Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Acute and two-week effects of neotame, stevia rebaudioside M and sucrose-sweetened biscuits on postprandial appetite and endocrine response in adults with overweight/obesity-a randomised crossover trial from the SWEET consortium.

BACKGROUND: Sweeteners and sweetness enhancers (S&SE) are used to replace energy yielding sugars and maintain sweet taste in a wide range of products, but controversy exists about their effects on appetite and endocrine responses in reduced or no added sugar solid foods. The aim of the current study was to evaluate the acute (1 day) and repeated (two-week daily) ingestive effects of 2 S&SE vs. sucrose formulations of biscuit with fruit filling on appetite and endocrine responses in adults with overweight and obesity. METHODS: In a randomised crossover trial, 53 healthy adults (33 female, 20 male) with overweight/obesity in England and France consumed biscuits with fruit filling containing 1) sucrose, or reformulated with either 2) Stevia Rebaudioside M (StRebM) or 3) Neotame daily during three, two-week intervention periods with a two-week washout. The primary outcome was composite appetite score defined as [desire to eat + hunger + (100 - fullness) + prospective consumption]/4. FINDINGS: Each formulation elicited a similar reduction in appetite sensations (3-h postprandial net iAUC). Postprandial insulin (2-h iAUC) was lower after Neotame (95% CI (0.093, 0.166); p < 0.001; d = -0.71) and StRebM (95% CI (0.133, 0.205); p < 0.001; d = -1.01) compared to sucrose, and glucose was lower after StRebM (95% CI (0.023, 0.171); p < 0.05; d = -0.39) but not after Neotame (95% CI (-0.007, 0.145); p = 0.074; d = -0.25) compared to sucrose. There were no differences between S&SE or sucrose formulations on ghrelin, glucagon-like peptide 1 or pancreatic polypeptide iAUCs. No clinically meaningful differences between acute vs. two-weeks of daily consumption were found. INTERPRETATION: In conclusion, biscuits reformulated to replace sugar using StRebM or Neotame showed no differences in appetite or endocrine responses, acutely or after a two-week exposure, but can reduce postprandial insulin and glucose response in adults with overweight or obesity. FUNDING: The present study was funded by the Horizon 2020 program: Sweeteners and sweetness enhancers: Impact on health, obesity, safety and sustainability (acronym: SWEET, grant no: 774293).

Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

Role of UDP-Glycosyltransferase (ugt) Genes in Detoxification and Glycosylation of 1-Hydroxyphenazine (1-HP) in Caenorhabditis elegans.

Caenorhabditis elegans is a useful model organism to study the xenobiotic detoxification pathways of various natural and synthetic toxins, but the mechanisms of phase II detoxification are understudied. 1-Hydroxyphenazine (1-HP), a toxin produced by the bacterium Pseudomonas aeruginosa, kills C. elegans. We previously showed that C. elegans detoxifies 1-HP by adding one, two, or three glucose molecules in N2 worms. Our current study evaluates the roles that some UDP-glycosyltransferase (ugt) genes play in 1-HP detoxification. We show that ugt-23 and ugt-49 knockout mutants are more sensitive to 1-HP than reference strains N2 or PD1074. Our data also show that ugt-23 knockout mutants produce reduced amounts of the trisaccharide sugars, while the ugt-49 knockout mutants produce reduced amounts of all 1-HP derivatives except for the glucopyranosyl product compared to the reference strains. We characterized the structure of the trisaccharide sugar phenazines made by C. elegans and showed that one of the sugar modifications contains an N-acetylglucosamine (GlcNAc) in place of glucose. This implies broad specificity regarding UGT function and the role of genes other than ogt-1 in adding GlcNAc, at least in small-molecule detoxification.

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.

Mutasynthesis generates nine new pyrroindomycins.

Pyrroindomycins (PYRs) represent the only spirotetramate natural products discovered in nature, and possess potent activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Their unique structure and impressive biological activities make them attractive targets for synthesis and biosynthesis; however, the discovery and generation of new PYRs remains challenging. To date, only the initial components A and B have been reported. Herein, we report a mutasynthesis approach for the generation of nine new PYRs with varying acyl modifications on their deoxy-trisaccharide moieties. This was achieved by blocking the formation of the acyl group 1,8-dihydropyrrolo[2,3-b]indole (DHPI) via gene pyrK1 inactivation and supplying chemical acyl precursors. The gene pyrK1 encodes a DUF1864 family protein that probably catalyzes the oxidative transformation of L-tryptophan to DHPI, and its deletion results in the abolishment of DHPI-containing PYRs and the accumulation of three new PYRs either without acyl modification or with DHPI replaced by benzoic acid and pyrrole-2-carboxylic acid. Capitalizing on the capacity of the ΔpyrK1 mutant to produce new PYRs, we have successfully developed a mutasynthesis strategy for the generation of six novel PYR analogs with various aromatic acid modifications on their deoxy-trisaccharide moieties, showcasing the potential for generating structurally diverse PYRs. Overall, this research contributes significantly to understanding the biosynthesis of PYRs and offers valuable perspectives on their structural diversity.

Characterization of a GH20 ß-N-Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto-N-triose II.

ß-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family ß-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 ß-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.

2'-Fucosyllactose Promotes Colonization of Akkermansia muciniphila and Prevents Colitis In Vitro and in Mice.

Akkermansia muciniphila is a potential candidate for ulcerative colitis prevention. Considering that it utilizes 2'-fucosyllactose (2'FL) for growth, 2'FL can be used to enrich the abundance of A. muciniphila in feces. However, whether the crosswalk between 2'FL and A. muciniphila can promote the intestinal colonization of A. muciniphila remains unclear. In this study, we explored the effect and the underlying mechanism of 2'FL on the colonization of A. muciniphila in vitro and in vivo as well as its alleviating effect on colitis. Our results revealed that 2'FL can serve as a carbon source of A. muciniphila to support the growth and increase cell-surface hydrophobicity and the expression of the genes coding fibronectin-binding autotransporter adhesin to promote the adhesion to Caco2/HT29 methotrexate (MTX) cells but not of galactooligosaccharides (GOS) and glucose. Moreover, 2'FL could increase the host mucin formation to promote the adhesion of A. muciniphila to Caco2/HT29 MTX cells but not of GOS and glucose. Furthermore, 2'FL could significantly increase the colonization of A. muciniphila in the gut to alleviate colitis in mice. Overall, the interplay between A. muciniphila and 2'FL is expected to provide an advantageous ecological niche for A. muciniphila so as to confer further health benefits against colitis.

Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Metal-conjugated Maltotriose Chlorins as Novel Photosensitizers for Photodynamic Therapy.

BACKGROUND/AIM: Photodynamic therapy (PDT) is a relatively non-invasive anti-cancer therapy that employs a photosensitizer with a specific wavelength of light, causing a photochemical reaction that releases free radicals, thereby inducing tumor cell necrosis via oxidative stress. The oxygen molecule reaches the singlet excited state through efficient energy transfer from an excited triplet state of the photosensitizer. Heavy atoms are frequently introduced in photosensitizers for efficiently generating reactive oxygen species (ROS) in PDT, known as the heavy atom effect. However, metal-complexed photosensitizers often show low water-solubility. To overcome this limitation and produce ROS effectively, we focused on the better solubility of photosensitizers with heavy metals bound within the chlorin skeleton and conjugated with glucose in this study. MATERIALS AND METHODS: We established maltotriose (Mal3)-conjugation with heavy metallochlorins [M (Mal3-chlorin), M=Pt or Pd)] and evaluated its anti-tumor effect. RESULTS: M (Mal3-chlorin) showed effective ROS production and singlet oxygen induction. Consequently, these cytotoxic factors caused effective anti-tumor effects and induced morphological changes, followed by cell death in vitro. In a xenograft tumor mouse model, PDT with M (Mal3-chlorin) showed tumor growth suppression. CONCLUSION: M (Mal3-Chlorin) might be an excellent glucose-conjugated chlorin because of its strong anti-tumor PDT effect.

Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Physicochemical, microbiological and metabolomics changes in yogurt supplemented with lactosucrose.

Lactosucrose (LS) is a known prebiotic that has gained recognition for its low caloric content and various health benefits. However, its potential in food applications remains largely unexplored. In this study the effects of adding LS to milk at concentrations (0 %, 2 %, 5 % and 8 % w/v) for yogurt production, and the relevant changes in yogurt texture, microbial composition and metabolomics were investigated. Our findings revealed that LS played a role in promoting the formation of a structured gel during fermentation, resulting in increased elasticity and viscosity while reducing fluidity. Additionally incorporating high doses of LS into yogurt led to reduced post-acidification, enhanced survival of starter bacteria, improved water retention capacity and overall texture throughout a refrigerated storage period of 21 days. Notably higher concentrations of LS (8 % w/v) exhibited effects on enhancing yogurt quality. Furthermore, untargeted metabolomics analysis using UPLC Q TOF MS/MS revealed 45 differentially expressed metabolites, including up-regulated L-arginine, L-proline and L-glutamic acid along with the down-regulated glutathione, L-tyrosine, L-phenylalanyl and L-proline. These differential metabolites were primarily associated with amino acid metabolism such as thiamine metabolism, nicotinic acid salt and nicotinamide metabolism, and pyrimidine metabolism. As a result, the inclusion of LS in yogurt had an impact on the production of various beneficial metabolites in yogurt, highlighting the importance of combining prebiotic LS with probiotics to obtain desired physiological benefits of yogurt.

Total Synthesis of Vibrio Cholerae O43 Tetrasaccharide Repeating Unit.

Vibrio cholerae is a pathogen responsible for the deadly pandemic - cholera. The glycans present on the surface of various strains of V. cholerae are considered as potential vaccine candidates. The tetrasaccharide repeating unit (RU) of V. cholerae O43 is decorated with less-explored rare deoxy amino sugars like d-quinosamine and d-viosamine, along with a rare amino acid, N-acetyl-l-allothreonine. Herein, we report a detailed account of the total synthesis of V. cholerae O43 tetrasaccharide RU. In our earlier attempt, while a one-pot assembly of trisaccharide was successful, the final coupling with a fully functionalized d-viosamine donor was low yielding. The successful route involved employing the Fmoc-protected d-viosamine building block as a donor and a late-stage amide bond formation of the tetrasaccharide.

Additive-assisted synthesis of α-Kdo glycosides with peracetylated glycosyl ynenoate as a donor.

A DMF-modulated glycosylation approach for the stereoselective synthesis of α-Kdo glycosides with readily accessible peracetylated Kdo ynenoate as a donor was described. By utilizing this approach, we completed the synthesis of various linkage types of Kdo-Kdo disaccharides and the α-Kdo-containing protected trisaccharide variant relevant to the lipopolysaccharide of Coxiella burnetii strain Nine Mile.

Microbial Synthesis of Lacto-N-fucopentaose I with High Titer and Purity by Screening of Specific Glycosyltransferase and Elimination of Residual Lacto-N-triose II and Lacto-N-tetraose.

Lacto-N-fucopentaose I (LNFP I) has recently been approved as generally recognized as safe, demonstrating its great commercial potential in the food industry. Microbial synthesis through metabolic engineering strategies is an effective approach for large-scale production of LNFP I. Biosynthesis of LNFP I requires consideration of two key points: high titer with low byproduct 2'-fucosyllactose (2'-FL) generation and high purity with low lacto-N-triose II (LNTri II) and lacto-N-tetraose (LNT) residues. Herein, α1,2-fucosyltransferase from Thermoanaerobacterium sp. RBIITD was screened from 16 selected LNFP I-producing glycosyltransferase candidates, showing the highest in vivo LNFP I productivity. Chromosomal integration of wbgO enhanced the LNFP I production by improving the precursor conversion from LNTri II to LNT. The best engineered strain produced 4.42 and 35.1 g/L LNFP I in shake-flask and fed-batch cultivation, respectively. The residual LNTri II and LNT were eliminated by further cultivation with a recombinant strain coexpressing Bifidobacterium bifidum ß-N-acetylhexosaminidase and lacto-N-biosidase. A strategy for LNFP I biosynthesis with high yield and purity was finally realized, providing support for its practical application in large-scale production.

