Iron absorption from an iron-fortified follow-up formula with and without the addition of a synbiotic or a human-identical milk oligosaccharide: a randomized cross-over stable isotope study in young Thai children.

BACKGROUND: Previous studies showed that pre- and probiotics may enhance iron absorption. Probiotics combined with prebiotics (synbiotics), including human-identical milk oligosaccharides (HiMO), are commonly added to infant and follow-up formula (FUF). Whether these additions enhance iron absorption from iron-fortified commercial milk formula is uncertain. OBJECTIVE: We determined the effect of adding: 1) a synbiotic (galacto-oligosaccharides (GOS)+Limosilactobacillus reuteri [L. reuteri]), or 2) the HiMO 2'-fucosyllactose (2'FL) to iron-fortified FUF on iron absorption in young Thai children. METHODS: In a randomized, controlled, single-blinded (participants) cross-over study, 82 Thai children aged 8-14 months were enrolled to consume single servings (235ml) of FUF with isotopically labelled ferrous sulfate (2.2 mg iron) with: 1) the synbiotic (400 mg/100 ml GOS and L. reuteri DSM 17938); 2) the 2'FL (100 mg/100 ml); and 3) without synbiotic and without 2'FL (control) in random order and a 3-day wash-out period between administrations. Fractional iron absorption (FIA[%]) was assessed by measuring erythrocyte incorporation of isotopic labels 14 days (n=26) and 28 days (n=76) after consumption of the last test FUF. RESULTS: Median (IQR) FIA from iron-fortified FUF with the synbiotic (8.2[5.2, 12.9]%) and with 2'FL (8.4[5.5, 14.1]%) did not differ from the control FUF (8.1[4.8,14.7]%) (synbiotic vs. control, P=0.24; 2'FL vs. control, P=0.95). FIA from all FUF did not differ when measured after 14 and 28 days of erythrocyte incorporation (Time, P=0.368; FUF, P=0.435; Time x FUF, P=0.937). Fecal pH and hemoglobin were negatively associated with FIA. CONCLUSION: In young Thai children, the addition of a synbiotic (GOS+L. reuteri) or 2'FL to iron-fortified FUF did not impact FIA from a single serving. The trial was registered at ClinicalTrial.gov as NCT04774016. The study was registered at clinicaltrials.gov as NCT04774016.

Recent advances of 3-fucosyllactose in health effects and production.

Human milk oligosaccharides (HMOs) have been recognized as gold standard for infant development. 3-Fucosyllactose (3-FL), being one of the Generally Recognized as Safe HMOs, represents a core trisaccharide within the realm of HMOs; however, it has received comparatively less attention in contrast to extensively studied 2'-fucosyllactose. The objective of this review is to comprehensively summarize the health effects of 3-FL, including its impact on gut microbiota proliferation, antimicrobial effects, immune regulation, antiviral protection, and brain maturation. Additionally, the discussion also covers the commercial application and regulatory approval status of 3-FL. Lastly, an organized presentation of large-scale production methods for 3-FL aims to provide a comprehensive guide that highlights current strategies and challenges in optimization.

Surface Proteins of Bifidobacterium Bifidum DNG6 Growing in 2'-FL Alleviating LPS-Induced Intestinal Barrier Injury in vitro.

Human milk oligosaccharides (HMOs) promote the growth and adhesion of bifidobacteria, thus exerting multiple biological functions on intestinal epithelial cells. Bacterial surface proteins play an important role in bacterial-host intestinal epithelial interactions. In this study, we aim to investigate the effects of surface proteins extracted from Bifidobacterium bifidum DNG6 (B. bifidum DNG6) consuming 2'-fucosyllactose (2'-FL) on Caco-2 cells monolayer barrier injury induced by lipopolysaccharide, compared with lactose (Lac) and galacto-oligosaccharides (GOS). Our results indicated that 2'-FL may promote the surface proteins of B. bifidum DNG6 to improve intestinal barrier injury by positively regulating the NF-κB signaling pathway, reducing inflammation(TNF-α reduced to 50.34%, IL-6 reduced to 22.83%, IL-1ß reduced to 37.91%, and IL-10 increased to 63.47%)and strengthening tight junction (ZO-1 2.39 times, Claudin-1 2.79 times, and Occludin 4.70 times). The findings of this study indicate that 2'-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface protein. The findings of this research will also provide theoretical support for the development of synbiotic formulations.

Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

Human milk oligosaccharides regulate human macrophage polarization and activation in response to Staphylococcus aureus.

Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is known about their effect on cells from the immune system. In this study, we investigated the direct effect of three structurally different HMOs on human derived macrophages before challenge with Staphylococcus aureus (S. aureus). The study demonstrates that individual HMO structures potently affect the activation, differentiation and development of monocyte-derived macrophages in response to S. aureus. 6´-Sialyllactose (6'SL) had the most pronounced effect on the immune response against S. aureus, as illustrated by altered expression of macrophage surface markers, pointing towards an activated M1-like macrophage-phenotype. Similarly, 6'SL increased production of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, IFN-γ and IL-1ß, when exposing cells to 6'SL in combination with S. aureus compared with S. aureus alone. Interestingly, macrophages treated with 6'SL exhibited an altered proliferation profile and increased the production of the classic M1 transcription factor NF-κB. The HMOs also enhanced macrophage phagocytosis and uptake of S. aureus. Importantly, the different HMOs did not notably affect macrophage activation and differentiation without S. aureus exposure. Together, these findings show that HMOs can potently augment the immune response against S. aureus, without causing inflammatory activation in the absence of S. aureus, suggesting that HMOs assist the immune system in targeting important pathogens during early infancy.

2'-Fucosyllactose Inhibits Human Norovirus Replication in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Currently, there are no targeted antivirals for the treatment of HuNoV infection. Histo-blood group antigens (HBGAs) on the intestinal epithelium are cellular attachment factors for HuNoVs; molecules that block the binding of HuNoVs to HBGAs thus have the potential to be developed as antivirals. Human milk oligosaccharides (HMOs) are glycans in human milk with structures analogous to HBGAs. HMOs have been shown to act as decoy receptors to prevent the attachment of multiple enteric pathogens to host cells. Previous X-ray crystallography studies have demonstrated the binding of HMO 2'-fucosyllactose (2'FL) in the same pocket as HBGAs for some HuNoV strains. We evaluated the effect of 2'FL on the replication of a globally dominant GII.4 Sydney [P16] HuNoV strain using human intestinal enteroids (HIEs) from adults and children. A significant reduction in GII.4 Sydney [P16] replication was seen in duodenal and jejunal HIEs from multiple adult donors, all segments of the small intestine from an adult organ donor and in two pediatric duodenal HIEs. However, 2'FL did not inhibit HuNoV replication in two infant jejunal HIEs that had significantly lower expression of α1-2-fucosylated glycans. 2'FL can be synthesized in large scale, and safety and tolerance have been assessed previously. Our data suggest that 2'FL has the potential to be developed as a therapeutic for HuNoV gastroenteritis.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Construction and application of 3-fucosyllactose whole-cell biosensor for high-throughput screening of overproducers.

Biosensor-based high-throughput screening is efficient for improving industrial microorganisms. There is a severe shortage of human milk oligosaccharides (HMOs) biosensors. This study established a 3-fucosyllactose (3-FL, a kind of HMOs) whole-cell biosensor by coupling cell growth with production. To construct and optimize the biosensor, an Escherichia coli 3-FL producer was engineered by deleting the manA, yihS and manX genes, directing the mannose flux solely to 3-FL synthesis. Then, an α-L-fucosidase was introduced to hydrolyze 3-FL to fucose which was used as the only carbon source for cell growth. Using the biosensor, the 3-FL production of a screened mutant was improved by 25 % to 42.05 ± 1.28 g/L. The productivity reached 1.17 g/L/h, the highest level reported by now. The csrB mutant obtained should be a new clue for the 3-FL overproduction mechanism. In summary, this study provided a novel approach to construct HMOs biosensors for strain improvement.

Rcs signal transduction system in Escherichia coli: Composition, related functions, regulatory mechanism, and applications.

The regulator of capsule synthesis (Rcs) system, an atypical two-component system prevalent in numerous gram-negative bacteria, serves as a sophisticated regulatory phosphorylation cascade mechanism. It plays a pivotal role in perceiving environmental stress and regulating the expression of downstream genes to ensure host survival. During the signaling transduction process, various proteins participate in phosphorylation to further modulate signal inputs and outputs. Although the structure of core proteins related to the Rcs system has been partially well-defined, and two models have been proposed to elucidate the intricate molecular mechanisms underlying signal sensing, a systematic characterization of the signal transduction process of the Rcs system remains challenging. Furthermore, exploring its corresponding regulator outputs is also unremitting. This review aimed to shed light on the regulation of bacterial virulence by the Rcs system. Moreover, with the assistance of the Rcs system, biosynthesis technology has developed high-value target production. Additionally, via this review, we propose designing chimeric Rcs biosensor systems to expand their application as synthesis tools. Finally, unsolved challenges are highlighted to provide the basic direction for future development of the Rcs system.

3-Fucosyllactose-mediated modulation of immune response against virus infection.

Viral pathogens, particularly influenza and SARS-CoV-2, pose a significant global health challenge. Given the immunomodulatory properties of human milk oligosaccharides, in particular 2'-fucosyllactose and 3-fucosyllactose (3-FL), we investigated their dietary supplementation effects on antiviral responses in mouse models. This study revealed distinct immune modulations induced by 3-FL. RNA-sequencing data showed that 3-FL increased the expression of interferon receptors, such as Interferon Alpha and Beta Receptor (IFNAR) and Interferon Gamma Receptor (IFNGR), while simultaneously downregulating interferons and interferon-stimulated genes, an effect not observed with 2'-fucosyllactose supplementation. Such modulation enhanced antiviral responses in both cell culture and animal models while attenuating pre-emptive inflammatory responses. Nitric oxide concentrations in 3-FL-supplemented A549 cells and mouse lung tissues were elevated exclusively upon infection, reaching 5.8- and 1.9-fold increases over control groups, respectively. In addition, 3-FL promoted leukocyte infiltration into the site of infection upon viral challenge. 3-FL supplementation provided protective efficacy against lethal influenza challenge in mice. The demonstrated antiviral efficacy spanned multiple influenza strains and extended to SARS-CoV-2. In conclusion, 3-FL is a unique immunomodulator that helps protect the host from viral infection while suppressing inflammation prior to infection.

Effects of 2'-fucosyllactose on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage.

2'-Fucosyllactose (2'-FL) is postulated to provide health benefits and promote the growth of probiotics. This work was undertaken to study the effects of 2'-FL on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage. Yogurts were produced containing 2'-FL (0 or 2 g/L) and Bifidobacterium strains of human origin (Bifidobacterium longum subsp. longum BB536 or Bifidobacterium longum subsp. infantis ATCC 15697) at a concentration of at least 109 CFU/mL. All yogurts were stored at 4°C for 5 weeks. Results showed that 2'-FL was stable in yogurts for at least 5 weeks of cold storage, and the addition of 2'-FL did not significantly alter yogurt fermentation parameters, associated metabolites, and the viability of mixed yogurt starter cultures and Bifidobacterium strains (p > 0.05). The addition of bifidobacteria had a negative impact (p < 0.05) on the survival rate of starter cultures, Streptococcus thermophilus and Lactobacillus delbureckii subsp. bulgaricus. Meanwhile, it is difficult to maintain a high survival rate of bifidobacteria in final yogurt products, and the addition of 2'-FL could not enhance the viability of bifidobacteria. B. longum BB536 survived at a level higher than 106 CFU/g for 28 days, while B. infantis ATCC15697 maintained this level for only 7 days. In summary, this study has shown the impact of 2'-FL and bifidobacterial species on yogurt properties, and results suggest that it is promising to use 2'-FL in yogurt products as a prebiotic. PRACTICAL APPLICATION: Yogurt is known for its beneficial effects on human health and nutrition. This study reported the production of symbiotic yogurt containing bifidobacteria and 2'-fucosyllactose (2'-FL) as a functional food for specified health uses. The viability of yogurt starter cultures and probiotic bifidobacterial strains was analyzed in this study. Moreover, this research demonstrated that 2'-FL could be added to yogurt without affecting the characteristics of yogurt significantly.

Ameliorating effect of 2'-fucosyllactose and 6'-sialyllactose on lipopolysaccharide-induced intestinal inflammation.

Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2'-FL and 6'-SL cotreatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal-inflamed suckling mice were treated with various mixtures of 2'-FL and 6'-SL. A 2'-FL:6'-SL ratio of 5:1 was identified as the most effective pretreatment HMO mixture in vitro; thus, this ratio was selected and used for low-, middle-, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as BW loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter, Mageeibacillus, and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus, and Saccharofermentans on short-chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggest that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development.

A 21-day safety evaluation of biotechnologically produced 3-fucosyllactose (3-FL) in neonatal farm piglets to support use in infant formulas.

