Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger ß5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

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.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Recent Progress in Human Milk Oligosaccharides and Its Antiviral Efficacy.

Gastrointestinal (GI)-associated viruses, including rotavirus (RV), norovirus (NV), and enterovirus, usually invade host cells, transmit, and mutate their genetic information, resulting in influenza-like symptoms, acute gastroenteritis, encephalitis, or even death. The unique structures of human milk oligosaccharides (HMOs) enable them to shape the gut microbial diversity and endogenous immune system of human infants. Growing evidence suggests that HMOs can enhance host resistance to GI-associated viruses but without a systematic summary to review the mechanism. The present review examines the lactose- and neutral-core HMOs and their antiviral effects in the host. The potential negative impacts of enterovirus 71 (EV-A71) and other GI viruses on children are extensive and include neurological sequelae, neurodevelopmental retardation, and cognitive decline. However, the differences in the binding affinity of HMOs for GI viruses are vast. Hence, elucidating the mechanisms and positive effects of HMOs against different viruses may facilitate the development of novel HMO derived oligosaccharides.

Diurnal rhythmicity of infant fecal microbiota and metabolites: A randomized controlled interventional trial with infant formula.

Microbiota assembly in the infant gut is influenced by diet. Breastfeeding and human breastmilk oligosaccharides promote the colonization of beneficial bifidobacteria. Infant formulas are supplemented with bifidobacteria or complex oligosaccharides, notably galacto-oligosaccharides (GOS), to mimic breast milk. To compare microbiota development across feeding modes, this randomized controlled intervention study (German Clinical Trial DRKS00012313) longitudinally sampled infant stool during the first year of life, revealing similar fecal bacterial communities between formula- and breast-fed infants (N = 210) but differences across age. Infant formula containing GOS sustained high levels of bifidobacteria compared with formula containing B. longum and B. breve or placebo. Metabolite and bacterial profiling revealed 24-h oscillations and circadian networks. Rhythmicity in bacterial diversity, specific taxa, and functional pathways increased with age and was strongest following breastfeeding and GOS supplementation. Circadian rhythms in dominant taxa were further maintained ex vivo in a chemostat model. Hence, microbiota rhythmicity develops early in life and is impacted by diet.

Potential biological functions and future perspectives of sialylated milk oligosaccharides.

Sialyllactoses (SLs) primarily include sialylated human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). First, the safety assessment of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) revealed low toxicity in various animal models and human participants. SLs constitute a unique milk component, highlighting the essential nutrients and bioactive components crucial for infant development, along with numerous associated health benefits for various diseases. This review explores the safety, biosynthesis, and potential biological effects of SLs, with a specific focus on their influence across various physiological systems, including the gastrointestinal system, immune disorders, rare genetic disorders (such as GNE myopathy), cancers, neurological disorders, cardiovascular diseases, diverse cancers, and viral infections, thus indicating their therapeutic potential.

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls.

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

A receptor required for chitin perception facilitates arbuscular mycorrhizal associations and distinguishes root symbiosis from immunity.

Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.

Production of prebiotic enriched maple syrup through enzymatic conversion of sucrose into fructo-oligosaccharides.

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products` prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible.

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property.

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.

Alginate oligosaccharide assimilation by gut microorganisms and the potential role in gut inflammation alleviation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. IMPORTANCE: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.

Effect of inulin, galacto-oligosaccharides, and polyphenols on the gut microbiota, with a focus on Akkermansia muciniphila.

Inulins, galacto-oligosaccharides (GOS) and polyphenols are considered to stimulate the growth of Akkermansia muciniphila (A. muciniphila) in the gut. We performed a meta-analysis of six microbiome studies (821 stool samples from 451 participants) to assess the effects of inulin, GOS, and polyphenols on the abundance of A. muciniphila in the gut. The intervention of GOS increased the relative abundance of A. muciniphila in healthy participants. Additionally, metabolic pathways associated with carbohydrate metabolism and short-chain fatty acid release were enriched following the GOS intervention. Furthermore, after the GOS intervention, the coexisting microbial communities of A. muciniphila, such as Eubacterium hallii and Bacteroides, exhibited an enhanced correlation with A. muciniphila. In conclusion, our findings suggest that GOS may promote the growth of A. muciniphila in the gut by modulating the gut microbiota composition.

Engineering Escherichia coli for Highly Efficient Biosynthesis of Lacto-N-difucohexaose II through De Novo GDP-l-fucose Pathway.

Lacto-N-difucohexaose II (LNDFH II) is a typical fucosylated human milk oligosaccharide and can be enzymatically produced from lacto-N-tetraose (LNT) by a specific α1,3/4-fucosyltransferase from Helicobacter pylori DMS 6709, referred to as FucT14. Previously, we constructed an engineered Escherichia coli BL21(DE3) with a single plasmid for highly efficient biosynthesis of LNT. In this study, two additional plasmids harboring the de novo GDP-L-fucose pathway module and FucT14, respectively, were further introduced to construct the strain for successful biosynthesis of LNDFH II. FucT14 was actively expressed, and the engineered strain produced LNDFH II as the major product, lacto-N-fucopentaose (LNFP) V as the minor product, and a trace amount of LNFP II and 3-fucosyllactose as very minor products. Additional expression of the α1,3-fucosyltransferase FutM1 from a Bacteroidaceae bacterium from the gut metagenome could obviously enhance the LNDFH II biosynthesis. After optimization of induction conditions, the maximum titer reached 3.011 g/L by shake-flask cultivation. During the fed-batch cultivation, LNDFH II was highly efficiently produced with the highest titer of 18.062 g/L and the productivity yield of 0.301 g/L·h.

Implementation of a Quorum-Sensing System for Highly Efficient Biosynthesis of Lacto-N-neotetraose in Engineered Escherichia coli MG1655.

Lacto-N-neotetraose (LNnT), a prominent neutral human milk oligosaccharide (HMO), serves as a pivotal structural element in complex HMO biosynthesis. Given its promising health effects for infants, the biosynthesis of LNnT is garnering greater interest. Using a previously engineered strain as a chassis, a highly effective LNnT producer was constructed. First, LNnT synthesis in Escherichia coli MG1655 was achieved by introducing ß1,3-N-acetylglucosaminyltransferase LgtA and ß1,4-galactosyltransferase CpsIaJ, coupled with the optimization of enzyme expression levels using various promoters. Subsequently, ugd underwent disruption, and the galE gene was enhanced by replacing its promoter with PJ23119 or Ptac. Then, a lux-type quorum sensing (QS) system was applied to achieve varied metabolic regulation. Additionally, systematic optimization of the QS promoters was conducted to further improve the LNnT titer in the shake flask. Finally, the extracellular titer of LNnT was 20.33 g/L, accompanied by a productivity of 0.41 g/L/h.