Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serA^T410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serA^T410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA^* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.
Culture Media ; Ergothioneine/metabolism* ; Escherichia coli/metabolism* ; Fermentation ; Histidine/metabolism* ; Metabolic Engineering

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies/*genetics ; Antibodies/immunology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Histidine-tRNA Ligase/biosynthesis ; Histidine-tRNA Ligase/*genetics ; Histidine-tRNA Ligase/immunology ; Open Reading Frames/genetics ; Prokaryotic Cells/metabolism

Animals ; Carrier Proteins/metabolism* ; Histidine/metabolism* ; Humans ; Methylation ; Methyltransferases/metabolism* ; Neoplasm Proteins/metabolism* ; Neoplasms/metabolism* ; Protein Processing, Post-Translational

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Histidine-tRNA Ligase ; biosynthesis ; genetics ; immunology ; Humans ; Open Reading Frames ; genetics ; Prokaryotic Cells ; metabolism

Bacterial Outer Membrane Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli Proteins ; chemistry ; genetics ; metabolism ; Glutamic Acid ; genetics ; metabolism ; Histidine ; genetics ; Hydrogen-Ion Concentration ; Ion Channel Gating ; genetics ; Lipid Bilayers ; metabolism ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Porins ; chemistry ; genetics ; metabolism

Glimepiride, a third generation sulfonylurea, is an antihyperglycemic agent widely used to treat type 2 diabetes mellitus. In this study, an untargeted urinary metabolomic analysis was performed to identify endogenous metabolites affected by glimepiride administration. Urine samples of twelve healthy male volunteers were collected before and after administration of 2 mg glimepiride. These samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then subjected to multivariate data analysis including principal component analysis and orthogonal partial least squares discriminant analysis. Through this metabolomic profiling, we identified several endogenous metabolites such as adenosine 3′, 5′-cyclic monophosphate (cAMP), quercetin, tyramine, and urocanic acid, which exhibit significant metabolomic changes between pre- and posturine samples. Among these, cAMP, which is known to be related to insulin secretion, was the most significantly altered metabolite following glimepiride administration. In addition, the pathway analysis showed that purine, tyrosine, and histidine metabolism was affected by pharmacological responses to glimepiride. Together, the results suggest that the pharmacometabolomic approach, based on LC-MS/MS, is useful in understanding the alterations in biochemical pathways associated with glimepiride action.
Adenosine ; Diabetes Mellitus, Type 2 ; Histidine ; Humans* ; Insulin ; Least-Squares Analysis ; Male ; Mass Spectrometry* ; Metabolism ; Metabolomics* ; Principal Component Analysis ; Quercetin ; Statistics as Topic ; Tyramine ; Tyrosine ; Urocanic Acid ; Volunteers

OBJECTIVETo analyze the phenotype and genotype of a family with inherited dysfibrinogenemia.

METHODSLaboratory tests including activated particle thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and the activity of protein C (PC), protein S(PS) and antithrombin (AT) were conducted in the proband and 4 family members. The activity and antigen of fibrinogen in plasma were measured by functional and immunoturbidimetry assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes were analyzed by direct sequencing.

RESULTSThe proband had normal APTT and PT, but prolonged TT. Her plasma fibrinogen levels were extremely reduced, which was also found in her mother. The sequencing results of the proband revealed heterozygous g.5678 G>A in the exon 8 of FGG gene originating from her mother, which caused Arg275His missense mutation.

CONCLUSIONDysfibrinogenemia in the family is caused by Arg275His in the beta chain of fibrinogen and it is the first report on a Chinese family with inherited dysfibrinogenemia.

Adult ; Afibrinogenemia ; blood ; genetics ; Amino Acid Substitution ; Arginine ; genetics ; DNA Mutational Analysis ; Female ; Fibrinogen ; genetics ; metabolism ; Histidine ; genetics ; Humans ; Male ; Pedigree ; Phenotype

The rapid increase in the prevalence of metabolic syndrome, which is associated with a state of elevated systemic oxidative stress and inflammation, is expected to cause future increases in the prevalence of diabetes and cardiovascular diseases. Oxidation of polyunsaturated fatty acids and sugars produces reactive carbonyl species, which, due to their electrophilic nature, react with the nucleophilic sites of certain amino acids. This leads to formation of protein adducts such as advanced glycoxidation/lipoxidation end products (AGEs/ALEs), resulting in cellular dysfunction. Therefore, an effective reactive carbonyl species and AGEs/ALEs sequestering agent may be able to prevent such cellular dysfunction. There is accumulating evidence that histidine containing dipeptides such as carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-methyl-L-histidine) detoxify cytotoxic reactive carbonyls by forming unreactive adducts and are able to reverse glycated protein. In this review, 1) reaction mechanism of oxidative stress and certain chronic diseases, 2) interrelation between oxidative stress and inflammation, 3) effective reactive carbonyl species and AGEs/ALEs sequestering actions of histidine-dipeptides and their metabolism, 4) effects of carnosinase encoding gene on the effectiveness of histidine-dipeptides, and 5) protective effects of histidine-dipeptides against progression of metabolic syndrome are discussed. Overall, this review highlights the potential beneficial effects of histidine-dipeptides against metabolic syndrome. Randomized controlled human studies may provide essential information regarding whether histidine-dipeptides attenuate metabolic syndrome in humans.
Amino Acids ; Anserine ; Carbohydrates ; Cardiovascular Diseases ; Carnosine ; Chronic Disease ; Dipeptides ; Fatty Acids, Unsaturated ; Histidine ; Humans ; Inflammation ; Metabolism ; Oxidative Stress ; Prevalence ; Sequestering Agents

Methyl parathion hydrolase (MPH) is a novel member of organophosphorus hydrolase. In this study, mpd gene was expressed in Escherichia coli DH5alpha with its native promoter. MPH was purified to homogeneity. Results show that metal-chelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis showed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2:1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. Experiment results suggested that aspartate, glutamate, arginine and lysine are not important for enzyme activity. But DEPC, which can modify histidine residue, inactivate the enzyme activity greatly, and the inactivation rate is 9.6 h(-1). This result reflects that histidine residues are essential for enzyme activity. All these results provide basic data for MPH structure and molecular evolution research.
Aryldialkylphosphatase ; chemistry ; Enzyme Activation ; drug effects ; Escherichia coli ; genetics ; metabolism ; Histidine ; chemistry ; Phosphoric Monoester Hydrolases ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics