Lacto-N-triose II (LNT II), characterized by the trisaccharide structure GlcNAc(β1-3)Gal(β1-4)Glc, is a fundamental neutral Human Milk Oligosaccharide (HMO). Beyond its intrinsic biological value, LNT II serves as the indispensable structural backbone for the biosynthesis of more complex Type I and Type II HMOs, including Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). CD BioGlyco provides a specialized LNT II production service powered by advanced Synthetic Glycobiology. By utilizing engineered microbial factories and cell-coupled biocatalytic strategies, we overcome the limitations of traditional extraction and chemical synthesis. Our platform delivers high-purity LNT II that facilitates groundbreaking research in infant nutrition, microbiome modulation, and therapeutic development.
Core Technologies
CD BioGlyco leverages a multi-dimensional engineering approach to ensure high-titer LNT II biosynthesis:
Glycosyltransferase Engineering
We utilize highly active β-1,3-N-acetylglucosaminyltransferases (e.g., LgtA from Neisseria meningitidis) optimized for regioselective attachment of GlcNAc to lactose.
UDP-GlcNAc Flux Optimization
Our strains feature refactored hexosamine biosynthetic pathways, overexpressing glmS, glmM, and glmU to ensure a saturating supply of nucleotide sugar donors.
Transporter Engineering
We overexpress endogenous efflux transporters such as setA and ydeA, which significantly enhances the extracellular accumulation of LNT II while reducing intracellular metabolic burden.
Cell-Coupled Biocatalysis
We employ a two-step strategy using engineered E. coli coupled with energy-generating yeast modules to facilitate high-efficiency conversion of GlcNAc and lactose into LNT II.
Building the Foundations of Gut Health: Advanced Glycobiology for High-Purity LNT II.
The LNT II production service is a comprehensive solution designed to meet the growing global demand for HMO core structures. Our service scope covers the entire development spectrum, beginning with custom microbial factory development. We offer the engineering of specific bacterial hosts (e.g., E. coli or Bacillus subtilis) that are tailored to your preferred carbon sources and production environment. Our process development and scale-up service assists clients in transitioning from milligram-scale research to metric-ton manufacturing, providing the necessary documentation for regulatory compliance and safety assessments.
Furthermore, we provide biocatalytic synthesis of LNT II derivatives, allowing for the modification of the triose backbone for specialized pharmaceutical applications. Our analytical characterization suite ensures that every milligram of LNT II delivered is of the highest structural integrity, supporting clinical trials and nutritional studies. We also offer strategic co-production, where LNT II is synthesized alongside other HMOs to create customized prebiotic blends. By integrating metabolic flux optimization with high-performance purification, CD BioGlyco empowers researchers and manufacturers to access high-quality LNT II that was previously difficult or too expensive to acquire. Our scope is built on the principle of sustainability, utilizing renewable feedstocks like GlcNAc from chitin to produce bio-identical milk oligosaccharides.
Workflow
1. Metabolic Pathway Design
We begin by designing an optimized metabolic chassis using genome-scale metabolic models and constraint-based flux balance analysis (FBA). This computational approach allows us to simulate carbon and energy flow through the native host metabolism and identify key nodes that compete with our target biosynthetic pathway. Based on these models, we strategically inactivate specific genes to eliminate undesirable side reactions. For instance, we knock out lacZ to prevent the hydrolytic cleavage of the precious acceptor substrate, lactose, into glucose and galactose. Simultaneously, we inactivate nanE or related genes in the N-acetylneuraminic acid salvage pathway to prevent the degradation and diversion of GlcNAc, a critical precursor, thereby channeling maximum flux toward the synthesis of the target HMO backbone.
2. Strain Construction & Integration
Following the in silico design, we engineer the production strain using precise genome editing. The key heterologous biosynthetic modules, encoding enzymes for precursor activation and the specific glycosyltransferase responsible for forming the target linkage, are assembled and integrated directly into the host chromosome. We employ CRISPR/Cas9 technology for high-efficiency, markerless integration at predetermined neutral sites. This method ensures stable, long-term genetic inheritance without the need for antibiotic selection, a crucial feature for industrial fermentation where plasmid instability and the use of selective agents are major concerns. The result is a robust, plasmid-free production strain with consistent performance over many generations.
3. Fermentation Optimization
We develop the bioprocess using high-throughput, parallel miniature bioreactor systems that mimic the conditions of larger vessels. This platform enables rapid, systematic screening of dozens of cultivation parameters. A primary focus is optimizing the dynamic feeding strategy for the two key substrates: lactose (acceptor) and GlcNAc (donor precursor). We test various feeding profiles, including pulsed, exponential, and pH-stat feeds, to carefully balance the growth phase with the production phase. The goal is to maintain non-inhibitory, yet non-limiting, concentrations of both substrates to maximize the specific productivity and final titer while minimizing the formation of metabolic by-products.
4. High-Titer Biosynthesis
Once optimized at the bench scale, the fermentation process is transferred to our pilot and production-scale bioreactors. We implement a high-cell-density fed-batch strategy, which involves an initial batch phase for rapid biomass accumulation, followed by a controlled feed phase that sustains the cells in a productive state for an extended period. Advanced process control loops continuously adjust nutrient feeds, pH, dissolved oxygen, and agitation to maintain optimal physiological conditions. This scalable approach reliably drives the synthesis of the target oligosaccharide to industrially relevant concentrations, consistently achieving high titers that often exceed 30 grams per liter, ensuring cost-effective manufacturing.
5. Multi-Stage Purification
The harvest broth undergoes a stringent, sequential purification process tailored to the physicochemical properties of the target molecule. The first step involves centrifugal separation or microfiltration to remove biomass. The clarified supernatant is then treated with activated carbon in a decolorization step to adsorb pigments and hydrophobic impurities. The core purification employs high-resolution ion-exchange chromatography, which selectively captures the charged oligosaccharide away from neutral sugars, residual salts, and other process-related impurities. Each step is optimized for yield and purity, resulting in a final product that meets rigorous quality standards with a chemical purity exceeding 98%.
6. Structural Characterization
Rigorous analytical control is applied to every production batch. We use a combination of orthogonal techniques for definitive verification. Liquid chromatography-mass spectrometry (LC-MS) provides accurate mass confirmation and assesses purity by detecting any residual substrates or related isomers. The conclusive proof of structure is obtained through proton nuclear magnetic resonance (1H-NMR) spectroscopy. This technique specifically confirms the presence and configuration of the characteristic β-1,3 glycosidic linkage by identifying the unique anomeric proton signals in the spectrum. Together, these analyses provide a comprehensive quality profile, guaranteeing the identity, linkage specificity, and high purity of the final product.
Publication Data
Journal: Scientific Reports
DOI: 10.1038/s41598-021-02741-x
IF: 3.9
Published: 2021
Results: In this study, the authors explored the utilization of HMOs by bacterial strains isolated from infant feces, focusing on their role in shaping the infant gut microbiota. They isolated twelve strains from genera including Bifidobacterium, Enterococcus, and Lactobacillus, and assessed their growth on major HMOs like 2'-fucosyllactose, LNT, and LNnT. Results revealed that only B. species could metabolize HMOs; specifically, Bifidobacterium infantis efficiently consumed all tested oligosaccharides, while B. dentium strains utilized only LNT and LNnT. Interestingly, B. dentium hydrolyzed these tetra-saccharides into galactose and LNTII, but solely consumed the galactose moiety, excreting LNT II into the environment. The researchers further characterized two intracellular β-galactosidases from B. dentium responsible for this cleavage, providing mechanistic insights into HMO degradation. These findings emphasize the specialized adaptation of bifidobacteria to HMOs and suggest potential cross-feeding dynamics in the infant gut ecosystem.
Application
Metabolic Engineering Precursors
LNT II is a critical starting material for the enzymatic synthesis of sialylated and fucosylated HMOs, facilitating the production of a wide range of human-identical glycans.
Diagnostic Glycan Arrays
High-purity LNT II is used to functionalize diagnostic surfaces for the detection of carbohydrate-binding antibodies and lectins in clinical samples.
Skin Microbiome Balancing
Exploring the application of LNT II in topical formulations to promote the growth of skin-protective commensal bacteria while inhibiting the colonization of opportunistic pathogens.
Nutraceutical Development
Development of premium prebiotic ingredients for functional foods and beverages that target the enhancement of the gut-brain axis and overall systemic wellness.
Advantages
Unmatched Regioselectivity
Our proprietary β-1,3-N-acetylglucosaminyltransferases ensure 100% correct linkage, preventing the formation of isomeric impurities common in chemical synthesis.
Plasmid-Free Stability
We utilize genomic integration technologies to create stable production strains that do not require antibiotic selection, making them ideal for manufacturing.
Optimized Efflux Systems
By engineering specific transporters like setA, we achieve higher extracellular yields and simplified downstream processing by keeping the product out of the cytoplasm.
High Molar Conversion
Our cell-coupled biocatalytic systems achieve exceptionally high conversion rates from lactose and GlcNAc, minimizing raw material waste and reducing costs.
Frequently Asked Questions
Customer Review
"The LNT II from CD BioGlyco is the cleanest we have ever tested. Its high purity was essential for our enzymatic elongation studies toward complex HMOs."
– Dr. F.G., Principal Scientist, Nutritional Research
"CD BioGlyco's ability to provide large-scale LNT II without plasmid instability issues has been a significant advantage for our commercialization timeline."
– Manager H.S., R&D, Functional Ingredients
"We observed excellent selectivity for Bifidobacterium in our in vitro models using CD BioGlyco's LNT II. Their structural validation is truly world-class."
– Dr. Y.Z., Senior Investigator, Microbiome Studies
"A reliable source for high-purity LNT II was missing in the industry until now. CD BioGlyco's Synthetic Glycobiology platform is a game-changer."
– Dr. E.R., Associate Professor, Carbohydrate Chemistry
Associated Services
Dextran Production Service
High-quality ɑ-1,6-glucan synthesis for pharmaceutical and clinical applications.
Curdlan Production Service
Production of β-1,3-glucans for specialized food textures and immunological research.
Succinoglycan Production Service
Advanced synthesis of acidic exopolysaccharides for industrial and biomedical use.
CD BioGlyco is a premier leader in the production of LNT II, providing the foundational building blocks for the next generation of HMO innovation. By utilizing state-of-the-art metabolic engineering and transporter optimization, we offer a high-purity, scalable, and sustainable supply of LNT II for global researchers and manufacturers. Our expertise ensures that your projects are supported by the highest quality glycans and comprehensive technical documentation. To discuss your LNT II requirements or to learn more about our synthetic glycobiology platform, please contact us. We are ready to support your project from concept to commercial scale.
Reference
Moya-Gonzálvez, E.M.; et al. Infant-gut associated Bifidobacterium dentium strains utilize the galactose moiety and release lacto-N-triose from the human milk oligosaccharides lacto-N-tetraose and lacto-N-neotetraose. Scientific reports. 2021, 11(1): 23328. (Open Access)
For research use only. Not intended for any clinical use.
