L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.
Humans ; Methionine ; Methylenetetrahydrofolate Reductase (NADPH2) ; Carbon ; Cysteine ; Escherichia coli/genetics* ; Hydroxymethyl and Formyl Transferases ; Carrier Proteins ; Escherichia coli Proteins

OBJECTIVE: To explore the clinical features and genetic basis of a child with Galactosemia. METHODS: A child who had presented at the Children's Hospital Affiliated to Zhengzhou University on November 20, 2019 was selected as the study subject. Clinical data of the child was collected. Whole exome sequencing was carried out for the child. Candidate variants were validated by Sanger sequencing. RESULTS: Clinical manifestations of the child have included anemia, feeding difficulty, jaundice, hypomyotonia, abnormal liver function and coagulation abnormality. Tandem mass spectrometry showed increased citrulline, methionine, ornithine and tyrosine. Urine organic acid analysis showed increased phenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 4-hydroxyphenylpyruvate and N-acetyltyrosine. Genetic testing revealed that the child has harbored compound heterozygous variants of the GALT gene, namely c.627T>A (p.Y209*) and c.370G>C (p.G124R), which were respectively inherited from her healthy parents. Among these, c.627T>A (p.Y209*) was known as a likely pathogenic variant, while c.370G>C (p. G124R) was unreported previously and also predicted as a likely pathogenic variant(PM1+PM2_Supporting+PP3_Moderate+PPR). CONCLUSION Above discovery has expanded the spectrum of the GALT gene variants underlying Galactosemia. Patients with thrombocytopenia, feeding difficulties, jaundice, abnormal liver function and coagulation abnormality without obvious causes should be analyzed by screening of metabolic diseases in combination with genetic testing.
Child ; Female ; Humans ; Galactosemias/genetics* ; Genetic Testing ; Health Status ; Methionine ; Muscle Hypotonia ; Mutation

Circadian Clocks/genetics* ; Reactive Oxygen Species ; Methionine ; Gastrointestinal Microbiome ; Gastrointestinal Tract ; Racemethionine ; Gene Expression

OBJECTIVE: To explore the role of the microbiota in drug naïve first-onset schizophrenia patients and to seek evidence from multidimensional longitudinal analyses of the intestinal microbiome and clinical phenotype with antipsychotic drugs (APDs) therapy. METHODS: In this study, 28 drug naïve first onset schizophrenia patients and age-, gender- and education-matched 29 healthy controls were included, and the patients were treated with APDs. We collected fecal and serum samples at baseline and after 6 weeks of treatment to identify the different microbiota strains and analyse their correlation with clinical symptoms and serum metabolites. The 16S rRNA genes of the gut microbiota were sequenced, and the diversity and relative abundance at the phylum and genus levels were analyzsed in detail. The PANSS score, BMI changed value, and serum metabolome were included in the data analyses. RESULTS: A multiomics study found a potential connection among the clinical phenotype, microbiota and metabolome. The species diversity analyses revealed that the alpha diversity index (chao1, ACE, and goods_coverage) in the schizophrenia APDs group was significantly lower than that in the control group, and the schizophrenia group had clear demarcation from the control group. The microbiota composition analysis results showed that the relative abundance of the genera of Bacteroides, Streptococcus, Romboutsia, and Eubacterium ruminantium group significantly changed after APDs treatment in the schizophrenia patients. These strains could reflect the APDs treatment effect. More genera had differences between the patient and control groups. The LEfSe analysis showed that Prevotella_9 and Bacteroides were enriched in schizophrenia, while Blautia, Dialister, and Roseburia were enriched in the control group. The correlation analysis between microbiota and clinical symptoms showed that Bifidobacterium in schizophrenia was positively correlated with the PANSS reduction rate of the general psychopathology scale. The BMI changed value was positively correlated with the alteration of Clostridium_sensu_stricto_1 during treatment and the baseline abundance of Bacteroides. Moreover, metabolomic data analysis revealed a significant correlation between specific genera and metabolites, such as L-methionine, L-proline, homovanillic acid, N-acetylserotonin, and vitamin B6. CONCLUSION Our study found some microbiota features in schizophrenia patients and healthy controls, and several strains were correlated with APDs effects. Furthermore, the multiomics analysis implies the intermediate role of microbiota between antipsychotic effects and serum metabolites and provides new evidence to interpret the difference from multiple levels in the pathogenesis and pharmacological mechanism of schizophrenia.
Humans ; Antipsychotic Agents ; Homovanillic Acid ; Metabolomics/methods* ; Methionine ; Microbiota ; Proline ; RNA, Ribosomal, 16S/genetics* ; Schizophrenia ; Vitamin B 6 ; Feces

