Lacto-N-neotetraose (LNnT) is a core tetrasaccharide and one of the most significant neutral Human Milk Oligosaccharide (HMO), characterized by its Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc structure. As a Type II isomer, LNnT serves as a vital prebiotic that selectively stimulates the growth of Bifidobacterium, particularly species that are essential for the maturation of the infant's immune system. Beyond its role in gut colonization, LNnT acts as a decoy receptor for respiratory and enteric pathogens, reducing the risk of infections. CD BioGlyco specializes in the high-titer production of LNnT using an advanced Synthetic Glycobiology platform. By integrating heterologous glycosyltransferases into optimized microbial hosts, we provide a sustainable, high-purity alternative to mammalian milk extraction, enabling the large-scale application of LNnT.
Core Technologies
CD BioGlyco utilizes a multi-layered engineering strategy to overcome the metabolic bottlenecks of LNnT biosynthesis:
Combinatorial Glycosyltransferase Expression
We deploy highly efficient β-1,3-N-acetylglucosaminyltransferases (LgtA) and β-1,4-galactosyltransferases (LgtB) derived from Neisseria meningitidis, optimized for sequential glycan elongation.
Metabolic Flux Balancing
Using CRISPR/Cas9 genomic integration, we balance the intracellular pools of UDP-GlcNAc and UDP-Gal to prevent the accumulation of intermediate triose structures like LNT II.
Host Chassis Refactoring
Our Escherichia coli and Saccharomyces cerevisiae platforms are engineered to be deficient in β-galactosidases (lacZ) and catabolic pathways for N-acetylglucosamine to ensure maximum carbon redirection toward LNnT.
Enhanced Precursor Transport
We overexpress lactose permeases (e.g., Lac12 or LacY) to facilitate efficient substrate uptake, coupled with optimized efflux transporters to simplify downstream product recovery.
Engineering the Gold Standard of Human-Identical Milk Oligosaccharides
At CD BioGlyco, our LNnT production service is designed to empower the next generation of nutritional and medical innovations. Our scope of service is comprehensive, covering every stage from strain development to industrial-scale supply. We understand that the transition from a laboratory concept to a market-ready product requires more than just a manufacturer; it requires a scientific partner. To this end, we offer custom pathway optimization, where our specialists refactor metabolic routes to suit specific client feedstocks, such as glucose, glycerol, or lactose.
Our process development and optimization service ensures that LNnT production is not only high-yield but also economically sustainable. We provide extensive analytical services, including quantitative glycomics and impurity profiling, which are crucial for the safety documentation required by global health authorities. For clients targeting specialized markets, we offer sub-page entry options for the co-production of LNnT with other HMOs like 2'-FL to create optimized, bio-mimetic blends. Furthermore, our scope includes pilot-scale prototyping for clinical trial material, ensuring that our clients have access to the same high-quality LNnT from the R&D phase through to commercial launch. By leveraging our deep expertise in synthetic glycobiology, CD BioGlyco provides a robust, scalable, and regulatory-ready supply of LNnT that meets the most demanding industrial standards.
Workflow
Genetic Pathway Assembly
We initiate the biosynthetic pathway for LNnT by de novo designing and synthesizing codon-optimized gene clusters tailored for high expression in our selected microbial chassis. These clusters encompass all necessary enzymes: the specific galactosyltransferases and N-acetylglucosaminyltransferases required to construct the LNnT backbone, as well as the accessory enzymes for UDP-sugar donor (UDP-Gal, UDP-GlcNAc) regeneration. Crucially, we employ chromosomal integration techniques to insert these complete operons into specific, high-stability genomic loci, thereby creating a robust, plasmid-free production strain. This strategy eliminates issues of plasmid loss, antibiotic marker usage, and genetic instability, forming a reliable genetic foundation for scalable industrial fermentation.
Multimodule Reprogramming
The biosynthetic pathway is treated as a system of interconnected functional modules (e.g., lactose uptake, UDP-sugar precursor synthesis, and sequential glycosyltransferase activities). We employ an iterative synthetic biology approach to reprogram this system. Using libraries of tunable promoters and ribosome binding sites (RBS), we systematically and independently adjust the expression level of each module. The optimization goal is to achieve perfect stoichiometric balance, ensuring that the flux of activated sugar donors precisely matches the catalytic capacity of the transferases at each step. This fine-tuning minimizes the accumulation of metabolic intermediates (like LNT or other isomers) and directs the maximum possible carbon flux from the starting substrate, lactose, towards the complete LNnT molecule, thereby maximizing the molar yield.
High-Density Fermentation
LNnT production is scaled through a precision-controlled, high-cell-density fed-batch fermentation process. We utilize a tiered bioreactor platform, from 5L development units to 10,000L industrial-scale fermenters. The process is centered on optimizing critical parameters, particularly oxygen transfer rates (OTR) through tailored agitation and aeration strategies, and dynamic feeding profiles for carbon sources (e.g., glycerol) and key precursors. By maintaining cells in a state of balanced growth and high productivity over an extended period, we maximize volumetric productivity. This scalable approach reliably achieves high LNnT titers, ensuring cost-effectiveness and meeting commercial production targets.
