Human Milk Oligosaccharides Research Articles

Metabolic Engineering of Escherichia coli BL21(DE3) for 2'-Fucosyllactose Synthesis in a Higher Productivity.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharides (HMOs). 2'-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2'-FL. Here, we aimed to construct a recombinant E. coli BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene lacY reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the manC-manB and gmd-wcaG gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the pgi gene. This further improved 2'-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in Neisseria sp., which exhibited a higher capacity for 2'-FL production. Fed-batch fermentation produced 141.27 g/L 2'-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2'-FL levels, indicating the potential of engineered E. coli BL21 (DE3) strains for use in the industrial production of 2'-FL.

Profiles of 71 Human Milk Oligosaccharides and Novel Sub-Clusters of Type I Milk: Results from the Ulm SPATZ Health Study

Background/Objectives: Although approximately 160 human milk oligosaccharides (HMOs) have been identified, current studies on HMO quantitation are limited to the 10–19 most abundant HMOs. We assessed the variations in the relative concentrations of 71 HMO structures over lactation in human milk samples by an advanced liquid chromatography–mass spectrometry approach. Methods: Samples were collected from 64 mothers at 6 weeks, 6 months, and 12 months of lactation in the Ulm SPATZ Health Study, a German birth cohort. In this longitudinal study, we fitted linear mixed-effect models to analyze changes in the log2-transformed and standardized HMO concentration over time. Based on the profile of 71 HMOs, we also fitted a group-based multi-trajectory (GBMT) model to cluster mothers secreting cluster type I milk, who account for the majority of lactating mothers. Results: We found that 52 HMOs had a decreasing trend (regression coefficients ranging from −1.41 to −0.17) and 9 had an increasing trend (regression coefficients ranging from 0.25 to 0.64) during lactation, and the findings were statistically significant after multiple testing corrections. Using human milk samples of 49 mothers with type I milk, we further identified two novel sub-clusters with distinct longitudinal trajectories of concentrations of 71 HMOs during lactation: Type I-a (N = 20) and I-b (N = 29). These sub-clusters were not associated with maternal non-genetic characteristics. Conclusions: Our findings extend existing knowledge about the structural diversity of HMOs and their variations over lactation. These may pave the way to investigate the potential nutritional benefits of various HMOs on infant health and early life development in the future.

Interactions of human milk oligosaccharides with the immune system

Human milk oligosaccharides (HMOs) are abundant, diverse and complex sugars present in human breast milk. HMOs are well-characterized barriers to microbial infection and by modulating the human microbiome they are also thought to be nutritionally beneficial to the infant. The structural variety of over 200 HMOs, including neutral, fucosylated and sialylated forms, allows them to interact with the immune system in various ways. Clinically, HMOs impact allergic diseases, reducing autoimmune and inflammatory responses, and offer beneficial support to the preterm infant immune health. This review examines the HMO composition and associated immunomodulatory effects, including interactions with immune cell receptors and gut-associated immune responses. These immunomodulatory properties highlight the potential for HMO use in early stage immune development and for use as novel immunotherapeutics. HMO research is rapidly evolving and promises innovative treatments for immune-related conditions and improved health outcomes.

Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

Free oligosaccharides in human milk have many biological functions for infant health. The reducing end of most human milk oligosaccharides is lactose, and caprine milk was reported to contain oligosaccharides structurally similar to those present in human milk. The structures of oligosaccharides were traditionally determined using nuclear magnetic resonance spectroscopy or enzyme digestion followed by various detection methods, e.g., liquid. Mass spectrometry has much higher sensitivity than nuclear magnetic resonance spectroscopy and enzyme digestion. However, conventional mass spectrometry methods only determine part of the structures of oligosaccharides, i.e., compositions and linkage positions. In this study, we used the latest developed mass spectrometry method, namely logically derived sequence tandem mass spectrometry, to determine the complete structures (i.e., composition, linkage positions, anomericities, and stereoisomers) of free neutral trisaccharides in caprine colostrum and mature milk. The high sensitivity of mass spectrometry enables us to discover oligosaccharides of low abundance. Isomers of (Hex)2HexNAc, (Hex)3, and (Hex)2Fuc which have not been reported before were identified. Many of them do not have lactose at the reducing end. Instead, the reducing end is either Glcβ-(1–4)-Glc or Glcβ-(1–4)-GlcNAc. These unusual oligosaccharides are higher in concentration and more structurally diverse in caprine colostrum than that in caprine mature milk and human milk. The structural diversity indicates more complicated biosynthetic pathways of caprine milk compared to that of human milk.

