Structural identification of carbohydrate isomers using ambient infrared-assisted dissociation.

Informative dissociation of carbohydrates using an infrared (IR) irradiation system is demonstrated under ambient conditions without the instrumentation of a mass spectrometer. Structural identification of carbohydrates and associated conjugates is essential for understanding their biological functions, but identification remains challenging. Herein, an easy and rugged method is reported for the structural identification of model carbohydrates, including Globo-H, three trisaccharide isomers (nigerotriose/laminaritriose/cellotriose), and two hexasaccharide isomers (laminarihexaose/isomaltohexaose). For Globo-H, the numbers of cross-ring cleavages increased by factors of 4.4 and 3.4 upon ambient IR exposure, compared to an untreated control and a collision-induced dissociation (CID) sample. Moreover, 25-82% enhancement in the numbers of glycosidic bond cleavages upon ambient IR exposure was also obtained compared to untreated and CID samples. Unique features of first-generation fragments produced by ambient IR facilitated the differentiation of three trisaccharide isomers. Semi-quantitative analysis was achieved (coefficient of determination (R2) of 0.982) in a mixture of two hexasaccharide isomers via unique features generated upon ambient IR. Photothermal and radical migration effects induced by ambient IR were postulated as responsible for promoting carbohydrate fragmentation. This easy and rugged method could be a universally applicable protocol and complementary to other techniques for detailed structural characterization of carbohydrates.

Production of 2'-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

Structural characterization of a novel fucosylated trisaccharide prepared from bacterial exopolysaccharides and evaluation of its prebiotic activity.

Fucosylated oligosaccharides have promising prospects in various fields. In this study, a fucosylated trisaccharide (GFG) was separated from the acidolysis products of exopolysaccharides from Clavibacter michiganensis M1. Structural characterization demonstrated that GFG consists of glucose, galactose, and fucose, with a molecular weight of 488 Da. Nuclear magnetic resonance analysis showed that it has a different structure than that of 2'-fucosyllactose (2'-FL), even though they have the same monosaccharide composition. In vitro prebiotic experiments were conducted to evaluate the differences in the utilization of three selected carbohydrates by fourteen bacterial strains. In comparison with 2'-FL, GFG could be utilized by more beneficial bacteria, leading to generate more short-chain fatty acids. Moreover, GFG could not promote the proliferation of Escherichia coli. This work describes a novel fucosylated oligosaccharide and its preparation method, and the obtained trisaccharide may serve as a promising candidate for fucosylated human milk oligosaccharides.

Total Synthesis of Trisaccharide Repeating Unit of Staphylococcus aureus Strain M.

An efficient total synthesis of a conjugation-ready trisaccharide repeating unit of Staphylococcus aureus strain M is reported here. The main challenges involved in this synthesis are the procurement of rare sugars (d-FucNAc and d-GalNAcA) and installation of consecutive 1,2-cis-glycosidic linkages between them. Stereoselective 1,2-cis glycosylation with the linker acceptor was achieved with easily accessible benzylidene protected d-galactosamine thioglycoside by employing a DMF modulated preactivation glycosylation method. The consecutive 1,2-cis linkages were installed with the help of solvent participation. The carboxylic acid functionality was introduced via postglycosylation oxidation on the disaccharide moiety. The total synthesis of trisaccharide repeating unit was accomplished with the longest linear sequence of 24 steps in 4.5% overall yield.

Synthesis of Dimeric Lewis A and Lewis B-Lewis A Tumor-Associated Carbohydrate Antigen Oligosaccharide Fragments.

Despite the interesting potential of tumor-associated carbohydrate antigens (TACAs) dimLea and LebLea to develop anticancer immunotherapies, little research has been conducted on these antigens. In our quest to discover fragments of these TACAs that could be targeted for the development of anticancer therapeutics, we report the synthesis of eight tri- to pentasaccharide fragments of these oligosaccharides. Unforeseen synthetic challenges are reported such as the incompatibility of a bromoalkyl glycoside in the reduction conditions needed to reduce a trichloroacetamide, the mismatched reactivities in a 2 + 1 synthetic strategy, and the surprising greater reactivity of a C-4 GlcNAc hydroxyl group versus that of the galactosyl OH-3 in the selective glycosylation of a trisaccharide diol. The desired final compounds were eventually obtained following a stepwise approach as nonyl or 9-aminononyl glycosides after one-step deprotection reactions in dissolving metal conditions. The 9-aminononyl glycosides will be conjugated to carrier proteins and the nonyl pentasaccharide glycoside will be used as a soluble inhibitor in binding experiments. In contrast, the nonyl tetrasaccharide glycosides are poorly soluble in water and their use in biochemical experiments will be limited.

