Modifying lactose permease through rational design and directed evolution, overexpressing permease to improve intracellular lactose supply.

• Genetic engineering

Deletion of specific coding genes enables simultaneous consumption of glucose and lactose. Screening genes related to LNT metabolic pathway and inactivating genes related to the bypass pathway.

• Adjust dynamic balance

Inhibiting or increasing the expression of genes necessary for homologous or heterologous pathway balance in strains such as Escherichia coli and Bacillus subtilis to reduce the impact on cell growth, etc.

CD BioGlyco uses the mature experience and technology accumulated by researchers to meet clients’ needs. If you have needs in LNT production, please contact us directly.

References

1. Deng, J.; et al. Recent advances and challenges in microbial production of human milk oligosaccharides. Systems Microbiology and Biomanufacturing. 2021, 1(1), 1-14.
2. Phipps, K.R.; et al. Preclinical safety evaluation of the human-identical milk oligosaccharide lacto-N-tetraose. Regulatory Toxicology and Pharmacology. 2018, 99: 260-273.