Optimization, validation, and greenness assessment of a capillary zone electrophoresis method for analysis of inorganic and organic anions in Human Milk Oligosaccharides

Optimization, validation, and greenness assessment of a capillary zone electrophoresis method for analysis of inorganic and organic anions in Human Milk Oligosaccharides

Characterization of Bifidobacterium bifidum growth and metabolism on whey protein phospholipid concentrate.

Whey protein phospholipid concentrate (WPPC) is a co-product generated during the manufacture of whey protein isolate. WPPC is depleted of simple sugars but contains numerous glycoconjugates embedded in the milk fat globule membrane, suggesting this fraction may serve as a carbon source for growth of bifidobacteria commonly enriched in breast fed infants. In this work, we demonstrate that WPPC can serve as a sole carbon source for the growth of Bifidobacterium bifidum, a species common to the breastfed infant and routinely used as a probiotic. Growth on WPPC fractions resulted in expression of key extracellular glycosyl hydrolases in B. bifidum associated with the catabolism of glycoproteins. Interestingly, this included induction of fucosidase genes in B. bifidum linked to catabolism of fucosylated human milk oligosaccharides even though the WPPC glycan possesses little fucose. Additional growth studies revealed that WPPC-glycan components N-acetylglucosamine or N-acetylgalactosamine were required for pre-activation of B. bifidum toward rapid growth on the fucosylated human milk oligosaccharides. Growth on WPPC fractions also resulted in expression of extracellular sialidases in B. bifidum which promoted a consistent release of sialic acid, a well-known component of bovine milk oligosaccharides and glycoconjugates with potential impacts on gut microbial ecology and host cognition. These studies suggest WPPC may serve as a promising bioactive component to facilitate probiotic activity for use in infant formulas and other synbiotic applications.

The Bifidogenic Effect of 2'Fucosyllactose Is Driven by Age-Specific Bifidobacterium Species, Demonstrating Age as an Important Factor for Gut Microbiome Targeted Precision Medicine.

The human gut microbiota develops in concordance with its host over a lifetime, resulting in age-related shifts in community structure and metabolic function. Little is known about whether these changes impact the community's response to microbiome-targeted therapeutics. Providing critical information on this subject, faecal microbiomes of subjects from six age groups, spanning from infancy to 70-year-old adults (n = six per age group) were harvested. The responses of these divergent communities to treatment with the human milk oligosaccharide 2'-fucosyllactose (2'FL), fructo-oligosaccharides (FOS), and lactose was investigated using the Ex vivo SIFR® technology that employs bioreactor fermentation and is validated to be predictive of clinical findings. Additionally, it was evaluated whether combining faecal microbiomes of a given age group into a single pooled microbiome produced similar results as the individual microbiomes. First, marked age-dependent changes in community structure were identified. Bifidobacterium levels strongly declined as age increased, and Bifidobacterium species composition was age-dependent: B. longum, B. catenulatum/pseudocatenulatum, and B. adolescentis were most prevalent for breastfed infants, toddlers/children, and adults, respectively. Metabolomic analyses (LA-REIMS) demonstrated that these age-dependent differences particularly impacted treatment effects of 2'FL (more than FOS/lactose). Further analysis revealed that while 2'FL enhanced production of short-chain fatty acids (SCFAs) and exerted potent bifidogenic effects, regardless of age, the specific Bifidobacterium species enhanced by 2'FL, as well as subsequent cross-feeding interactions, were highly age-dependent. Furthermore, single-pooled microbiomes produced results that were indicative of the average treatment response for each age group. Nevertheless, pooled microbiomes had an artificially high diversity, thus overestimating treatment responses (especially for infants), did not recapitulate interindividual variation, and disallowed for the correlative analysis required to unravel mechanistic actions. Age is an important factor in shaping the gut microbiome, with the dominant taxa and their metabolites changing over a lifetime. This divergence affects the response of the microbiota to therapeutics, demonstrated in this study using 2'FL. These results evidence the importance of screening across multiple age groups separately to provide granularity of how therapeutics impact the microbiome and, consequently, human health.

