Intestinal persistence of Bifidobacterium infantis is determined by interaction of host genetics and antibiotic exposure.

Probiotics have gained significant attention as a potential strategy to improve health by modulating host-microbe interactions, particularly in situations where the normal microbiota has been disrupted. However, evidence regarding their efficacy has been inconsistent, with considerable interindividual variability in response. We aimed to explore whether a common genetic variant that affects the production of mucosal α(1,2)-fucosylated glycans, present in around 20% of the population, could explain the observed interpersonal differences in the persistence of commonly used probiotics. Using a mouse model with varying α(1,2)-fucosylated glycans secretion (Fut2WT or Fut2KO), we examined the abundance and persistence of Bifidobacterium strains (infantis, breve, and bifidum). We observed significant differences in baseline gut microbiota characteristics between Fut2WT and Fut2KO littermates, with Fut2WT mice exhibiting enrichment of species able to utilize α(1,2)-fucosylated glycans. Following antibiotic exposure, only Fut2WT animals showed persistent engraftment of Bifidobacterium infantis, a strain able to internalize α(1,2)-fucosylated glycans, whereas B. breve and B. bifidum, which cannot internalize α(1,2)-fucosylated glycans, did not exhibit this difference. In mice with an intact commensal microbiota, the relationship between secretor status and B. infantis persistence was reversed, with Fut2KO animals showing greater persistence compared to Fut2WT. Our findings suggest that the interplay between a common genetic variation and antibiotic exposure plays a crucial role in determining the dynamics of B. infantis in the recipient gut, which could potentially contribute to the observed variation in response to this commonly used probiotic species.

The Importance of the FUT2 rs602662 Polymorphism in the Risk of Cardiovascular Complications in Patients after Kidney Transplantation.

The FUT2 gene encodes an enzyme called α-1,2-fucosyltransferase, which is involved in the formation of blood group antigens AB0(H) and is also involved in the processes of vitamin B12 absorption and its transport between cells. FUT2 gene polymorphisms are associated with vitamin B12 levels in the body. Vitamin B12 deficiency associated with hyperhomocysteinemia is a major risk factor for cardiovascular diseases (CVDs), which are one of the main causes of death in patients after kidney transplantation. The aim of our study was to determine the impact of the rs602662 (G>A) polymorphism of the FUT2 gene on the functionality of transplanted kidneys and the risk of CVD in patients after kidney transplantation. The study included 402 patients treated with immunosuppression (183 patients taking cyclosporine (CsA) and 219 patients taking tacrolimus (TAC)). The analysis of the FUT2 rs602662 (G>A) polymorphism was performed using real-time PCR. Patients with CsA were more likely to be underweight (1.64% vs. 0.91%) and obese (27.87% vs. 15.98%), while those taking TAC were more likely to be of normal weight (39.27%) or overweight (43.84%). No statistically significant differences were observed comparing the mean blood pressure, both systolic and diastolic. The renal profile showed a higher median urea nitrogen concentration in patients with CsA (26.45 mg/dL (20.60-35.40) vs. 22.95 mg/dL (17.60-33.30), p = 0.004). The observed frequency of rs602662 alleles of the FUT2 gene was similar in the analyzed groups. The A allele was present in 43.7% of patients with CsA and 41.1% of those taking TAC (OR = 0.898; 95% CI: 0.678-1.189; p = 0.453). In the group with CsA, the GG genotype was present in 32.2% of patients, the GA in 48.1% and the AA in 19.7%. A similar distribution was obtained in the TAC group: GG-33.8%, GA-50.2%, and AA-16.0%. An association of genotypes containing the G allele with a higher incidence of hypertension was observed. The G allele was present in 65% of people with hypertension and in 56% of patients with normal blood pressure (p = 0.036). Moreover, the evaluation of the renal parameters showed no effect of the FUT2 polymorphism on the risk of organ rejection because the levels of creatinine, eGFR, potassium, and urea nitrogen were prognostic of successful transplantation. Our results suggest that the rs6022662 FUT2 polymorphism may influence the risk of cardiovascular diseases.

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.

Protein O-fucosyltransferase 1 promotes PD-L1 stability to drive immune evasion and directs liver cancer to immunotherapy.

BACKGROUND AND AIMS: The immunosuppressive tumor microenvironment (TME) plays an essential role in cancer progression and immunotherapy response. Despite the considerable advancements in cancer immunotherapy, the limited response to immune checkpoint blockade (ICB) therapies in patients with hepatocellular carcinoma (HCC) remains a major challenge for its clinical implications. Here, we investigated the molecular basis of the protein O-fucosyltransferase 1 (POFUT1) that drives HCC immune evasion and explored a potential therapeutic strategy for enhancing ICB efficacy. METHODS: De novo MYC/Trp53-/- liver tumor and the xenograft tumor models were used to evaluate the function of POFUT1 in immune evasion. Biochemical assays were performed to elucidate the underlying mechanism of POFUT1-mediated immune evasion. RESULTS: We identified POFUT1 as a crucial promoter of immune evasion in liver cancer. Notably, POFUT1 promoted HCC progression and inhibited T-cell infiltration in the xenograft tumor and de novo MYC/Trp53-/- mouse liver tumor models. Mechanistically, we demonstrated that POFUT1 stabilized programmed death ligand 1 (PD-L1) protein by preventing tripartite motif containing 21-mediated PD-L1 ubiquitination and degradation independently of its protein-O-fucosyltransferase activity. In addition, we further demonstrated that PD-L1 was required for the tumor-promoting and immune evasion effects of POFUT1 in HCC. Importantly, inhibition of POFUT1 could synergize with anti-programmed death receptor 1 therapy by remodeling TME in the xenograft tumor mouse model. Clinically, POFUT1 high expression displayed a lower response rate and worse clinical outcome to ICB therapies. CONCLUSIONS: Our findings demonstrate that POFUT1 functions as a novel regulator of tumor immune evasion and inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC.