OBJECTIVE: To summarize newborn screening for methionine adenosyltransferase I/III (MAT I/III) deficiency in Quanzhou region of Fujian Province. METHODS: A total of 364 545 neonates were screened for inherited metabolic diseases by tandem mass spectrometry. High-throughput next generation sequencing combined with Sanger sequencing was used to detect potential variants in newborns with MAT I/III deficiency. Pathogenicity of suspected variants was predicted by using MutationTaster and HSF software. RESULTS: Three newborns were identified with MAT I/III deficiency by newborn screening, which yielded an incidence rate of 1 in 121 515. Amino acid and acylcarnitine analysis suggested that the serum methionine of the three patients have increased to various extents. All patients showed normal growth and development during follow-up, and were found to carry MAT1A gene variants including two missense variants [c.776C>T (p.Ala259Val) and c.791G>A (p.Arg264His)] and a synonymous variant [c.360C>T (p.Cys120Cys)]. Among these, c.776C>T (p.Ala259Val) and c.791G>A (p.Arg264His) were known to be pathogenic, whereas c.360C>T (p.Cys120Cys) was a novel variant. Bioinformatics analysis suggested that this variant may alter RNA splicing and affect the structure and function of the MAT1A protein. CONCLUSION A systematic review of newborn screening for MAT I/III deficiency was provided. Discovery of the novel variant has enriched the variant profile of the MAT1A gene and provided a basis for the diagnosis of this disease.
Amino Acid Metabolism, Inborn Errors ; diagnosis ; China ; Genetic Variation ; Humans ; Infant, Newborn ; Methionine Adenosyltransferase ; deficiency ; genetics ; Neonatal Screening

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
Animals ; Apoptosis ; Diet ; Endothelial Cells ; Erectile Dysfunction/prevention & control* ; Fibrosis ; Humans ; Hyperhomocysteinemia/complications* ; Male ; Methionine ; Oxidative Stress ; Penis/pathology* ; Rats ; Rats, Sprague-Dawley ; Rats, Transgenic ; Signal Transduction/genetics* ; Tissue Kallikreins/genetics*

OBJECTIVETo detect potential mutations of MAT1A gene in a child suspected with simple hypermethioninemia by MS/MS neonatal screening.

METHODSClinical data of the child was collected. Genomic DNA was extracted by a standard method and subjected to targeted sequencing using an Ion AmpliseqInherited Disease Panel. Detected mutations were verified by Sanger sequencing.

RESULTSThe child showed no clinical features except evaluated methionine. A novel compound mutation of the MAT1A gene, i.e., c.345delA and c.529C>T, was identified in the child. His father and mother were found to be heterozygous for the c.345delA mutation and c.529C>T mutation, respectively.

CONCLUSIONThe compound mutation c.345delA and c.529C>T of the MAT1A gene probably underlie the disease in the child. The semi-conductor sequencing has provided an important means for the diagnosis of hereditary diseases.

Amino Acid Metabolism, Inborn Errors ; genetics ; pathology ; Base Sequence ; DNA Mutational Analysis ; methods ; Family Health ; Fathers ; Female ; Genetic Predisposition to Disease ; genetics ; Glycine N-Methyltransferase ; deficiency ; genetics ; Heterozygote ; Humans ; Infant, Newborn ; Infant, Newborn, Diseases ; genetics ; pathology ; Male ; Methionine Adenosyltransferase ; genetics ; Mothers ; Mutation