Personalized modeling of gut microbiome metabolism throughout the first year of life

BackgroundEarly-life exposures including diet, and the gut microbiome have been proposed to predispose infants towards multifactorial diseases later in life. Delivery via Cesarian section disrupts the establishment of the gut microbiome and has been associated with negative long-term outcomes. Here, we hypothesize that Cesarian section delivery alters not only the composition of the developing infant gut microbiome but also its metabolic capabilities. To test this, we developed a metabolic modeling workflow targeting the infant gut microbiome.MethodsThe AGORA2 resource of human microbial genome-scale reconstructions was expanded with a human milk oligosaccharide degradation module. Personalized metabolic modeling of the gut microbiome was performed for a cohort of 20 infants at four time points during the first year of life as well as for 13 maternal gut microbiome samples.ResultsHere we show that at the earliest stages, the gut microbiomes of infants delivered through Cesarian section are depleted in their metabolic capabilities compared with vaginal delivery. Various metabolites such as fermentation products, human milk oligosaccharide degradation products, and amino acids are depleted in Cesarian section delivery gut microbiomes. Compared with maternal gut microbiomes, infant gut microbiomes produce less butyrate but more L-lactate and are enriched in the potential to synthesize B-vitamins.ConclusionsOur simulations elucidate the metabolic capabilities of the infant gut microbiome demonstrating they are altered in Cesarian section delivery at the earliest time points. Our workflow can be readily applied to other cohorts to evaluate the effect of feeding type, or maternal factors such as diet on host-gut microbiome inactions in early life.

FUNCTIONS AND INDIVIDUAL MECHANISMS OF INFLUENCE OF BIFIDOBACTERIA ON THE STATE OF HEALTH OF INFANTS (Literary review)

Bifidobacteria are members of the human intestinal microbiota, and some strains may have health-promoting effects. The genus Bifidobacterium belongs to the phylum Actinobacteria. Firmicutes, Bacteroidetes, and Actinobacteria are the most abundant phyla in the human gut microbiota, with Firmicutes and Bacteroidetes predominating in adults and Actinobacteria in breast-fed infants, where Bifidobacteria can reach levels exceeding 90% of the total bacterial population. They are among the first microbial colonizers of the newborn gut and play a key role in the development of its physiology, including the maturation of the immune system and the utilization of food components. In fact, some nutrients, such as human milk oligosaccharides, are important factors in the development of bifidobacteria. There is now well-documented scientific evidence of the efficacy of bifidobacteria-containing products in some intestinal and extraintestinal pathologies. In this review, we will focus on the role of bifidobacteria as members of the human intestinal microbiota and their use as probiotics for disease prevention and treatment.

Evaluating clinical opinions on the efficacy and tolerability of a novel human milk oligosaccharide-based human milk fortifier containing lactoferrin, docosahexaenoic acid and arachidonic acid

Background: To gather and analyse the opinions of healthcare professionals regarding the effectiveness and tolerability of a novel human milk oligosaccharide (HMO)-based fortifier containing lactoferrin, docosahexaenoic acid (DHA) and arachidonic acid (ARA) in neonatal nutrition. Methods: A cross-sectional survey was conducted among neonatologists and paediatricians who have utilised the novel HMO-based human milk fortifier (HMoF) in their clinical practice. Participants were asked to provide their feedback on various parameters including nutritional adequacy, growth outcomes, tolerance, and overall clinical efficacy. Results: Majority of respondents reported positive outcomes in terms of enhanced growth parameters, improved gastrointestinal tolerance, and overall health of the infants. The fortifier was particularly noted for its role in supporting the overall growth of the infants including weight gain, length gain and head circumference gain due to the presence of HMOs, lactoferrin, DHA, and ARA. Conclusions: The novel HMO-based human milk fortifier containing lactoferrin, DHA, and ARA has garnered a favourable and positive opinion by healthcare professionals and demonstrates potential benefits in enhancing neonatal nutrition and health outcomes.

Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in Escherichia coli.

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved de novo biosynthesis of DFL; however, substantial residual intermediates of fucosyllactoses (FL) were detected. In this study, DFL biosynthesis was optimized, and residual FL were reduced by regulating metabolic pathways. Different plasmid combinations were used to regulate gene expression, achieving an optimal flux balance between 2'-FL and DFL. The expression level of key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) was then enhanced by increasing plasmid copy number and integrating fucTa gene into the chromosome. Exocytosis of 2'-FL was reduced by deleting the sugar efflux transporter setA gene, thereby minimizing residual FL. Finally, strain BSF41 produced 55.3 g/L of DFL with only 2.59 g/L of residual FL in a 5 L fermentor, representing the highest reported titer to date. This study provides an important foundation for advancing the biosynthesis of fucosylated HMOs.