3-Fucosyllactose (3-FL) is one of the most abundant fucosylated oligosaccharides in human breast milk and is an approved infant formula ingredient world-wide. 3-FL functions as a prebiotic to promote early microbial colonization of the gut, increase pathogen resistance and modulate immune responses. To investigate safety and potential gut microbiota effects, 3-FL was fed for 21-days to farm piglets beginning on Postnatal Day (PND) 2. Fructooligosaccharide (FOS), an approved infant formula ingredient, was used as a reference control. Standard toxicological endpoints were evaluated, and the gut microbiota were assessed. Neither 3-FL (245.77 and 489.72 mg/kg/day for males and 246.57 and 494.18 mg/kg/day for females) nor FOS (489.44 and 496.33 mg/kg/day males and females, respectively) produced any adverse differences in growth, food intake or efficiency, clinical observations, or clinical or anatomic pathology changes. Differences in the gut microbiota after 3-FL consumption (versus control and FOS groups) included the absence of Bifidobacterium species from the piglets, enrichment of Prevotellamassilia timonensis, Blautia species, Mediterranea massiliensis, Lachnospiraceae incertae sedis, and Eubacterium coprostanoligens and lower relative abundance of Allisonella histaminiformans and Roseburia inulinivorans. This study further supports the safe use of 3-FL produced using biotechnology as a nutritional ingredient in foods.

A human milk oligosaccharide prevents intestinal inflammation in adulthood via modulating gut microbial metabolism.

Observational evidence suggests that human milk oligosaccharides (HMOs) promote the growth of commensal bacteria in early life and adulthood. However, the mechanisms by which HMOs benefit health through modulation of gut microbial homeostasis remain largely unknown. 2'-fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and contributes to the essential health benefits associated with human milk consumption. Here, we investigated how 2'-FL prevents colitis in adulthood through its effects on the gut microbial community. We found that the gut microbiota from adult mice that consumed 2'-FL exhibited an increase in abundance of several health-associated genera, including Bifidobacterium and Lactobacillus. The 2'-FL-modulated gut microbial community exerted preventive effects on colitis in adult mice. By using Bifidobacterium infantis as a 2'-FL-consuming bacterial model, exploratory metabolomics revealed novel 2'-FL-enriched secretory metabolites by Bifidobacterium infantis, including pantothenol. Importantly, pantothenate significantly protected the intestinal barrier against oxidative stress and mitigated colitis in adult mice. Furthermore, microbial metabolic pathway analysis identified 26 dysregulated metabolic pathways in fecal microbiota from patients with ulcerative colitis, which were significantly regulated by 2'-FL treatment in adult mice, indicating that 2'-FL has the potential to rectify dysregulated microbial metabolism in colitis. These findings support the contribution of the 2'-FL-shaped gut microbial community and bacterial metabolite production to the protection of intestinal integrity and prevention of intestinal inflammation in adulthood.IMPORTANCEAt present, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether human milk oligosaccharides could serve as effective therapeutics for gastrointestinal-related diseases. Results from the present study uncover 2'-FL-driven alterations in bacterial metabolism and identify novel B. infantis-secreted metabolites following the consumption of 2'-FL, including pantothenol. This work further demonstrates a previously unrecognized role of pantothenate in significantly protecting the intestinal barrier against oxidative stress and mitigating colitis in adult mice. Remarkably, 2'-FL-enhanced bacterial metabolic pathways are found to be dysregulated in the fecal microbiota of ulcerative colitis patients. These novel metabolic pathways underlying the bioactivities of 2'-FL may lay a foundation for applying individual oligosaccharides for prophylactic intervention for diseases associated with impaired intestinal homeostasis.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Efficient Biosynthesis of Difucosyllactose via De Novo GDP-l-Fucose Pathway in Metabolically Engineered Escherichia coli.

Difucosyllactose (DFL) is an important component of human milk oligosaccharides (HMOs) and has significant benefits for the growth and development of infants. So far, a few microbial cell factories have been constructed for the production of DFL, which still have problems of low production and high cost. Herein, a high-level de novo pathway DFL-producing strain was constructed by multistep optimization strategies in Escherichia coli BL21star(DE3). We first efficiently synthesized the intermediate 2'-fucosyllactose (2'-FL) in E. coli BL21star(DE3) by the advisable stepwise strategy. The truncated α-1,3/4-fucosyltransferase (Hp3/4FT) was then introduced into the engineered strain to achieve de novo biosynthesis of DFL. ATP-dependent protease (Lon) and GDP-mannose hydrolase (NudK) were deleted, and mannose-6-phosphate isomerase (ManA) was overexpressed to improve GDP-l-fucose accumulation. The regulator RcsA was overexpressed to fine-tune the expression level of pathway genes, thereby increasing the synthesis of DFL. The final strain produced 6.19 g/L of DFL in the shake flask and 33.45 g/L of DFL in the 5 L fermenter, which were the highest reported titers so far. This study provides a more economical, sustainable, and effective strategy to produce the fucosylated human milk oligosaccharides (HMOs).

Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.

Introduction: Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2'-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

Influence of microbially fermented 2´-fucosyllactose on neuronal-like cell activity in an in vitro co-culture system.

Scope: 2´-Fucosyllactose (2´-FL), the most abundant oligosaccharide in human milk, plays an important role in numerous biological functions, including improved learning. It is not clear, however, whether 2´-FL or a cleavage product could influence neuronal cell activity. Thus, we investigated the effects of 2´-FL, its monosaccharide fucose (Fuc), and microbial fermented 2´-FL and Fuc on the parameters of neuronal cell activity in an intestinal-neuronal transwell co-culture system in vitro. Methods: Native 13C-labeled 2´-FL and 13C-Fuc or their metabolites, fermented with Bifidobacterium (B.) longum ssp. infantis and B. breve, which were taken from the lag-, log- and stationary (stat-) growth phases of batch cultures, were applied to the apical compartment of the co-culture system with Caco-2 cells representing the intestinal layer and all-trans-retinoic acid-differentiated SH-SY5Y (SH-SY5YATRA) cells mimicking neuronal-like cells. After 3 h of incubation, the culture medium in the basal compartment was monitored for 13C enrichment by using elemental analysis isotope-ratio mass spectrometry (EA-IRMS) and effects on cell viability, plasma, and mitochondrial membrane potential. The neurotransmitter activation (BDNF, GABA, choline, and glutamate) of SH-SY5YATRA cells was also determined. Furthermore, these effects were also measured by the direct application of 13C-2´-FL and 13C-Fuc to SH-SY5YATRA cells. Results: While no effects on neuronal-like cell activities were observed after intact 2´-FL or Fuc was incubated with SH-SY5YATRA cells, supernatants from the stat-growth phase of 2´-FL, fermented by B. longum ssp. infantis alone and together with B. breve, significantly induced BDNF release from SH-SY5YATRA cells. No such effects were found for 2´-FL, Fuc, or their fermentation products from B. breve. The BDNF release occurred from an enhanced vesicular release, which was confirmed by the use of the Ca2+-channel blocker verapamil. Concomitant with this event, 13C enrichment was also observed in the basal compartment when supernatants from the stat-growth phase of fermentation by B. longum ssp. infantis alone or together with B. breve were used. Conclusion: The results obtained in this study suggest that microbial products of 2´-FL rather than the oligosaccharide itself may influence neuronal cell activities.

Efficient production of 2'-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli.

BACKGROUND: The biosynthesis of human milk oligosaccharides (HMOs) using several microbial systems has garnered considerable interest for their value in pharmaceutics and food industries. 2'-Fucosyllactose (2'-FL), the most abundant oligosaccharide in HMOs, is usually produced using chemical synthesis with a complex and toxic process. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce 2'-FL, but the low stoichiometric yields (2'-FL/glucose or glycerol) are still far from meeting the requirements of industrial production. The sufficient carbon flux for 2'-FL biosynthesis is a major challenge. As such, it is of great significance for the construction of recombinant strains with a high stoichiometric yield. RESULTS: In the present study, we designed a 2'-FL biosynthesis pathway from fructose with a theoretical stoichiometric yield of 0.5 mol 2'-FL/mol fructose. The biosynthesis of 2'-FL involves five key enzymes: phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-D-mannose 4,6-dehydratase (Gmd), and GDP-L-fucose synthase (WcaG), and α-1,2-fucosyltransferase (FucT). Based on starting strain SG104, we constructed a series of metabolically engineered E. coli strains by deleting the key genes pfkA, pfkB and pgi, and replacing the original promoter of lacY. The co-expression systems for ManB, ManC, Gmd, WcaG, and FucT were optimized, and nine FucT enzymes were screened to improve the stoichiometric yields of 2'-FL. Furthermore, the gene gapA was regulated to further enhance 2'-FL production, and the highest stoichiometric yield (0.498 mol 2'-FL/mol fructose) was achieved by using recombinant strain RFL38 (SG104ΔpfkAΔpfkBΔpgi119-lacYΔwcaF::119-gmd-wcaG-manC-manB, 119-AGGAGGAGG-gapA, harboring plasmid P30). In the scaled-up reaction, 41.6 g/L (85.2 mM) 2'-FL was produced by a fed-batch bioconversion, corresponding to a stoichiometric yield of 0.482 mol 2'-FL/mol fructose and 0.986 mol 2'-FL/mol lactose. CONCLUSIONS: The biosynthesis of 2'-FL using recombinant E. coli from fructose was optimized by metabolic engineering strategies. This is the first time to realize the biological production of 2'-FL production from fructose with high stoichiometric yields. This study also provides an important reference to obtain a suitable distribution of carbon flux between 2'-FL synthesis and glycolysis.