Impact of fucosyltransferase 1-mediated epidermal blood group antigen H on anti-inflammatory response in atopic dermatitis.

The presence of the blood group H2 antigen on the membrane of red blood cells determines blood type O in individuals and this H2 antigen serves as a precursor to the A and B antigens expressed in blood types A and B, respectively. However, the specific involvement of ABH antigens in skin diseases is unknown. Therefore, we aim to investigate the expression of ABH antigens in skin tissue of patients with atopic dermatitis (AD) and MC903-induced AD-like mice. We demonstrated that the expression of ABH antigen is primarily located in the granular and horny layers of the skin in healthy control individuals. However, in patients with AD, the expression of the ABH antigen was absent or diminished in these layers, while the H2 antigen expression increased in the spinous layers of the affected skin lesions. Then, we investigated the biological function of blood group H antigen mediated by fucosyltransferase 1 (Fut1) in the skin, utilizing an AD mouse model induced by MC903 in wild-type (WT) and Fut1-knockout mice. After the application of MC903, Fut1-deficient mice, with no H2 antigen expression on their skin, exhibited more severe clinical signs, increased ear swelling, and elevated serum IgE levels compared with those of WT mice. Additionally, the MC903-induced thickening of both the epidermis and dermis was more pronounced in Fut1-deficient mice than that in WT mice. Furthermore, Fut1-deficient mice showed a significantly higher production of interleukin-4 (IL-4) and IL-6 in skin lesions compared with that of their WT counterparts. The expression of chemokines, particularly Ccl2 and Ccl8, was notably higher in Fut1-deficient mice compared with those of WT mice. The infiltration of CD4+ T cells, eosinophils, and mast cells into the lesional skin was significantly elevated in Fut1-deficient mice compared with that in WT mice. These findings demonstrate the protective role of H2 antigen expression against AD-like inflammation and highlight its potential therapeutic impact on AD through the regulation of blood group antigens.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.

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.

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds.

The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy-Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy-Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy-Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.

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.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Using Tumor Marker Gene Variants to Improve the Diagnostic Accuracy of DUPAN-2 and Carbohydrate Antigen 19-9 for Pancreatic Cancer.

PURPOSE: Circulating carbohydrate antigen 19-9 (CA19-9) levels reflect FUT3 and FUT2 fucosyltransferase activity. Measuring the related glycan, DUPAN-2, can be useful in individuals unable to synthesize CA19-9. We hypothesized that similar to CA19-9, FUT functional groups determined by variants in FUT3 and FUT2 influence DUPAN-2 levels, and having tumor marker reference ranges for each functional group would improve diagnostic performance. MATERIALS AND METHODS: Using a training/validation study design, FUT2/FUT3 genotypes were determined in 938 individuals from Johns Hopkins Hospital: 607 Cancer of the Pancreas Screening (CAPS) study subjects with unremarkable pancreata and 331 with pancreatic ductal adenocarcinoma (PDAC). Serum DUPAN-2 and CA19-9 levels were measured by immunoassay. RESULTS: In controls, three functional FUT groups were identified with significant differences in DUPAN-2 levels: FUT3-intact, FUT3-null/FUT2-intact, and FUT3-null/FUT2-null. DUPAN-2 training set diagnostic cutoffs for each FUT group yielded higher diagnostic sensitivity in the validation set for patients with stage I/II PDAC than uniform cutoffs (60.4% [95% CI, 50.2 to 70.0] v 39.8% [30.0 to 49.8]), at approximately 99% (96.7 to 99.6) specificity. Combining FUT/CA19-9 and FUT/DUPAN-2 tests yielded 78.4% (72.3 to 83.7) sensitivity for stage I/II PDAC, at 97.7% (95.3 to 99.1) specificity in the combined sets, with higher AUC (stage I/II: 0.960 v 0.935 for CA19-9 + DUPAN-2 without the FUT test; P < .001); for stage I PDAC, sensitivity was 62.0% (49.1 to 73.2; AUC, 0.919 v 0.883; P = .03). CA19-9 levels in FUT3-null/FUT2-null PDAC subjects were higher than in FUT3-null/FUT2-intact subjects (median/IQR; 24.9/57.4 v <1/2.3 U/mL; P = .0044). In a simulated CAPS cohort, AUC precision recall (AUCPR) scores were 0.51 for CA19-9 alone, 0.64 for FUT/CA19-9, 0.73 for CA19-9/DUPAN-2, and 0.84 for FUT/CA19-9/DUPAN-2. CONCLUSION: Using a tumor marker gene test to individualize CA19-9 and DUPAN-2 reference ranges achieves high diagnostic performance for stage I/II pancreatic cancer.

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.

Fucosyltransferase 8 regulates adult neurogenesis and cognition of mice by modulating the Itga6-PI3K/Akt signaling pathway.

Fucosyltransferase 8 (Fut8) and core fucosylation play critical roles in regulating various biological processes, including immune response, signal transduction, proteasomal degradation, and energy metabolism. However, the function and underlying mechanism of Fut8 and core fucosylation in regulating adult neurogenesis remains unknown. We have shown that Fut8 and core fucosylation display dynamic features during the differentiation of adult neural stem/progenitor cells (aNSPCs) and postnatal brain development. Fut8 depletion reduces the proliferation of aNSPCs and inhibits neuronal differentiation of aNSPCs in vitro and in vivo, respectively. Additionally, Fut8 deficiency impairs learning and memory in mice. Mechanistically, Fut8 directly interacts with integrin α6 (Itga6), an upstream regulator of the PI3k-Akt signaling pathway, and catalyzes core fucosylation of Itga6. Deletion of Fut8 enhances the ubiquitination of Itga6 by promoting the binding of ubiquitin ligase Trim21 to Itga6. Low levels of Itga6 inhibit the activity of the PI3K/Akt signaling pathway. Moreover, the Akt agonist SC79 can rescue neurogenic and behavioral deficits caused by Fut8 deficiency. In summary, our study uncovers an essential function of Fut8 and core fucosylation in regulating adult neurogenesis and sheds light on the underlying mechanisms.

Highly-efficient in vivo production of lacto-N-fucopentaose V by a regio-specific α1,3/4-fucosyltransferase from Bacteroides fragilis NCTC 9343.

Lacto-N-fucopentaose V (LNFP V) is a typical human milk pentasaccharide. Multi-enzymatic in vitro synthesis of LNFP V from lactose was reported, however, microbial cell factory approach to LNFP V production has not been reported yet. In this study, the biosynthetic pathway of LNFP V was examined in Escherichia coli. The previously constructed E. coli efficiently producing lacto-N-tetraose was used as the starting strain. GDP-fucose pathway module and a regio-specific glycosyltransferase with α1,3-fucosylation activity were introduced to realize the efficient synthesis of LNFP V. The α1,3/4-fucosyltransferase from Bacteroides fragilis was selected as the best enzyme for in vivo biosynthesis of LNFP V from nine candidates, with the highest titer and the lowest by-product accumulation. A beneficial variant K128D was obtained to further enhance LNFP V titer using computer-assisted site-directed mutagenesis. The final strain EW10 could produce 25.68 g/L LNFP V by fed-batch cultivation, with the productivity of 0.56 g/L·h.

Core fucosylation of maternal milk N-glycans imparts early-life immune tolerance through gut microbiota-dependent regulation in RORγt+ Treg cells.

Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.

FUT4 promotes the progression of Cholangiocarcinoma by modulating epithelial-mesenchymal transition.

Cholangiocarcinoma (CCA) is a common gastrointestinal malignancy characterized by a poor prognosis. Considering its prevalence, exploring its underlying molecular biological mechanisms is of paramount clinical importance. In this study, bioinformatics techniques were utilized to analyze CCA sample data obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The analysis revealed a notable upregulation in FUT4 expression in CCA samples. To further investigate the functional implications of FUT4, in vivo and in vitro experiments were conducted, which demonstrated that FUT4 overexpression significantly enhances the proliferative and migratory capabilities of tumor cells. Subsequent sequencing analysis unveiled a correlation between FUT4 and epithelial-mesenchymal transition (EMT). Indeed, the pioneering discovery of elevated FUT4 expression in CCA was highlighted in this study. Further investigations into the function of FUT4 in CCA provided initial insights into its role in driving cancer progression via EMT. These findings present promising avenues for the diagnosis and treatment of CCA.[Figure: see text].

High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model.

Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.

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.

Protein O-Fucosyltransferase Is Required for the Efficient Invasion of Hepatocytes by Plasmodium berghei Sporozoites.

The O-fucosylation of the thrombospondin type I repeat (TSR) domain is important for TSR-containing proteins' optimal folding and stability. However, the importance of Plasmodium O-fucosyltransferase 2 (POFut2) remains unclear due to two different reports. Here, we disrupted the POFut2 gene in Plasmodium berghei and demonstrated that POFut2 KO parasites develop normally in blood and mosquito stages but show reduced infectivity in mice. We found that the reduced infectivity of POFut2 KO sporozoites was due to a diminished level of TRAP that affected the parasite gliding motility and hepatocyte infectivity. Using all-atom MD simulation, we also hypothesize that O-fucosylation impacts the TSR domain's stability more than its heparin binding capacity.