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).

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.

Con A lectin binding by synthetic bivalent arabinomannan tri- and pentasaccharides reveals connectivity-dependent functional valencies.

Lectin Con A, with specificity to interact with α-d-mannopyranoside, achieves tight binding affinity with the aid of optimal multivalent ligand valencies, distances and orientations between the ligands. A series of synthetic arabinomannans, possessing arabinan core and mannan at the non-reducing ends, is studied to assess the above constraints involved with lectin binding in this report. Trisaccharides, with (1 â†’ 2)(1 â†’ 3), (1 â†’ 2)(1 â†’ 5) and (1 â†’ 3)(1 â†’ 5) glycosidic bond connectivities, and a pentasaccharide with mannopyranosides at the non-reducing ends are synthesized. The binding affinities of the mannose bivalent ligands are studied with tetrameric Con A lectin by isothermal titration calorimetry (ITC). Among the derivatives, trisaccharide with (1 â†’ 2)(1 â†’ 3) glycosidic bond connectivity and the pentasaccharide undergo lectin interaction, clearly fulfilling the bivalent structural and functional valencies. Remaining oligosaccharides exhibit only a functional monovalency, defying the bivalent structural valency. The trisaccharide fulfilling the structural and functional valencies represent the smallest bivalent ligand, undergoing the lectin interaction in a trans-mode.

Next-generation prebiotics: Maillard-conjugates of 2'-fucosyllactose and lactoferrin hydrolysates beneficially modulate gut microbiome composition and health promoting activity in a murine model.

Current prebiotics are predominantly carbohydrates. However, great competition exists among gut microbes for the scarce protein in the colon, as most consumed protein is digested and absorbed in the small intestine. Herein we evaluated in-vivo novel next-generation prebiotics: protein-containing-prebiotics, for selectively-targeted delivery of protein to colonic probiotics, to boost their growth. This system is based on micellar-particles, composed of Maillard-glycoconjugates of 2'-Fucosyllactose (2'-FL, human-milk-oligosaccharide) shell, engulfing lactoferrin peptic-then-tryptic hydrolysate (LFH) core. This core-shell structure lowers protein-core digestibility, while the prebiotic glycans are hypothesized to serve as molecular-recognition ligands for selectively targeting probiotics. To study the efficacy of this novel prebiotic, we fed C57BL/6JRccHsd mice with either 2'-FL-LFH Maillard-glycoconjugates, unconjugated components (control), or saline (blank). Administration of 2'-FL-LFH significantly increased the levels of short-chain-fatty-acids (SCFAs)-producing bacterial families (Ruminococcaceae, Lachnospiraceae) and genus (Odoribacter) and the production of the health-related metabolites, SCFAs, compared to the unconjugated components and to saline. The SCFAs-producing genus Prevotella significantly increased upon 2'-FL-LFH consumption, compared to only moderate increase in the unconjugated components. Interestingly, the plasma-levels of inflammation-inducing lipopolysaccharides (LPS), which indicate increased gut-permeability, were significantly lower in the 2'-FL-LFH group compared to the unconjugated-components and the saline groups. We found that Maillard-glycoconjugates of 2'-FL-LFH can serve as novel protein-containing prebiotics, beneficially modulating gut microbial composition and its metabolic activity, thereby contributing to host health more effectively than the conventional carbohydrate-only prebiotics.