Evaluation and Mechanistic Investigation of Human Milk Oligosaccharide against SARS-CoV-2.

Four human milk oligosaccharides (HMOs), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 2'-fucosyllactose (2'-FL), and 3-fucosyllactose (3-FL), were assessed for their possible antiviral activity against the SARS-CoV-2 spike receptor binding domain (RBD) in vitro. Among them, only 2'-FL/3-FL exhibited obvious antibinding activity against direct binding and trans-binding in competitive immunocytochemistry and enzyme-linked immunosorbent assays. The antiviral effects of 2'-FL/3-FL were further confirmed by pseudoviral assays with three SARS-Cov-2 mutants, with a stronger inhibition effect of 2'-FL than 3-FL. Then, 2'-FL/3-FL were studied with molecular docking and microscale thermophoresis analysis, showing that the binding sites of 2'-FL on RBD were involved in receptor binding, in addition to a tighter bond between them, thus enabling 2'-FL to be more effective than 3-FL. Moreover, the immunomodulation effect of 2'-FL was preliminary evaluated and confirmed in a human alveolus chip. These results would open up possible applications of 2'-FL for the prevention of SARS-CoV-2 infections by competitive binding inhibition.

Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids.

Efficient formate dehydrogenase (FDH)-based cofactor regeneration systems are widely used for biocatalytic processes due to their ready availability, low reduction potential, and production of only benign byproducts. However, FDHs are usually specific to NAD+, and NADPH regeneration with formate is challenging. Herein, an FDH with a preference for NAD+ from Azospirillum palustre (ApFDH) was selected owing to its high activity. By static and dynamic structural analyses, a beneficial substitution, D222Q, was identified for cofactor-preference switching. However, its total activity was substantially decreased by 90% owing to the activity-specificity trade-off. Subsequently, a semirational library was designed and screened, which yielded a variant ApFDHD222Q+A199G+H380S with satisfactory activity and NADP+ specificity. Our analysis of dynamical cross-correlations revealed a substitution combination that brought balance to the dynamical correlation network. This combination successfully overcame the activity-specificity-stability trade-off and resulted in a beneficial outcome. The substitution combination (D222Q-A199G/H380S-C256A/C146S) enabled the simultaneous improvement of activity, specificity, and stability and was successfully applied to other 17 FDHs. Finally, by employing engineered ApFDH, an NADPH regeneration system was developed, optimized, and utilized for the asymmetric biosynthesis of l-phosphinothricin.

Loss of Phosphomannose Isomerase Impairs Growth, Perturbs Cell Wall Integrity, and Reduces Virulence of Fusarium oxysporum f. sp. cubense on Banana Plants.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, necessitating urgent measures to control this disease. However, the molecular mechanisms underlying Foc TR4 virulence remain elusive. Phosphomannose isomerase is a key enzyme involved in the biosynthesis of GDP mannose, an important precursor of fungal cell walls. In this study, two phosphomannose isomerases were identified in the Foc TR4 genome, of which only Focpmi1 was highly expressed throughout all developmental stages. Generated null mutants in Foc TR4 showed that only the ΔFocpmi1 mutant required exogenous mannose for growth, indicating that Focpmi1 is the key enzyme involved in GDP mannose biosynthesis. The Focpmi1 deficient strain was unable to grow without exogenous mannose and exhibited impaired growth under stress conditions. The mutant had reduced chitin content in its cell wall, rendering it vulnerable to cell wall stresses. Transcriptomic analysis revealed up- and down-regulation of several genes involved in host cell wall degradation and physiological processes due to the loss of Focpmi1. Furthermore, Focpmi1 was also found to be crucial for Foc TR4 infection and virulence, making it a potential antifungal target to address the threats posed by Foc TR4.

Emulsions containing composite (clove, oregano, and cinnamon) essential oils: Phase inversion preparation, physicochemical properties and antibacterial mechanism.

Natural essential oils (EOs), especially those combining different individual EOs (also termed composite EOs) with enhanced performance, are becoming healthy, market-sought food preservatives/additives. This study aims to provide insights into the challenge regarding EOs processing due to their low solubility and the elusive mechanism under the enhanced bio-reactivity of composite EOs. A unique oil/water interacting network was created by phase-inversion processing, which enhances EO solubilization and emulsification to form composite EO formulations (EOFs) containing ordinary cinnamon, oregano and clove EOs. These EOFs mainly contained cinnamaldehyde, carvacrol and eugenol and exhibited excellent post-storage stability. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability of EOFs (at 15.880 µL/mL) was > 88%, and the Ferric reducing antioxidant power (FRAP) was 1.8 mM FeSO4·7H2O. The minimum inhibitory concentration (MIC) of EOFs against E. coli and S. aureus was ∼7.940 µL/mL. The EOFs could cause quick deterioration of bacterial structures, demonstrating high efficacy in bacteria-killing and anti-biofilm formation.

[Glycosylation, glycan receptors recognition of SARS-CoV-2 and discoveries of glycan inhibitors against SARS-CoV-2].

COVID-19 represents the most serious public health event in the past few decades of the 21st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.

Novel Insights Into the Sulfated Glucuronic Acid-Based Anti-SARS-CoV-2 Mechanism of Exopolysaccharides From Halophilic Archaeon Haloarcula hispanica.

The pandemic caused by SARS-CoV-2 is the most widely spread disease in the 21st century. Due to the continuous emergence of variants across the world, it is necessary to expand our understanding of host-virus interactions and explore new agents against SARS-CoV-2. In this study, it was found exopolysaccharides (EPSs) from halophilic archaeon Haloarcula hispanica ATCC33960 can bind to the spike protein of SARS-CoV-2 with the binding constant KD of 2.23 nM, block the binding of spike protein to Vero E6 and bronchial epithelial BEAS-2B cells, and inhibit pseudovirus infection. However, EPSs from the gene deletion mutant △HAH_1206 almost completely lost the antiviral activity against SARS-CoV-2. A significant reduction of glucuronic acid (GlcA) and the sulfation level in EPSs of △HAH_1206 was clearly observed. Our results indicated that sulfated GlcA in EPSs is possible for a main structural unit in their inhibition of binding of SARS-CoV-2 to host cells, which would provide a novel antiviral mechanism and a guide for designing new agents against SARS-CoV-2.

Recombinant Arthromyces ramosus Peroxidase Has Similar Substrate Specificity Profiles as, but a Catalytic Efficiency up to 11-Fold Higher than, Horseradish Peroxidase.

Fungal peroxidases are valuable enzymes. Arthromyces ramosus peroxidase (ARP) and horseradish peroxidase (HRP) share a conserved catalytic site. Both native ARP and recombinant ARP (rARP) were not commercially available. The substrate specificity and kinetic parameters of rARP and HRP were not well compared, particularly relevent to structure-activity relationship. In this work, rARP expressed by Komagataella phaffii had a production yield of 6.2 mg/L, up to 155-fold higher than ARP and other recombinant peroxidases, and a specific activity of 3240 units/mg toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), up to 29-fold higher than HRP and other peroxidases. The Michaelis constant (Km) and first-order rate constant (kcat) of rARP showed 10-fold substrate affinity and consequently 6-fold catalytic efficiency of HRP toward ABTS. Under optimal conditions, rARP shared similar substrate specificity profiles as commercial HRP; the second-order rate constants (kapp) of rARP showed 2-11-fold catalytic efficiency of HRP toward well-known peroxidase substrates. rARP's higher catalytic efficiency was also in agreement with the shorter binding distance of H/N-His56 in rARP/substrate in comparison to that of HRP/substrate, as illustrated by docking simulation. The rARP had similar substrate specificity profiles as, but higher specific activity and catalytic efficiency than, HRP, which merits its further structure-functional characterization and applications.

Galactofuranose (Galf)-containing sugar chain contributes to the hyphal growth, conidiation and virulence of F. oxysporum f.sp. cucumerinum.

The ascomycete fungus Fusarium oxysporum f.sp. cucumerinum causes vascular wilt diseases in cucumber. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. BLASTp searches of the Aspergillus fumigatus UgmA and galatofuranosyltransferases (Galf-transferases) sequences in the F. oxysporum genome identified two genes encoding putative UDP-galactopyranose mutase (UGM), ugmA and ugmB, and six genes encoding putative Galf-transferase homologs. In this study, the single and double mutants of the ugmA, ugmB and gfsB were obtained. The roles of UGMs and GfsB were investigated by analyzing the phenotypes of the mutants. Our results showed that deletion of the ugmA gene led to a reduced production of galactofuranose-containing sugar chains, reduced growth and impaired conidiation of F. oxysporum f.sp. cucumerinum. Most importantly, the ugmA deletion mutant lost the pathogenicity in cucumber plantlets. Although deletion of the ugmB gene did not cause any visible phenotype, deletion of both ugmA and ugmB genes caused more severe phenotypes as compared with the ΔugmA, suggesting that UgmA and UgmB are redundant and they can both contribute to synthesis of UDP-Galf. Furthermore, the ΔgfsB exhibited an attenuated virulence although no other phenotype was observed. Our results demonstrate that the galactofuranose (Galf) synthesis contributes to the cell wall integrity, germination, hyphal growth, conidiation and virulence in Fusarium oxysporum f.sp. cucumerinum and an ideal target for the development of new anti-Fusarium agents.

Combined Effect of the Essential Oil and Collagen Film on the Quality of Pacific Mackerel (Pneumatophorus japonicus) Fillet During Cold Storage.

Essential oils (EOs) and collagen have received recent attention in the seafood industry due to their abilities of antibacterial and seafood preservation individually. However, to the authors' best knowledge, very few publications address the issue of the combined effect of EOs and collagen on seafood preservation. Pacific mackerel is one of the most economically valuable fish species in China and easy to deteriorate during storage. Therefore, present study investigated the effect of combined EOs (cinnamon, oregano, and clove) and collagen on the quality of Pacific mackerel during cold storage. A suite of microbiological, physical, and chemical properties that are indicative of quality was measured. From the results, mackerel fillets treated with an EO-collagen film had a smaller increase in microbial counts compared with control. Furthermore, total volatile basic nitrogen (TVB-N), thiobarbituric acid related substance, and pH of mackerel fillet were lower when treated with an EO-collagen film and somewhat lower when treated with collagen alone. According to texture measurements of muscle, samples treated with EO-collagen film began to deteriorate in 8 d, versus only 4 d for control samples. EOs likely contributed to antibacterial and antioxidative activity, and the collagen film isolated muscle from air, which in turn reduced oxidation and retained the quality. Consequently, combination of EOs and collagen film efficiently extends shelf-life of Pacific mackerel during storage.

Protein O-mannosylation affects protein secretion, cell wall integrity and morphogenesis in Trichoderma reesei.

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Enhanced glycosylation of an S-layer protein enables a psychrophilic methanogenic archaeon to adapt to elevated temperatures in abundant substrates.

Adaptation to higher temperatures would increase the environmental competitiveness of psychrophiles, organisms that thrive in low-temperature environments. Methanolobus psychrophilus, a cold wetland methanogen, 'evolved' as a mesophile, growing optimally at 30 °C after subculturings, and cells grown with ample substrates exhibited higher integrity. Here, we investigated N-glycosylation of S-layer proteins, the major archaeal envelope component, with respect to mesophilic adaptation. Lectin affinity enriched a glycoprotein in cells grown at 30 °C under ample substrate availability, which was identified as the S-layer protein Mpsy_1486. Four N-glycosylation sites were identified on Mpsy_1486, which exhibited different glycosylation profiles, with N94 only found in cells cultured at 30 °C. An N-linked glycosylation inhibitor, tunicamycin, reduced glycosylation levels of Mpsy_1486 and growth at 30 °C, thus establishing a link between S-layer protein glycosylation and higher temperature adaptation of the psychrophilic archaeon M. psychrophilus.

Genetical and O-glycoproteomic analyses reveal the roles of three protein O-mannosyltransferases in phytopathogen Fusarium oxysporum f.sp. cucumerinum.

Protein O-mannosyltransferases (PMTs) have been identified in fungi but not in plants and nematodes, which makes PMTs become attractive targets for developing a new strategy against phytopathogens. Three PMTs have been identified in Fusarium oxysporum, a fungal pathogen that causes vascular wilt in a broad range of economical crops. By deletion or suppression of the pmt genes, we showed that all mutants displayed retarded growth, reduced conidiation, cell wall defects, ER stress and attenuated virulence in F. oxysporum f.sp. cucumerinum. In addition, the Δpmt1 exhibited reduced thermotolerance, while the Δpmt4 and the pmt2 conditional mutant exhibited abnormal polarized growth. Comparative glycoproteome analysis of these pmt mutants revealed that PMTs preferentially modified random coils with flanking regions rich in Ser, Thr, Ala, Glu, Asp and Lys at the stem region of membrane proteins, the N-terminal region close to signal peptide of secreted proteins, or surface of soluble proteins. PMT1 specifically acted on nuclear proteins and proteins that are responsible for protein folding, which might contribute to thermotolerance. PMT4 specifically acted on the membrane and soluble proteins in secretory pathways, especially the GPI anchoring pathway, which might contribute to synthesis and transportation of GPI anchored proteins and thus polarized growth. PMT2 was responsible for modification of proteins that are required for protein folding and cell wall synthesis, which might make PMT2 essential. Our results gave an insight to understanding of the roles of each O-mannosyltransferase in F. oxysporum f.sp. cucumerinum and provide a new perspective to prevent Fusarium wilt.

[Cloning and identification of cellulase genes from uncultured microorganisms in pulp sediments from paper mill effluent].

The metagenomic DNA of pulp sediments from paper mill effluent was extracted and purified. The 16S rDNA was amplified using the purified metagenomic DNA as template and a 16S rDNA library was prepared. Sequence analysis of 16S rDNA clones showed that diverse of uncultured bacteria inhabit in this environment, which can be classified into 4 clusters as Spirochaetes, Proteobacteria, Bacteroidetes and Firmicutes. A metagenomic library containing 10000 clones was constructed into cosmid vector, and the capacity of inserted DNA of which was 3.53 x 10(8) bp. Functional screening of the library resulted in isolation of two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressing beta-glucosidase activity. One clone expressing strongest enzyme activity from each activity category was chosen to be further analyzed. Three novel cellulase genes designated as umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expression. The umcel5L encodes an endoglucanase belonging to glycosyl hydrolase family 5, which is most related to an endoglucanase from Bradyrhizobium japonicum at 43% identity and 59% similarity. The umcel5M encodes a cellodextrinase belonging to glycosyl hydrolase family 5, which is most similar to a cellodextrinase from Fibrobacter succinogenes at 48% identity and 69% similarity. The umbgl3D encodes a putative beta-glucosidase belonging to glycosyl hydrolase family 3, which shares highest homology with a beta-glucosidase from Thermotoga maritima at 46% identity and 61% similarity. It is the first time to reveal the bacterial diversity of pulp sediments from paper mill effluent and clone novel cellulase genes from the bacteria by culture-independent method.