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on ß→α Loop 5.

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on ß→α loop 5 of the catalytic (ß/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

Clinical effects of combined Lactobacillus paracasei and kestose on canine atopic dermatitis.

Probiotics and prebiotics are viable bacteria with beneficial effects on the host and components that selectively act on the beneficial commensal bacteria, respectively. The combined use of probiotics and prebiotics is termed synbiotics. Probiotic intake improves dysbiosis in the intestinal microbiota and can positively affect canine atopic dermatitis (CAD). However, clinical studies on improvements in CAD using synbiotics remain limited. In this study, 15 dogs with CAD who received prednisolone, a synthetic glucocorticoid (GC) used in the treatment of CAD, for more than 90 days were continuously treated with Lactobacillus paracasei M-1 from fermented food as a probiotic, and trisaccharide kestose as a prebiotic, for 90 days to determine their synbiotic effects on CAD. The CAD symptoms were evaluated using the canine atopic dermatitis lesion index (CADLI) and pruritus visual analog scores (PVAS) at 30, 60 and 90 days after synbiotic administration. The total prednisolone use for 90 days pre- and post-administration was also evaluated. Synbiotic administration significantly reduced the CADLI (pre: median, 28.0 [22.0-32.0]; 30 days: median, 20.0 [20.0-28.0]; 60 days: median, 20.0 [10.0-21.0]; 90 days: median, 12.0 [10.0-19.0]) and PVAS (pre: median, 6.0 [5.0-7.0]; 30 days: median, 3.0 [3.0-3.5]; 60 days: median, 3.0 [3.0-3.5]; 90 days: median, 2.0 [2.0-3.5]) scores, and reduced the total prednisone use over 90 days (pre: 112.0 [25-450] mg; post: 80.0 [18.-300.0] mg; p⟨0.001) in the 15 dogs. Thus, the synbiotic activity of L. paracasei M-1 and trisaccharide kestose can improve CAD.

Gas-Phase Behavior of Galactofuranosides upon Collisional Fragmentation: A Multistage High-Resolution Ion Mobility Study.

Carbohydrates are ubiquitous in nature but are among the least conserved biomolecules in life. These biopolymers pose a particular challenge to analytical chemists because of their high diversity and structural heterogeneity. In addition, they contain many isomerisms that complicate their structural characterization, notably by mass spectrometry. The tautomerism of the constitutive subunits is of particular interest. A given cyclized monosaccharide unit can take two forms: a most common 6-membered ring (pyranose, p) and a more flexible 5-membered ring (furanose, f). The tautomers impact the biological properties of polysaccharides, resulting in interesting properties of the derived oligosaccharides. From an analytical point of view, the impact of tautomerism on the gas-phase behavior of ions has scarcely been described in the literature. In this work, we study the behavior of Galf-containing oligosaccharides, ionized as [M+Li]+ species, under collisional dissociation (CID) conditions using high-resolution and multistage ion mobility (IMS) on a Cyclic IMS platform. In the first part of this work, we studied whether disaccharidic fragments released from Galf-containing (Gal)1(Man)2 trisaccharides (and their Galp counterpart) would match the corresponding disaccharide standards, and─despite the fragments generally being a good match─we showed the possibility of Galf migrations and other unidentified alterations in the IMS profile. Next, we expanded on these unknown features using multistage IMS and molecular dynamics, unveiling the contributions of additional gas-phase conformers in the profile of fragments from a Galf-containing trisaccharide compared with the corresponding disaccharides.

De novo biosynthesis of 2'-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1,2-fucosyltransferase from Azospirillum lipoferum.

Human milk oligosaccharides are complex, indigestible oligosaccharides that provide ideal nutrition for infant development. Here, 2'-fucosyllactose was efficiently produced in Escherichia coli by using a biosynthetic pathway. For this, both lacZ and wcaJ (encoding ß-galactosidase and UDP-glucose lipid carrier transferase, respectively) were deleted to enhance the 2'-fucosyllactose biosynthesis. To further enhance 2'-fucosyllactose production, SAMT from Azospirillum lipoferum was inserted into the chromosome of the engineered strain, and the native promoter was replaced with a strong constitutive promoter (PJ23119). The titer of 2'-fucosyllactose was increased to 8.03 g/L by introducing the regulators rcsA and rcsB into the recombinant strains. Compared to wbgL-based strains, only 2'-fucosyllactose was produced in SAMT-based strains without other by-products. Finally, the highest titer of 2'-fucosyllactose reached 112.56 g/L in a 5 L bioreactor by fed-batch cultivation, with a productivity of 1.10 g/L/h and a yield of 0.98 mol/mol lactose, indicating a strong potential in industrial production.