Role of maternal milk in providing a healthy intestinal microbiome for the preterm neonate.

The immature gastrointestinal tract of preterm neonates leads to a delayed and distinctive establishment of the gut microbiome, making them susceptible to potentially pathogenic bacteria and increasing the risk of infections. Maternal milk, recognized as the optimal source of nutrition, plays a multifaceted role in modulating the gut microbiome of premature newborns. Human milk oligosaccharides, acting as prebiotics, provide essential nourishment for key bacteria such as Bifidobacterium, contributing to the proliferation of beneficial bacterial populations. Additionally, maternal milk is rich in Immunoglobulins that stimulate immune cell responses, providing protective effects on the infant's gut mucosa. Moreover, bioactive proteins such as secretory immunoglobulin A (SIgA), lactoferrin, lysozyme, and mucins play a crucial role in defending against pathogens and regulating the immune system at the cellular level. These proteins contribute not only to infection prevention but also emphasize the impact of breast milk in fortifying the body's innate defenses. This multifaceted role of maternal milk, including essential nutrients, beneficial bacteria, and bioactive proteins, highlights the importance of promoting the mother's own milk feeding in the Neonatal Intensive Care Unit (NICU). It not only optimizes the long-term outcomes and well-being of preterm infants but also provides a holistic approach to their health and development. IMPACT: This article contributes to the current understanding of the relationship between breastfeeding and the intestinal microbiota. Fill gaps in existing literature about the subject. Provides new insights for future research.

Absolute quantification of eight human milk oligosaccharides in breast milk to evaluate their concentration profiles and associations with infants' neurodevelopmental outcomes.

Human milk oligosaccharides (HMOs) have been positively associated with child neurodevelopment in some cohort studies. However, there is a lack of consistency in the association between HMOs and benefits to infants' brains. Moreover, the quantification methods for HMOs have not yet been standardized. In this study, we developed a quantification method for evaluating eight HMOs (2'-fucosyllactose [2'-FL], 3'-fucosyllactose [3'-FL], 3'-sialyllactose [3'-SL], 6'-sialyllactose [6'-SL], lactosialyltetrasaccharide a [LSTa], lactosialyltetrasaccharide b [LSTb], lactosialyltetrasaccharide c [LSTc], and disialyllacto-N-tetraose [DSLNT]) in breast milk. After validating the method, we applied it to 1-month breast milk samples (n=150) from the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study to assess HMO profiles in breast milk and their possible association with changes in head circumference z-score (ΔHCZ) and neurodevelopmental scores of children (as measured by the Ages and Stages Questionnaire, Third Edition). The validation demonstrated that the method had relative standard deviation ≤ 12.7% of precision and 79.5-110.9% of accuracy. Using this method, eight HMO levels (2'-FL, 0-4.74 mg/mL; 3'-FL, 0.02-1.52 mg/mL; 3'-SL, 0.07-0.32 mg/mL; 6'-SL, 0.01-0.70 mg/mL; LSTa, 0.002-0.043 mg/mL; LSTb, 0.02-0.31 mg/mL; LSTc, 0.001-0.47 mg/mL; and DSLNT, 0.09-0.71 mg/mL [min-max, all participants]) and the ratio of low secretors (16.0%) in the Japanese cohort were obtained. The obtained HMO levels in breast milk were subjected to multivariate analysis to screen for HMOs showing a positive association with ΔHCZ and neurodevelopmental scores. The results proposed that ΔHCZ was positively associated with LSTb and 2'-FL levels, whereas neurodevelopmental scores were positively associated with 2'-FL levels (among all participants) and 3'-SL and DSLNT levels (among secretor participants). This study showed that the developed method provides HMO profiles in Japanese breast milk, as well as additional information on the associations between specific HMOs and neurodevelopment, reinforcing the sum of evidence for the role of HMOs in the brain.

Structural elucidation and characterization of GH29A α-l-fucosidases and the effect of pH on their transglycosylation.

GH29A α-l-fucosidases (EC3.2.1.51) catalyze the release of α-l-fucosyl moieties from the nonreducing end of glycoconjugates by hydrolysis and some also catalyze transglycosylation. The latter is particularly interesting with regard to designing enzymatic synthesis of human milk oligosaccharides (HMOs). We combined the bioinformatics tool conserved unique peptide patterns (CUPP) and phylogenetic clustering to discover new microbial GH29A α-l-fucosidases of the underexplored CUPP group GH29:13.1. Three uncharacterized bacterial enzymes (EaGH29, SeGH29, and PmGH29) and two previously identified GH29A α-l-fucosidases (BF3242 and TfFuc1) were selected for reaction optimization, biochemical, and structural characterization. Kinetics, pH-temperature optima, and substrate preference for 2-chloro-4-nitrophenyl-α-l-fucopyranoside (CNP-α-l-Fuc) and 2'-fucosyllactose (2'FL) were determined. Transglycosylation was favored at high neutral to alkaline pH, especially for EaGH29, SeGH29, TfFuc1, and BF3242, mainly because hydrolysis was decreased. The α-l-fucosidases exhibited medium regioselectivity in transglycosylation, generally forming two out of five detected lacto-N-fucopentaose (LNFP) isomers from 2'FL and lacto-N-tetraose (LNT). Alkaline pH also affected the transglycosylation product regioselectivity of SeGH29, which was also affected by a Leu/Phe exchange in the acceptor binding site. New crystal structures of TfFuc1 and BF3242 showed congruence in active site topology between these two enzymes and contributed to understanding the function of GH29A α-l-fucosidases. Notably, the structural data provide new insight into the role of an Asn residue located between the two catalytic residues in the active site.

Engineered β1-3-N-Acetylglucosaminyltransferase Facilitating the One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides.

β1-3-linked N-acetylglucosaminide is a prevalent carbohydrate motif found in oligosaccharides, polysaccharides, glycoproteins, and glycolipids. It is a crucial component of human milk oligosaccharides (HMOs). Neisseria meningitidis β1-3-N-acetylglucosaminyltransferase (NmLgtA) catalyzes the formation of a glycosidic bond and has the potential for use in synthesizing HMOs. However, this application is hindered by challenges such as low levels of enzyme expression, poor stability, and significant aggregation. Since there is no available crystal structure for NmLgtA, we used its AlphaFold 2 predicted structure to identify potential unfavorable factors. We then modified the enzyme by removing the 17 N-terminal amino acids and substituting nine specific residues. The engineered NmLgtA-Opti exhibited improved thermal stability, increased soluble protein expression, complete relief from aggregation, and enhanced catalysis while maintaining its catalytic specificity and substrate promiscuity. Furthermore, NmLgtA-Opti maximizes substrate utilization and can be employed in a sequential one-pot multienzyme platform for high-yield production of HMOs.

Versatile Strategy for the Chemoenzymatic Synthesis of Branched Human Milk Oligosaccharides Containing the Lacto-N-Biose Motif.

Human milk oligosaccharides (HMOs) exhibit prebiotic, antimicrobial, and immunomodulatory properties and confer significant benefits to infants. Branched HMOs are constructed through diverse glycosidic linkages and prominently feature the lacto-N-biose (LNB, Gal-β1,3-GlcNAc) motif with fucose and/or sialic acid modifications, displaying structural complexity that surpasses that of N- and O-glycans. However, synthesizing comprehensive libraries of branched HMO is challenging due to this complexity. Although a few systematic synthetic strategies have emerged, many of them rely on labor-intensive chemical methodologies or exploit the substrate specificity of human N-acetylglucosaminyltransferase 2 (hGCNT2). In this study, we capitalized on the substrate promiscuities of hGCNT2 and bacterial glycosyltransferases (GTs) to construct a universal tetrasaccharide core in a highly efficient manner. This core was systematically and flexibly extended to generate diverse branched HMOs utilizing the promiscuity of bacterial GTs coupled with N-trifluoroacetyl glucosamine (GlcNTFA), which facilitated sugar chain elongation. The GlcNTFA residues were subsequently converted into various N-modified glucosamines through straightforward chemical manipulations to modulate the activities of additional GTs during glycan extension. These masked amino groups were ultimately reverted to N-acetyl groups, facilitating the synthesis of a broad range of asymmetric and multiantennary HMOs featuring LNB moieties, including many previously inaccessible structures.

Efficient and Cost-Effective Synthesis of N-Acetyllactosamine by Sequential Modular Enzymatic Cascade Reactions Involving NTP Regeneration.

Human milk oligosaccharides (HMOs) play important roles in the development of infants, which are the third most abundant component in human milk. N-Acetyllactosamine (LacNAc) is an important intermediate for the biosynthesis of other HMOs and antigens. Since currently appropriate synthetic methods for large-scale production of LacNAc are not available, it is urgently needed to develop an efficient and cost-effective synthetic pathway for LacNAc preparation. In this study, a cost-effective pathway of LacNAc synthesis involving regeneration of adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP) was established. After optimizing the reaction conditions, LacNAc was synthesized at a yield of >90% via a sequential one-pot multienzyme (OPME) method with crude enzymes at 100 mM substrates. Finally, LacNAc was produced efficiently and cost-effectively at a 5 L scale. This strategy would possibly meet the requirements of potential industrial production of LacNAc and would provide guidance for the production of other structurally complex HMOs or functional oligosaccharides in the future.

Combining 2'-fucosyllactose and galactooligosaccharides exerts anti-inflammatory effects and promotes gut health

This study investigated the potential of 2'-Fucosyllactose (2'-FL) and galactooligosaccharides (GOS) combinations as a novel and cost-effective substitute for human milk oligosaccharides (HMOs) in promoting gut health and reducing inflammation. In vitro studies using Caco-2 cells showed that 2'-FL and GOS combinations (H1: GOS:2'-FL ratio of 1.8:1; H2: ratio of 3.6:1) reduced lipopolysaccharide-induced inflammation by decreasing pro-inflammatory markers, while individual treatments had no significant effects. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, combined 2'-FL and GOS supplementation alleviated symptoms, improved gut permeability, and enhanced intestinal structure, with the GH1 group (H1 combo with DSS) being the most effective. 2'-FL and GOS combinations also enhanced short-chain fatty acid production in infant fecal batch fermentation and mouse fecal analysis, with GH1 showing the most promising results. GH1 supplementation altered gut microbiota in mice with DSS-induced colitis, promoting microbial diversity and a more balanced Firmicutes to Bacteroidota ratio. Infant formula products (IFPs) containing 2'-FL and GOS combinations (IFP2: 174 mg GOS and 95 mg 2'-FL per 14 g serving, 1.8:1 ratio; IFP3: 174 mg GOS and 48 mg 2'-FL per 14 g serving, 3.6:1 ratio) demonstrated gastrointestinal protective and anti-inflammatory properties in a coculture model of Caco-2 and THP-1 cells. These findings suggest that 2'-FL and GOS combinations have potential applications in advanced infant formulas and supplements to promote gut health and reduce inflammation.

De novo 2'-fucosyllactose biosynthesis using glucose as the sole carbon source by multiple engineered Bacillus subtilis.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered Bacillus subtilis strain that produces 2'-FL using glucose as the sole carbon source. First, a lactose biosynthesis module was constructed by expressing β-1,4-galactosyltransferase gene from Neisseria meningitidis. A 2'-FL titer of 2.53±0.07g/L was subsequently achieved using glucose as the sole carbon source by the combination of lactose and GDP-L-fucose (GDP-Fuc) biosynthesis modules. Introducing an exogenous nonphosphorylated transport system enhanced the supply of intracellular nonphosphorylated glucose, and the 2'-FL titer increased to 4.94±0.35g/L. Next, a transcription factor screening platform was designed. Based on this platform, the ligand of the transcription factor LacI was changed from isopropyl β-D-thiogalactoside to lactose. A lactose-responsive genetic circuit was then constructed and used for the dynamic regulation of metabolic fluxes between lactose and GDP-Fuc biosynthesis modules. Ultimately, the 2'-FL titer of the dynamically regulated strain improved by 107% to 9.67±0.65g/L in shake-flask, and the titer and yield in a 3-L bioreactor reached 30.1g/L and 0.15g/g using glucose as the sole carbon source. By using multidimensional engineering strategies, this study constructed a B. subtilis strain capable of efficiently producing 2'-FL with glucose as the sole carbon source, paving the way for the industrial production of 2'-FL with low cost in the future.

Sensitive and high-throughput isomer-specific analysis of human milk oligosaccharides using UPLC-MS/MS and its application to secretor status assignment

Sensitive and high-throughput isomer-specific analysis of human milk oligosaccharides using UPLC-MS/MS and its application to secretor status assignment