OBJECTIVETo observe the analgesic effect of electroacupuncture (EA) on collagen-induced arthritis (CIA) rats and its regulating effect on inflammation reaction and the endogenous opioid system of synovial tissues. Methods A total of 30 healthy male Wistar rats were randomly divided into a control group, a model group and an EA group, 10 rats in each one. The chronic pain model of CIA rats was made by cattle type-II collagen in the model group and EA group. Rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Kunlun" (BL 60) for 30 min from 16th day after model establishment, once a day for consecutive 10 days. Rats in the control group did not receive any treatment. Rats in the model group were treated with fixation as the EA group. Threshold of pain, arthritis index, paw swelling were measured before model establishment and 16 d, 20 d, 23 d and 25 d after model establishment. The levels of beta-endorphin (β-END), met-enkephalin (met-ENK), dynorphin A (Dyn A) were measured by radioimmunoassay; the mRNA expressions of mu opioid receptor (MOR), kappa opioid receptor (KOR) and delta opioid receptor (DOR) in synovial tissues of CIA rats were detected by I quantitative polymerase chain reaction (qPCR).

RESULTSCompared with the control group, threshold of pain was reduced (all P<0. 01), arthritis index was increased (all P<0. 01) and paw swelling was increased (all P<0. 01) in the model group on the 16th day, 20th day, 23rd day, 25th day after model establishment. Compared with the model group, the threshold of pain was increased in the EA group (all P<0. 01), arthritis index and paw swelling were reduced (all P<0. 01) on the 23rd day and 25th day after model establishment. Compared with the control group, the level of Dyn A in synovial tissues of CIA rats was increased in the model group (P<0. 01); the mRNA expressions of MOR, KOR and DOR were down-regulated lower than 0. 5 fold of normal level. Compared with the model group, the level of β-END in synovial tissues of the knee joint was increased in the EA group (P<0. 05), and the mRNA expressions of MOR, KOR and DOR in synovial tissues of CIA rats were up-regulated more than 2 folds of normal level.

CONCLUSIONThe intervention of EA on chronic pain of CIA rats is superior, which is likely to be related with effects of EA on anti-inflammation and up-regulation of synovial tissue β-END and MOR, KOR, DOR.

Acupuncture Analgesia ; Acupuncture Points ; Analgesics, Opioid ; immunology ; Animals ; Arthritis, Rheumatoid ; immunology ; therapy ; Cattle ; Chronic Pain ; immunology ; therapy ; Dynorphins ; genetics ; immunology ; Electroacupuncture ; Enkephalin, Methionine ; genetics ; immunology ; Humans ; Male ; Rats ; Rats, Wistar ; Receptors, Opioid, mu ; genetics ; immunology ; Synovial Fluid ; immunology ; beta-Endorphin ; genetics ; immunology

Dentin matrix protein 1 (DMP1) is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with αvβ3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo. We first demonstrated that a patient with the DMP1 M1V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressing (NLS)DMP1, in which the endoplasmic reticulum (ER) entry signal sequence of DMP1 was replaced by a nuclear localization signal (NLS) sequence, under the control of a 3.6 kb rat type I collagen promoter plus a 1.6 kb intron 1. We then crossbred the (NLS)DMP1 transgenic mice with Dmp1 null mice to express the (NLS)DMP1 in Dmp1-deficient genetic background. Although immunohistochemistry demonstrated that (NLS)DMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis.
Animals ; Cell Nucleus ; genetics ; Codon, Initiator ; genetics ; Collagen Type I ; genetics ; Endoplasmic Reticulum ; genetics ; Extracellular Matrix Proteins ; genetics ; Familial Hypophosphatemic Rickets ; genetics ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; Introns ; genetics ; Methionine ; genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation ; genetics ; Odontoblasts ; cytology ; Odontogenesis ; genetics ; Periodontal Diseases ; genetics ; Periodontal Ligament ; pathology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; genetics ; Tooth Abnormalities ; genetics ; Tooth Eruption ; genetics ; Transcription Factors ; genetics ; Transgenes ; genetics ; Valine ; genetics ; Young Adult

OBJECTIVETo study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead.

METHODSMale SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay.

RESULTSThe expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05).

CONCLUSIONMethionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Choline ; pharmacology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Male ; Methionine ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley