3'-Fucosyllactose (3'-FL) is a critical trisaccharide within the human milk oligosaccharide (HMO) family, distinguished by its unique α-1,3-glycosidic linkage. While 2'-FL often dominates the spotlight, 3'-FL is unique because its concentration in human breast milk actually increases over the course of lactation, suggesting a vital role in the later stages of infant development and immune maturation. CD BioGlyco specializes in the high-efficiency biosynthesis of 3'-FL through our advanced Synthetic Glycobiology Platform. We provide a scalable, sustainable alternative to traditional extraction methods, delivering human-identical milk oligosaccharides (HiMOs) that meet the rigorous purity standards required for nutritional, pharmaceutical, and diagnostic research. By re-engineering microbial metabolic flux, CD BioGlyco ensures that this high-value glycan is available for global innovation in gut health and beyond.
Core Technologies
Our production excellence is rooted in the strategic integration of several high-end synthetic biology tools:
α-1,3-Fucosyltransferase Optimization
We utilize a library of high-activity α-1,3-fucosyltransferases (α1,3-FT), specifically engineered for superior affinity toward lactose and GDP-L-fucose.
GDP-L-fucose Regeneration Modules
To overcome the cost-limiting nature of nucleotide sugar donors, we implement a de novo synthesis pathway (ManB, ManC, Gmd, and WcaG) that recycles metabolic intermediates.
Metabolic Flux Redirection
Using CRISPR/Cas9, we disable competing lactose-consuming enzymes (e.g., β-galactosidase) and fucose-degrading pathways to ensure 100% of the substrate is channeled toward 3'-FL.
Synthetic Glycobiology Chassis
We employ GRAS-certified bacterial and yeast strains, optimized for high-density fermentation and minimal byproduct secretion, ensuring a clean downstream profile.
Engineering the Future of Humanized Nutrition through Synthetic Glycobiology.
At CD BioGlyco, our 3'-FL production service is a flagship component of our broader HMO platform. Our service scope extends far beyond simple contract manufacturing; we provide a holistic partnership for glycan innovation. We offer custom microbial strain engineering, where we tailor the host organism to utilize specific, low-cost carbon sources such as glycerol or glucose. Our process development service assists clients in moving from laboratory proof-of-concept to pilot-scale production, providing all necessary documentation for regulatory filings.
Furthermore, we offer high-purity 3'-FL supply in quantities ranging from milligrams for laboratory assays to multi-kilogram batches for clinical trials. Our analytical suite includes quantitative glycomics, ensuring that every milligram of 3'-FL delivered meets the exact specifications for purity and bio-identity. We also provide isotope-labeled 3'-FL synthesis for pharmacokinetic and metabolic studies. By leveraging our deep expertise in synthetic glycobiology, CD BioGlyco supports the development of infant formulas, dietary supplements, and therapeutic agents that aim to mimic the complex immunological benefits of human milk. Our scope also includes the production of 3'-FL derivatives and analogs for specialized glyco-biology research, ensuring our clients stay at the forefront of carbohydrate science.
Workflow
Enzyme Engineering and Selection
We initiate enzyme discovery by screening a diverse library of α-1,3-fucosyltransferase (3-FucT) genes, primarily sourced from microbial organisms such as Helicobacter pylori and Bacteroides fragilis known for their activity. The most promising candidates are subjected to rational protein engineering and directed evolution campaigns. Key engineering goals include enhancing thermostability for robustness during fermentation, improving catalytic efficiency (kcat/Km) for higher productivity, and increasing solubility/expression in the chosen microbial host (e.g., E. coli or yeast). High-throughput screening assays are used to identify variants with superior performance, ultimately selecting a lead enzyme variant that ensures efficient and specific formation of the α-1,3 linkage onto lactose.
Pathway Integration
The biosynthetic pathway for 3'-FL is stably incorporated into the host cell's genome to ensure genetic stability without antibiotic selection. This involves the precise chromosomal integration of two coordinated modules: 1) the de novo GDP-L-fucose donor synthesis module (e.g., genes manB, manC, gmd, wcaG), and 2) the optimized α-1,3-fucosyltransferase gene. Using synthetic biology tools, such as promoters and ribosomal binding sites (RBS) of tunable strength, we meticulously balance the expression of all pathway genes. This fine-tuning is critical to prevent the accumulation of metabolic intermediates (like GDP-L-fucose or phosphorylated sugars) that could cause toxicity, inhibit growth, or lead to unwanted byproducts, thereby ensuring efficient flux towards 3'-FL.
Fermentation Optimization
Process development begins in controlled, small-scale bioreactors. A design of experiments (DoE) approach is employed to systematically evaluate and optimize critical process parameters. This includes determining the ideal cultivation temperature and pH profile for both cell growth and product synthesis. A key focus is optimizing the lactose feeding strategy, as lactose serves as the essential acceptor molecule for fucosylation. We test different feeding schedules (e.g., pulse, continuous, or pH-stat feeds) and concentrations to maintain non-inhibitory, non-repressing levels that maximize 3'-FL titers, yield, and productivity while minimizing residual lactose and diauxic effects.
Scale-Up Fermentation
The optimized process parameters are transferred to our pilot or production-scale fermenters. We implement advanced high-cell-density fed-batch strategies, precisely controlling the feed of carbon sources (e.g., glycerol or glucose) and nitrogen to achieve and maintain very high biomass concentrations. The process is carefully scaled by maintaining constant key parameters such as oxygen transfer rate (OTR), mixing time, and power input per volume. The objective is to reproducibly achieve industrially relevant 3'-FL concentrations with high volumetric productivity, ensuring the process is robust, economical, and ready for manufacturing.
Downstream Processing (DSP)
Following fermentation, the harvest broth undergoes a stringent, multi-step purification sequence designed to isolate 3'-FL from the complex biological matrix. The process typically starts with cell separation via microfiltration or centrifugation. The clarified broth then undergoes ultrafiltration (UF) to remove high molecular weight impurities like host cell proteins (HCP) and DNA fragments. Subsequent nanofiltration (NF) is employed to concentrate the product, remove salts, and eliminate smaller organic impurities and pigments. A final polishing step, often involving ion-exchange chromatography or activated carbon treatment, ensures the removal of any remaining charged or colored contaminants, resulting in a highly pure product solution.
Structural Confirmation
Rigorous analytical control is applied to every production batch. The primary structure and linkage specificity are confirmed using a combination of orthogonal techniques. Liquid chromatography-mass spectrometry (LC-MS) provides accurate mass confirmation and purity assessment. Proton nuclear magnetic resonance spectroscopy (1H-NMR) is the definitive method to verify the characteristic anomeric proton signal of the α-1,3 glycosidic linkage, unequivocally distinguishing 3'-FL from its isomer 2'-FL and other possible fucosylated by-products. This dual analysis guarantees the structural fidelity, linkage specificity, and isomeric purity of the final 3'-FL product, ensuring it meets all specifications.
Publication Data
Journal: Scientific Reports
DOI: 10.1038/s41598-020-72792-z
IF: 3.9
Published: 2020
Results: In this study, the authors investigated the strain-specific strategies of Bifidobacterium longum subsp. infantis, comparing the probiotic strain Bi-26 with the type strain 15697 in utilizing fucosylated HMOs, specifically 2'-FL, 3'-FL, and difucosyllactose (DFL). Through growth assays, metabolite profiling, and transcriptomic analyses, they demonstrated that Bi-26 exhibits faster growth rates, higher cell densities, and distinct fermentation end-products, such as increased lactate and pyruvate excretion, compared to 15697, which produced more acetate and formate. Genomic comparisons revealed that Bi-26 has a reduced genome, lacking certain transporters and glycosidases but retaining core metabolic genes, leading to a more efficient and specialized utilization of FLs. Transcriptome data indicated that Bi-26 activates broader regulatory networks in response to FLs, suggesting adaptive evolution for prioritizing these abundant HMOs. The findings highlight that even within the same subspecies, metabolic strategies can vary significantly, influencing ecological competitiveness in the infant gut microbiome. This work underscores the importance of strain-level differences in probiotic functionality and HMO metabolism.
Application
Metabolic Syndrome Studies
Evaluating the effect of 3'-FL on glucose metabolism and weight management by modulating the GLP-1 pathway via gut microbiota metabolites.
Diagnostic Glycan Standards
Used as a high-purity analytical standard for glycomics research and the calibration of mass spectrometry equipment in clinical labs.
Skin Microbiome Care
Incorporated into topical formulations to promote the growth of skin-protective commensals and reduce the colonization of harmful Staphylococcus species.
Vaccine Adjuvant Development
Exploring the use of fucosylated glycans as natural components to enhance the targeted delivery and efficacy of oral vaccines.
Advantages
Superior α-1,3-Linkage Specificity
Our proprietary enzymes ensure 100% regioselectivity, preventing the formation of unwanted isomers like 2'-FL, which is critical for accurate biological results.
Animal-Free Production
Our entire process is strictly microbial-based, ensuring that the 3'-FL is vegan-friendly, Halal, Kosher, and free from any bovine-derived contaminants.
Enhanced Metabolic Stability
By engineering the host to be deficient in fucose-processing enzymes, we achieve a high conversion rate of lactose to 3'-FL with minimal substrate waste.
Optimized Downstream Recovery
Our refined purification protocols achieve >99% purity, removing all traces of lactose and monosaccharides that could interfere with downstream applications.
Frequently Asked Questions
Customer Review
"The 3'-FL from CD BioGlyco showed remarkable consistency across different batches. It was crucial for our long-term study on gut colonization patterns."
– Dr. H.W., Lead Scientist, Pediatric Health
"CD BioGlyco's ability to scale production quickly allowed us to meet our clinical trial deadlines. Their technical support during the formulation phase was invaluable."
– Manager L.K., R&D, Functional Ingredients
"We were specifically looking for high-purity 3'-FL without any 2'-FL contamination. CD BioGlyco delivered exactly what we needed for our receptor binding assays."
– Dr. C.B., Principal Researcher, Immunology
"Excellent service and high-quality glycans. Their synthetic glycobiology platform is clearly a leader in the field of HMO production."
– Dr. S.J., Associate Professor, Microbiome Studies
Associated Services
Blood Group B Tri-saccharide Antigen Production Service
High-purity synthesis of B-antigen motifs for transfusion medicine research.
Blood Group A Hexa-saccharide Antigen Production Service
Advanced production of complex A-antigen structures for diagnostic development.
Blood Group B Hexa-saccharide Antigen Production Service
Specialized synthesis of large B-antigen glycans for immunological studies.
CD BioGlyco stands at the intersection of synthetic biology and human health, providing unparalleled expertise in the production of 3'-FL. Our platform overcomes the traditional barriers of glycan synthesis, offering a reliable, high-purity, and scalable source of this essential human milk oligosaccharide. By choosing CD BioGlyco, you are partnering with a specialist committed to precision, safety, and scientific excellence. To explore how our 3'-FL production capabilities can support your research or product development goals, please contact us. We offer customized solutions tailored to your specific purity and volume requirements.
Reference
Zabel, B.E.; et al. Strain-specific strategies of 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose assimilation by Bifidobacterium longum subsp. infantis Bi-26 and ATCC 15697. Scientific Reports. 2020, 10(1): 15919. (Open Access)
For research use only. Not intended for any clinical use.
