Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy.

The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus Enterococcus improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective E. faecalis was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.

Emerging Strategies to Combat ß-Lactamase Producing ESKAPE Pathogens.

Since the discovery of penicillin by Alexander Fleming in 1929 as a therapeutic agent against staphylococci, ß-lactam antibiotics (BLAs) remained the most successful antibiotic classes against the majority of bacterial strains, reaching a percentage of 65% of all medical prescriptions. Unfortunately, the emergence and diversification of ß-lactamases pose indefinite health issues, limiting the clinical effectiveness of all current BLAs. One solution is to develop ß-lactamase inhibitors (BLIs) capable of restoring the activity of ß-lactam drugs. In this review, we will briefly present the older and new BLAs classes, their mechanisms of action, and an update of the BLIs capable of restoring the activity of ß-lactam drugs against ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens. Subsequently, we will discuss several promising alternative approaches such as bacteriophages, antimicrobial peptides, nanoparticles, CRISPR (clustered regularly interspaced short palindromic repeats) cas technology, or vaccination developed to limit antimicrobial resistance in this endless fight against Gram-negative pathogens.

Genome Analysis of Enterococcus mundtii Pe103, a Human Gut-Originated Pectinolytic Bacterium.

Pectin exists in significant amounts in vegetables and fruits as a component of the plant cell wall. In human diet, pectin is not degraded by the human digestive enzymes due to its complex structure; only gut bacteria degrade pectin in the large intestine. To date, although pectin is one of the most important sources of dietary fiber in human diet, there have been only few reports on human gut-originated pectinolytic bacteria. In this study, the strain Enterococcus mundtii Pe103, a bacterium with pectin-degrading activity, was isolated from the feces of a healthy Korean adult female. Culture experiments revealed that it could grow on pectin as the sole carbon source by degrading pectin to approximately 35% within 13 h. We report the complete genome data of human gut E. mundtii Pe103. It consists of a circular chromosome (3,084,146 bps) and two plasmids (63,713 and 56,223 bps). Genomic analysis suggested that at least nine putative enzymes related to pectin degradation are present in E. mundtii Pe103. These enzymes may be involved in the degradation of pectin. The whole genome information of E. mundtii Pe103 could improve the understanding of the mechanism underlying the degradation of pectin by human gut microbiota.

Molecular mechanisms of vancomycin resistance.

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram-positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi-enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d-Ala-d-Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d-Ala-d-lac or d-Ala-d-Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects.

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases.

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2'') family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). The structure of the ternary complex of APH(2'')-IIIa with GDP and kanamycin was solved at 1.34 Šresolution and was compared with substrate-bound structures of APH(2'')-Ia, APH(2'')-IIa and APH(2'')-IVa. In contrast to the case for APH(2'')-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2'')-IIa, APH(2'')-IIIa and APH(2'')-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.

Enterococci Isolated from Trout in the Bukovec Water Reservoir and Cierny Váh River in Slovakia and Their Safety Aspect.

The aim of this study was to investigate enterococci as lactic acid bacteria and as part of Firmicutes phylum. We focused on the virulence factor, biofilm formation, and antibiotic resistance and also on lactic acid production and enterocin gene detection. Intestinal samples were taken from 50 healthy trout (3 Salmo trutta and 47 Salmo gairdneri) collected in April 2007, 2010, and 2015 from different locations at the Bukovec water reservoir and the Cierny Váh River in Slovakia. Twenty pure colonies were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification system based on protein fingerprints, and then seven identified strains were also phenotyped. Based on the identification methods used, the identified enterococci (7) belong taxonomically to four different enterococcal species: Enterococcus durans, E. faecium, E. mundtii, and E. thailandicus. They were hemolysis, DNase, and gelatinase negative with acceptable enzymatic activity. They did not form biofilm and were mostly susceptible to antibiotics. All strains produced lactic acid amounting to 1.78 ± 0.33 mmol/l on average and possessed the gene for enterocin A production. This is the first study reporting more detailed properties of enterococci from trout in Slovakian wild water sources, and it produces new possibilities for studying microbiota in trout.

Expression and characterization of a recombinant stevioside hydrolyzing ß-glycosidase from Enterococcus casseliflavus.

The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel ß-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants kcat/Km for pNPG and kcat/Km for stevioside of 8583 mM-1s-1 and 95.41 mM-1s-1, respectively. When compared to the stevioside hydrolyzing ß-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications.

Azoreductase activity of dye-decolorizing bacteria isolated from the human gut microbiota.

The gut microbiota enriches the human gene pool and contributes to xenobiotic metabolism. Microbial azoreductases modulate the reduction of azo-bonds, activating produgs and azo polymer-coated dosage forms, or degrading food additives. Here, we aimed to screen the healthy human gut microbiota for food colorant-reducing activity and to characterize factors modulating it. Four representative isolates from screened fecal samples were identified as E. coli (AZO-Ec), E. faecalis (AZO-Ef), E. avium (AZO-Ev) and B. cereus (AZO-Bc). Both AZO-Ef and AZO-Ev decolorized amaranth aerobically and microaerophilically while AZO-Ec and AZO-Bc had higher aerobic reduction rates. The isolates varied in their activities against different dyes, and the azo-reduction activity mostly followed zero-order reaction kinetics, with a few exceptions. Additionally, the isolates had different pH dependence, e.g., AZO-Ec was not affected by pH variation while AZO-Bc exhibited variable degradation kinetics at different pH levels. Cell-free extracts showed NADH-dependent enzymatic activities 14-19 times higher than extracellular fractions. FMN did not affect the reducing activity of AZO-Ef cell-free extract, whereas AZO-Ec, AZO-Ev and AZO-Bc had significantly higher reduction rates in its presence (P values = 0.02, 0.0001 and 0.02, respectively). Using Degenerate primers allowed the amplification of azoreductase genes, whose sequences were 98-99% similar to genes encoding FMN-dependent-NADH azoreductases.

CylA is a sequence-specific protease involved in toxin biosynthesis.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

Bio-guided Purification and Mass Spectrometry Characterisation Exploring the Lysozyme-like Protein from Enterococcus lactis Q1, an Unusual Marine Bacterial Strain.

Lactic acid bacteria produce various antibacterial peptides such as bacteriocins that are active against pathogenic and spoilage microorganisms. Very little attention has been paid to the production of lysozyme as an antimicrobial enzyme. The present work represents one of the few studies reporting lysozyme production by enterococci. Indeed, this study was first conducted to evaluate the antimicrobial activity of Enterococcus lactis Q1, an enterocin P-producing strain previously isolated from fresh shrimp (Penaeus vannamei), against multidrug-resistant clinical isolates. Results showed significant inhibitory activity (P < 0.05) towards diverse pathogens. The purification of the antimicrobial substances produced by Q1 strain leads to the isolation of two active fractions. The SDS-PAGE and mass spectrometry analyses of fraction number 2 (fraction 2) revealed the presence of a protein with molecular mass of 14.3 kDa. Additionally, the experimental results are consistent with mass spectra of industrial lysozyme (Fluka ref. 62970). The lysozyme produced by Enterococcus lactis Q1 strain was confirmed by a plate method against Micrococcus luteus ATCC 4698. Also, sensitivity of the Q1 strain to different concentrations of lysozyme was investigated. For the first time, this study shows that E. lactis Q1 produces lysozyme which could be an excellent candidate in food biopreservation or production of functional foods to promote health benefits.

Co-occurrence of antimicrobial resistance and virulence determinants in enterococci isolated from traditionally fermented fish products.

OBJECTIVES: Fermented foods frequently consumed in Northeast India can act as a reservoir for disseminating pathogenic organisms. Enterococci are often responsible for contamination of food products. This study investigated the antimicrobial resistance and co-existing virulence determinants of enterococci found in traditionally processed foods in India. METHODS: A total of 38 enterococci isolates identified as Enterococcus faecalis isolated from fermented fish samples from retail markets of Northeast India were selected for screening of pathogenic traits. RESULTS: Of the 38 isolates, 8 (21%) were able to hydrolyse gelatin and 13 (34%) showed protease activity. Screening for haemolytic activity of the isolates showed no positive test on sheep blood. The presence of virulence genes (gelE, agg, esp, cpd, efaAfs and cylA) was investigated by PCR. gelE, agg and esp were present in 17, 13 and 4 isolates, respectively. cpd and efaAfs were found in all isolates, whereas cylA was not detected. High resistance percentages to various antibiotics included kanamycin (63%), vancomycin and gentamicin (58%), tetracycline (53%) and rifampicin (50%). The vanA genotype was confirmed in 15 multidrug- and vancomycin-resistant strains. CONCLUSION: The simultaneous occurrence of virulence determinants and antimicrobial resistance in enterococci prevalent in the fermented fish products studied poses a potential threat of transmission to humans through the food chain. This study highlights the importance of E. faecalis as a reservoir of antimicrobial resistance and virulence factors and their potential transfer to humans. The findings reopen the issue of food safety regarding enterococci prevalent in traditionally processed fish products in the region.

Streptococcus endopeptidases promote HPV infection in vitro.

Both cervical and throat cancers are associated with human papillomavirus (HPV). HPV infection requires cleavage of the minor capsid protein L2 by furin. While furin is present in the vaginal epithelium, it is absent in oral epithelial basal cells where HPV infection occurs. The objective of this study was to investigate whether common oral bacteria express furin-like peptidases. By screening strains representing 12 oral Streptococcus and Enterococcus species, we identified that eight Streptococcus strains displayed high levels of furin-like peptidase activity, with S. gordonii V2016 the highest. We constructed null mutations for 14 genes encoding putative endopeptidases in S. gordonii V2016. Results showed that three endopeptidases, PepO, PulO, and SepM, had furin-like activities. All three mutants showed decreased natural transformation by chromosomal DNA, while the pepO mutant also showed reduced transformation by plasmid DNA, indicating involvement of these endopeptidases in competence development. The purified S. gordonii PepO protein promoted infection of epithelial 293TT cells in vitro by HPV16 pseudovirus. In conclusion, oral bacteria might promote HPV infection and contribute to HPV tissue tropism and subsequent carcinogenesis in the oral cavity and throat by providing furin-like endopeptidases.

Coumarin-based, switchable fluorescent substrates for enzymatic bacterial detection.

Enzymatically-switchable fluorescent substrates, such as the commercially available 4-methyl umbelliferones (4-MU) are used as standard indicators of enzymatic activity for the detection of various microorganisms and pathogens. However, a major disadvantage of 4-MU is its relatively high pKa leading to only partial dissociation of the fluorescent anion under the conditions where the enzymes are most effective (pH 6-6.5). Here we present a method for new, enzymatically-switchable, fluorescent substrates with improved photo-physico/chemical properties. The lead derivative, 4-AAU, shows excellent solubility in aqueous media (0.81 mg/mL) when compared to 4-MU (0.16 mg/mL), significantly improved quantum yield and wider dynamic range of its fluorescence properties. The corresponding bacterial substrate ß-4-AAUG showed superior selectivity in the detection of clinically relevant amounts of E. coli, Enterococcus and K. pneumonia (1 CFU). The fluorescence intensity of ß-4-AAUG was almost 5 times higher than that of the standard, the detection was possible in reasonably short time (∼ 2.5 h) and with excellent sensitivity.

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains.

Antibiotic resistance to aminoglycoside group of antibiotics mainly occurs by aminoglycoside kinases (AKs). Thus, targeting AKs from different multidrug resistant (MDR) strains could result into inhibition of AK enzymes and ultimately the resistance. Therefore, the present study aims to target one of these AKs that is APH(3')-Ia from Acinetobacter baumannii through structure based virtual screening (SBVS) and test the binding affinity of the most stable virtually screened inhibitor with AKs from Mycobacterium tuberculosis, Acinetobacter baumannii, Enterococcus gallinarum, and Escherichia coli. SBVS investigated ZINC71575479 as a most stable inhibitor with -8.92 kcal/mol of binding energy and 0.66 µM of inhibition constant. Molecular docking results revealed that the ZINC71575479 can efficiently bind to nucleotide triphosphate (NTP) binding site of different AKs which is a known drug target site. Sequence analysis study of different AKs showed no significant similarity for active site residues; however structure superimposition study showed conserved NTP-binding domain. Molecular dynamics (MD) simulations showed stable behavior of all docked complexes with notable conformational stability of salt bridges at NTP-binding site of different AKs. Binding energy calculations revealed the interactions between key residues from NTP- binding domain of different AKs with ZINC71575479. In order to validate the MD simulations and binding energy results, crystal structure complexed with tyrphostin AG1478 a known inhibitor of AKs was kept as control. Thus, this work demonstrates the binding efficiency of ZINC71575479 toward different AKs for circumventing aminoglycoside resistance and opens avenues for the development of new antibiotics that can target diverse MDR strains with aminoglycoside resistance.

Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 µg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at specific concentrations. HLAR in E. faecalis MCC3063 may be due to the combined expression of both the aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes which could be therapeutically challenging. A combined expression of both the genes in E. faecalis MCC3063 may yield HLAR which could be therapeutically challenging. The study highlights the significant alterations in the mRNA expression levels of aac(6')Ie-aph(2″)Ia and aph(3')IIIa in resistant pathogens, upon exposure to clinically vital aminoglycosides.

Characterization of a Glutamate Decarboxylase (GAD) from Enterococcus avium M5 Isolated from Jeotgal, a Korean Fermented Seafood.

To develop starters for the production of functional foods or materials, lactic acid bacteria producing γ-aminobutyric acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium L-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced 18.47 ± 1.26 mg/ml GABA when incubated for 48 h at 37°C in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax values of GAD were 3.26 ± 0.21 mM and 0.0120 ± 0.0001 mM/min, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin.

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s - either a ligand induced dimerization and/or compaction of the monomer are considered.

Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus TCCC11660.

Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of L-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo6+ and Mg2+ had positive effects, while Cu2+, Fe2+, Zn2+ and Co2+ showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the K m and V max values for the sole L-glutamate substrate were 5.26 and 3.45 µmol L-1 min-1, respectively.

Biosynthesis and role of 3-methylbutanal in cheese by lactic acid bacteria: Major metabolic pathways, enzymes involved, and strategies for control.

Branched chain aldehyde, 3-methylbutanal is associated as a key flavor compound with many hard and semi-hard cheese varieties. The presence and impact of this flavor compound in bread, meat, and certain beverages has been recently documented, however its presence and consequences regarding cheese flavor were not clearly reported. This paper gives an overview of the role of 3-methylbutanal in cheese, along with the major metabolic pathways and key enzymes leading to its formation. Moreover, different strategies are highlighted for the control of this particular flavor compound in specific cheese types.

Efficient chemoenzymatic synthesis of uridine 5'-diphosphate N-acetylglucosamine and uridine 5'-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes.

Uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5'-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.