Advanced Purification
Following fermentation, the harvest broth is processed through a proprietary, sequential downstream purification platform. An initial clarification step removes cellular biomass. The clarified stream then undergoes nanofiltration to concentrate the product and remove low molecular weight impurities, salts, and residual monosaccharides. Subsequent ion-exchange chromatography selectively isolates the charged LNnT molecule from other oligosaccharides and neutral impurities. A final polishing step involving activated carbon treatment effectively decolorizes the solution and removes trace organic contaminants. This integrated multi-step process is designed to rigorously remove host cell proteins, nucleic acids, and residual substrates, consistently yielding LNnT with exceptional purity.
Quality Control & Structural Verification
Stringent analytical protocols are applied to validate every production batch. We employ orthogonal high-resolution techniques for definitive characterization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides accurate molecular weight confirmation and quantifies purity by detecting any residual substrates or related oligosaccharide isomers. The definitive structural proof is delivered by proton nuclear magnetic resonance (1H-NMR) spectroscopy, which unequivocally identifies the unique anomeric proton signatures corresponding to the specific β-1,3 linkage (between GlcNAc and Gal) and the β-1,4 linkage (between Gal and Glc). This comprehensive analysis guarantees the correct structural identity, linkage specificity, and a final chemical purity exceeding 98% for all released products.
Publication Data
Journal: Scientific Reports
DOI: 10.1038/s41598-020-57860-8
IF: 3.9
Published: 2020
Results: In this scientific investigation, the authors explored the therapeutic potential of LNnT in enhancing wound healing by modulating type 2 immune responses. They conducted both in vitro and in vivo experiments, beginning with cell viability assays on human dermal fibroblasts to confirm LNnT's non-toxicity. Subsequently, a full-thickness wound model was established in mice, which received intradermal injections of LNnT at 100 μg or 200 μg doses, compared to a PBS control group. Macroscopic evaluations demonstrated that LNnT significantly accelerated wound closure rates by day 7, while histological analyses revealed improved healing scores, reduced epidermal thickness, and increased hair follicle formation. Stereological data indicated decreased neutrophil infiltration and elevated fibroblast and basal cell counts. Molecular analyses showed upregulated expression of key genes involved in type 2 immunity, including IL-10, IL-4, and IL-13, alongside temporal changes in collagen deposition. The study concludes that LNnT facilitates wound repair through its anti-inflammatory properties and induction of type 2 immune pathways, highlighting its promise for clinical applications in skin regeneration.
Application
Gut-Brain Axis Research
Researchers utilize our high-purity LNnT to study the influence of sialylated and neutral glycans on early-life cognitive development and neuroprotection.
Necrotizing Enterocolitis (NEC) Studies
Applied in clinical models to evaluate how LNnT strengthens the intestinal barrier and prevents the onset of life-threatening gut inflammation.
Dermatological Formulations
Emerging research explores the use of LNnT in skincare to balance the skin microbiome and protect against environmental pathogens.
Glycan Microarrays and Diagnostics
High-purity LNnT is used as a standard in carbohydrate microarrays to detect and quantify glycan-binding proteins in clinical research.
Advantages
Exceptional Structural Accuracy
Our engineered transferases ensure precise Type II structure formation, delivering LNnT that is chemically and biologically identical to human milk.
Plasmid-Free Stability
Our strains feature genomic integration of biosynthetic genes, eliminating the need for antibiotics and ensuring stability over hundreds of generations.
Multi-Host Versatility
We offer production in both bacterial (E. coli) and yeast (S. cerevisiae) chassis, allowing for flexibility based on regional regulatory preferences.
Minimal Impurity Profiles
Our purification protocols effectively eliminate LNT II (triose) and other intermediate glycans, ensuring a high-purity final product (>98%).
Frequently Asked Questions
Customer Review
"The LNnT from CD BioGlyco is exceptionally consistent. Their structural verification data were critical for our study on neonatal gut colonization."
– Dr. S.H., Head of Pediatric Research
"We found that CD BioGlyco's LNnT had the highest efficacy in stimulating Bifidobacterium growth compared to other commercial sources we tested."
– Dr. J.L., Senior Scientist, Microbiology
"A reliable supply of high-purity LNnT is essential for our research into the gut-brain axis. CD BioGlyco is our preferred partner."
– Dr. K.G., Associate Professor, Clinical Nutrition
Associated Services
Levan Production Service
High-molecular-weight fructans for specialized pharmaceutical study.
Trehalose Production Service
Production of the essential protective disaccharide for protein stabilization.
Chitinbiose Production Service
Precision synthesis of chitin-derived oligosaccharides for gut research.
CD BioGlyco is a global leader in the biosynthetic production of LNnT. By harnessing the power of synthetic glycobiology, we provide a high-quality, scalable, and bio-identical supply of this critical HMO. Our commitment to scientific precision and industrial reliability ensures that our clients are equipped to lead in the fields of infant nutrition and human health. For detailed product specifications, project quotes, or technical inquiries regarding our LNnT production capabilities, please contact us.
Reference
Farhadihosseinabadi, B.; et al. The in vivo effect of Lacto-N-neotetraose (LNnT) on the expression of type 2 immune response involved genes in the wound healing process. Scientific Reports. 2020, 10(1): 997. (Open Access)
For research use only. Not intended for any clinical use.