Elimination of Byproduct Generation and Enhancement of 2'-Fucosyllactose Synthesis by Expressing a Novel α1,2-Fucosyltransferase in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) is a kind of fucosylated human milk oligosaccharide (HMO), representing the most abundant oligosaccharide in breast milk. We conducted systematic studies on three canonical α1,2-fucosyltransferases (WbgL, FucT2, and WcfB) to quantify the byproducts in a lacZ- and wcaJ-deleted Escherichia coli BL21(DE3) basic host strain. Further, we screened a highly active α1,2-fucosyltransferase from Helicobacter sp. 11S02629-2 (BKHT), which exhibits high in vivo 2'-FL productivity without the formation of byproducts difucosyl lactose (DFL) and 3-FL. The maximum 2'-FL titer and yield reached 11.13 g/L and 0.98 mol/mol of lactose, respectively, in shake-flask cultivation, both approaching the theoretical maximum value. In a 5 L fed-batch cultivation, the maximum 2'-FL titer reached 94.7 g/L extracellularly with a yield of 0.98 mol of 2'-FL/mol of lactose and productivity of 1.14 g L-1 h-1. Our reported 2'-FL yield is the highest from lactose reported to date.

Deacetylation of Arabinofuranosylated Xylopyranosyl Residues Related to Plant Xylan: Significant Differences Between Xylan Deacetylases Classified into Various Carbohydrate Esterase Families.

A chemical synthesis of two novel phenyl glycosides of trisaccharides related to acetylarabinoxylan is described. The trisaccharides bear acetyl and arabinofuranosyl moieties at the non-reducing-end xylopyranosyl residue, which is substituted at positions 2 and 3. Both compounds were treated with various xylan deacetylases classified in different carbohydrate esterase (CE) families and significant differences between the families were found. While the arabinosylation hampers deacetylation by CE2-CE5 and CE12 family members, both epitopes are deesterified by CE1 and in particular CE6 enzymes. The 3-O-acetylated 2-O-arabinosylated compound is also processed by CE7 and majority of CE16 esterases, but not by a hitherto non-classified Flavobacterium johnsoniae acetylxylan esterase. The data suggests that a slow deesterification of the 2-O-acetylated 3-O-arabinosylated compound may be due to the acetyl group migration followed by deacetylation of this migration product.

Total Synthesis of Trisaccharide Repeating Unit of Staphylococcus aureus Type 8 (CP8) Capsular Polysaccharide.

Herein, we report a highly efficient total synthesis of Staphylococcus aureus type 8 trisaccharide repeating unit in a lesser number of steps and high stereoselectivity. The complex trisaccharide contains rare amino sugars, viz., d-fucosamine, l-fucosamine, and 2-acetamido d-mannuronic acid. The installation of consecutive sterically hindered 1,2-cis glycosidic linkages, especially ß-mannosylation, is the key challenge in this synthesis. The total synthesis of target molecule was completed via a longest linear sequence of 18 steps in 7.1% overall yield.

De novo biosynthesis of 2'-fucosyllactose in engineered Pichia pastoris.

PURPOSE: Pichia pastoris is well known for its ability to produce short and low-immunogenic humanized glycosyl chains onto recombinant glycoproteins, it was thus speculated to be applicable to synthesize oligosaccharides. In this study, generally recognized as safe (GRAS) microorganism Pichia pastoris GS115 was tested for its potential to be used as a new synthetic chassis to produce the most abundant human milk oligosaccharide 2'-fucosyllactose (2'-FL). METHODS: To enable the de novo synthesis of 2'-FL, lactose transporter lac12, two enzymes of gmd, gmer, and fucosyltransferases futC were integrated into the genome of P. pastoris, under the control of constitutive PGAP promoter. RESULTS: The resulting recombinant yeasts yielded up to 0.276 g/L through culture optimization in a 5 L bioreactor. CONCLUSION: To our knowledge, this is the first report of 2'-FL production in engineered Pichia pastoris. This work is a good starting point to produce 2'-FL using Pichia pastoris as a viable chassis.

Combinatorial metabolic engineering and tolerance evolving of Escherichia coli for high production of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL) is an important functional ingredient of advanced infant formula. Here, Escherichia coli MG1655 was engineered for achieving high 2'-FL production. The expressions of 2'-FL synthesis pathway genes were finely regulated with single or multi copies according to rate-limiting enzyme diagnosis. On this basic, the branch pathway genes were deleted, and the overexpression of the 2'-FL efflux protein SetA and the fructose-1,6-bisphosphatase GlpX were tuned. The resulting strain produced 46.06 ± 1.28 g/L 2'-FL in a 5-L fermenter. Furtherly, adaptive laboratory evolution was conducted. A rpoC gene mutation was obtained which could improve the cell tolerance and the 2'-FL production up to 61.06 ± 1.93 g/L, with the highest productivity of 1.70 g/L/h among E. coli strains by now. Taken together, this work provides a combinatorial strategy to improve 2'-FL accumulation including rational fine-tuning pathway genes expressions and irrational adaptive laboratory evolution. This study should be helpful for constructing high level 2'-FL producers.

Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli.

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

Biosynthesis of lactosucrose by a new source of ß-fructofuranosidase from Bacillus methanolicus LB-1.

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The ß-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B. methanolicus LB-1 under the optimized conditions: 10 g/L of glucose, 5 g/L of yeast extract, initial medium pH at 7.0, 37 °C, 24 h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45 kDa. Biosynthesis of LS was performed using the purified ß-fructofuranosidase, and production of LS reached 110 g/L under the optimized reaction conditions: pH at 7.0, 37 °C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28 h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30 min of elution, confirming that B. methanolicus LB-1 ß-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of ß-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

Effects of Bifidobacterium with the Ability of 2'-Fucosyllactose Utilization on Intestinal Microecology of Mice.

In breast milk, 2'-Fucosyllactose (2'FL) is the most abundant breast milk oligosaccharide and can selectively promote the proliferation of bifidobacteria. This study aimed to explore the effect of ifidobacterial with different utilization capacities of 2'FL on the intestinal microecology of mice. Furthermore, the effects of ifidobacterial with different 2'FL utilization capabilities on mice gut microbiota under the competitive pressure of 2'FL as a carbon source were explored. Compared with the control group, 2'FL, Bifidobacterium (B.) bifidum M130R01M51 + 2'FL, B. longum subsp. Longum CCFM752, and CCFM752 + 2'FL treatments significantly decreased the food intake. Moreover, the water intake, body weight, and fecal water content in all groups showed no significant difference compared with the control group. The combination of B. longum subsp. longum CCFM752 and 2'FL can significantly increase the levels of pro-inflammatory and anti-inflammatory factors. B. bifidum M130R01M51 and mixed strains combined with 2'FL significantly increased the contents of acetic acid and isobutyric acid. The results showed that B. bifidum M130R01M51, B. breve FHuNCS6M1, B. longum subsp. longum CCFM752, and B. longum subsp. infantis SDZC2M4 combined with 2'FL significantly increased the species richness of the gut microbiota. Moreover, B. longum subsp. longum CCFM752 and B. longum subsp. infantis SDZC2M4 significantly increased the abundance of Faecalibaculum and Bifidobacterium, respectively. In conclusion, exploring the impact on intestinal microecology can provide theoretical guidance for the development of personalized prebiotics for different bifidobacteria, which has the potential to improve the ecological imbalance of infant gut microbiota.

Human Milk Oligosaccharide 2'-Fucosyllactose Inhibits Ligand Binding to C-Type Lectin DC-SIGN but Not to Langerin.

Human milk oligosaccharides (HMOs) and their most abundant component, 2'-Fucosyllactose (2'-FL), are known to be immunomodulatory. Previously, it was shown that HMOs and 2'-FL bind to the C-type lectin receptor DC-SIGN. Here we show, using a ligand-receptor competition assay, that a whole mixture of HMOs from pooled human milk (HMOS) and 2'-FL inhibit the binding of the carbohydrate-binding receptor DC-SIGN to its prototypical ligands, fucose and the oligosaccharide Lewis-B, (Leb) in a dose-dependent way. Interestingly, such inhibition by HMOS and 2'-FL was not detected for another C-type lectin, langerin, which is evolutionarily similar to DC-SIGN. The cell-ligand competition assay using DC-SIGN expressing cells confirmed that 2'-FL inhibits the binding of DC-SIGN to Leb. Molecular dynamic (MD) simulations show that 2'-FL exists in a preorganized bioactive conformation before binding to DC-SIGN and this conformation is retained after binding to DC-SIGN. Leb has more flexible conformations and utilizes two binding modes, which operate one at a time via its two fucoses to bind to DC-SIGN. Our hypothesis is that 2'-FL may have a reduced entropic penalty due to its preorganized state, compared to Leb, and it has a lower binding enthalpy, suggesting a better binding to DC-SIGN. Thus, due to the better binding to DC-SIGN, 2'-FL may replace Leb from its binding pocket in DC-SIGN. The MD simulations also showed that 2'-FL does not bind to langerin. Our studies confirm 2'-FL as a specific ligand for DC-SIGN and suggest that 2'-FL can replace other DC-SIGN ligands from its binding pocket during the ligand-receptor interactions in possible immunomodulatory processes.