Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli / 生物工程学报

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.

Prevalence of thrombophilia-associated genetic risk factors in blood donors of a regional hospital in southern Brazil

ABSTRACT Introduction: Thromboembolic events occur due to an imbalance in the hemostasis and some factors associated with this condition can be inherited. In order to evaluate the frequency of genotypes considered to be common hereditary risk factors for thrombophilia associated with venous thrombosis (g.1691G>A and g.20210G>A) and hyperhomocysteinemia (g.677C>T and g.1298A>C), samples from voluntary healthy blood donors at the Hospital de Clínicas de Porto Alegre were tested. Methods: We examined 325 blood samples from blood donors collected from October 2017 to July 2018. Blood was collected on filter paper and the DNA was extracted for single nucleotide polymorphisms (SNPs) analysis using the qualitative real time polymerase chain reaction. Results: The calculated frequencies of each genetic variant in heterozygosity were 4% for the FV gene (g.1691G> A), 4% for the F2 gene (g.20210G> A) and 42% and 39% for methylenetetrahydrofolate reductase (MTHFR), g.677C>T and g.1298A>C, respectively. Only the genetic variants of MTHFR were found in homozygosity, with frequencies of 14% and 6% (g.677C>T and g.1298A>C), respectively. Discussion: Altogether, these results describe the frequencies of genetic variants associated with venous thrombosis and hyperhomocysteinemia in the analyzed group and are important to enhance our current knowledge about the genetic profiles of Brazilian blood donors.

Association between MTHFR gene polymorphism and primary hypertension in children / 中国当代儿科杂志

OBJECTIVES@#To study the distribution characteristics of methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism in children with primary hypertension, and to explore the association between MTHFR C677T gene polymorphism and H-type hypertension in children.@*METHODS@#A total of 121 children with primary hypertension who were hospitalized in the department of cardiovascular medicine from January to July 2021, newly diagnosed, and untreated were retrospectively selected as the subjects. The children were divided into three groups: CC genotype (19 children), CT genotype (51 children), and TT genotype (51 children). According to the serum homocysteine (Hcy) level, they were divided two groups: H-type hypertension (47 children) and simple hypertension (74 children). The medical data were compared between the groups. The association between MTHFR C677T gene polymorphism and H-type hypertension was evaluated.@*RESULTS@#The mutation frequency of T allele in children with primary hypertension was significantly higher than that in healthy adults in Beijing and Chinese Han adults (P<0.001). The serum Hcy level in the TT genotype group was significantly higher than that in the CC and CT genotype groups (P<0.001). The serum Hcy level in the H-type hypertension group was significantly higher than that in the simple hypertension group (P<0.001), and MTHFR C677T was mostly TT genotype, which was associated with the risk of H-type hypertension (OR=12.71, P<0.001). There was no significant difference in the incidence rate of target organ damage between the H-type hypertension and simple hypertension groups (P>0.05). However, multiple organ involvement was observed in the H-type hypertension group at diagnosis, accounting for 11% (5/47).@*CONCLUSIONS@#The mutation rate of MTHFR C677T T allele in children with primary hypertension is high and associated with the serum Hcy level. TT genotype is an independent risk factor for H-type hypertension in children, and it may be related to the severity of early target organ damage.

Correlation between Serum Homocysteine Level, MTHFR Gene Polymorphism and Patients with Hematological Diseases Complicated with Coronary Heart Disease / 中国实验血液学杂志

OBJECTIVE@#To observe the expression level of serum homocysteine (Hcy) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in patients with hematological diseases complicated with coronary heart disease, and analyze the relationship between serum Hcy level, MTHFR gene polymorphism and coronary heart disease.@*METHODS@#The medical records of 80 patients with coronary heart disease who completed treatment of hematological diseases during the period from March 2018 to March 2020 were selected as observation group. In addition, the medical records of 92 patients with hematological diseases who completed treatment in our hospital during the same period were selected as control group. Venous blood samples of the two groups were collected to detect serum Hcy level and MTHFR gene polymorphism. The serum Hcy levels of the two groups with different MTHFR genotypes were compared, and the effects of the above indicators on hematological diseases complicated with coronary heart disease were analyzed.@*RESULTS@#The detection rates of MTHFR gene TT and TC in the observation group were higher than those in the control group, while the distribution frequency of MTHFR genotype CC was lower (P<0.05). The serum Hcy levels of the patients with MTHFR genotype TT and TC in the observation group was higher than the control group (P<0.05). Binary logistic regression analysis showed that MTHFR gene TC/CC genotype serum Hcy overexpression may be influencing factor which induced coronary heart disease in patients with hematological diseases (OR=2.107/OR=1.634, P<0.05). ROC curves showed that the AUC of serum Hcy level of MTHFR gene TC/CC genotype and hematological disease complicated with coronary heart disease were both > 0.8. When MTHFR gene TC reaching the optimal threshold of 22.165 μmol/L, the sensitivity was 0.950 and the specificity was 0.837, While MTHFR gene CC reached the optimal threshold of 19.630 μmol/L, the sensitivity was 0.938 and the specificity was 0.826, the best predictive value could be obtained.@*CONCLUSION@#The changes of serum Hcy and MTHFR gene polymorphisms may be involved in the pathological process in patients with hematological diseases complicated with coronary heart disease. In the future, early detection of serum Hcy levels and MTHFR gene polymorphisms in patients with hematological diseases can be used to predict the risk of coronary heart disease.

Methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF) polymorphisms in Brazilian patients with Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC)

OBJECTIVE: The folate pathway is involved in hepatic carcinogenesis and angiogenesis. Polymorphisms in genes related to such processes, including methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF)] may play an important role in the development of hepatocellular carcinoma (HCC). The objective of this study was to evaluate MTHFR and VEGF polymorphisms in Brazilian patients with hepatitis C virus (HCV)-related HCC. METHODS: A total of 119 patients diagnosed with confirmed HCC and HCV were included in the study. SNP genotyping assays were performed using real-time PCR. VEGFA (rs2010963, rs3025039, and rs833061) and MTHFRC677T (rs1801133, rs1801131) polymorphisms were evaluated. RESULTS: The C alleles of MTHFR (rs1801131) and VEGF (rs2010963) were associated with protection against the development of multinodular HCC, while the T allele of MTHFR (rs1801133) was associated with a higher risk of multinodular presentation [p=0.04 OR 1.835 CI (1.022-3.297)]. Multivariate analysis revealed that the GG/GC genotypes of VEGF rs2010963 were independently associated with multinodular tumors at diagnosis (p=0.013; OR 4.78 CI (1.38-16.67)]. CONCLUSION: Our results suggest that these polymorphisms may increase the risk of rapid tumor progression in patients with HCV infection. This subgroup of patients with HCC and who present polymorphism is more likely to be diagnosed with multinodular disease and not be amenable to receiving curative treatments. These data must be validated in larger cohorts, and the screening intervals can be customized based on genetic history.

Trombosis senovenosa cerebral en un recién nacido con mutación MTHFR C677T tratado con enoxaparina / Cerebral sinovenous thrombosis in a newborn with mutation of MTHFR C677T treated with enoxaparin

Resumen: Introducción: La trombosis senovenosa cerebral neonatal (TSVC), es una patología rara y generalmente grave, de la cual se conoce poco sobre los mecanismos fisiopatológicos responsables y, aunque controvertido, se ha sugerido que la trombofilia genética, puede desempeñar un rol en la patogénesis. Debido a los temores de un sangrado intracraneal el tratamiento anticoagulante con heparina de bajo peso mole cular es controvertido. Objetivo: presentar un recién nacido con una trombosis senovenosa cerebral neonatal, discutir los factores de riesgo trombofílico, y el manejo con heparina de bajo peso molecu lar de la trombosis venosa cerebral. Caso Clínico: Recién nacido de término que debutó a los 8 días de vida con convulsiones clónicas, rechazo al pecho más hipoactividad motora. La neuroimagen con RM mostró una TSVC involucrando múltiples senos venosos, un infarto hemorrágico talámico dere cho y congestión venosa de la sustancia blanca frontal. El estudio de trombofilia puso de relieve una mutación homocigota del gen MTHFR C677T. El tratamiento con heparina de bajo peso molecular se asoció a repermeabilización del seno sagital superior a los 23 días de iniciada la terapia. Conclusio nes: La presentación clínica de la TSVC en el neonato es inespecífica, probablemente en relación con la extensión y gravedad de la lesión y el desarrollo de complicaciones asociadas, como infartos he morrágicos venosos intraparenquimatosos o hemorragia intraventricular. Estas complicaciones son detectables mediante Ecografia o Resonancia Magnética, y deben hacer sospechar una TSVC. En esta experiencia el tratamiento anticoagulante mostró ser seguro y prevenir la extensión de la trombosis.

Abstract: Introduction: Neonatal cerebral sinovenous thrombosis (CSNT) is a rare and generally serious con dition about which there is little knowledge of the responsible pathophysiological mechanisms and, although controversial, it has been suggested that genetic thrombophilia may play a role in its patho genesis. Out of concern for intracranial bleeding, the anticoagulant treatment with low-molecular- weight heparin is controversial. Objective: To present a case of a newborn with neonatal CSNT, to analyze the thrombophilic risk factors, and the management of cerebral venous thrombosis with low-molecular-weight heparin. Clinical Case: Full-term newborn who presented at eight days of life breastfeeding rejection, clonic seizures, and locomotor hypoactivity. The MRI neuroimaging showed a CSNT involving multiple venous sinuses, a right thalamic hemorrhagic infarction, and venous congestion in frontal white matter. Thrombophilia study highlighted a homozygous MTHFR C677T mutation. Treatment with low-molecular-weight heparin was associated with repermeabilization of the superior sagittal sinus after 23 days of starting therapy. Conclusions: The clinical presentation of CSNT in the neonate is nonspecific, probably related to the extent and severity of the injury and the development of associated complications, such as venous hemorrhagic infarctions and intraparenchymal or intraventricular hemorrhage. These complications are detected through ultrasound or MRI, and they should make us suspect a CSNT. In this experience, the anticoagulant treatment proved to be safe and prevents thrombus propagation.

Correlations of Methylenetetrahydrofolate Reductase Gene Polymorphism and Genomic DNA Hypomethylation Level with Ankylosing Spondylitis / 中国医学科学院学报

To investigate the relationship of both DNA methylation level and methylenetrahydrofolate reductase(MTHFR)gene polymorphism with ankylosing spondylitis(AS). Totally 200 Chinese AS patients with HLA-B27(+)and 120 healthy controls were included from Hunan Province.All the cases were diagnosed according to the 1984 modified New York criteria for AS.The DNA methylation was examined by cytosine extension method,while the MTHFR gene C677T polymorphism was analyzed by the polymerase chain reaction(PCR)and restriction fragment length polymorphism(RFLP).The plasma homocysteine(Hcy)level was examined by enzyme-linked immunosorbent assay(ELISA),while the red blood folate level was analyzed by the specific immunoassays. The ratio of the T/T genotype mutation in the AS group was significantly higher than in the control group(17.0% 5.0%;=9.874, =0.002).The plasma homocysteine concentration of AS group was(18.71 ± 2.42)μmol/L,which was significantly higher than that in normal control group [(10.97 ± 2.93)μmol/L](=24.402, <0.001).The plasma Hcy concentration of the T/T genotype [(21.70±1.80)μmol/L] was significantly higher than that of the C/C genotype[(18.31±1.94)μmol/L](=12.088, =0.01)and the C/T genotype [(17.80±2.18)μmol/L](=6.496, =0.01)in the AS group.The DNA methylation level of the T/T genotype in AS group was significantly lower than that in normal control group(=5.655, <0.001)and also significantly lower than those of the C/C genotype(=11.514, <0.001)and the C/T genotype in AS group(=10.287, <0.001). In the Han population in Hunan Province,the C677T polymorphism of the MTHFR gene is associated with the onset of AS.The T/T mutation at position 677 of the MTHFR gene is an important influencing factor for hyperhcyemia in the AS patients.The T/T mutation at position 677 of the MTHFR gene is associated with genomic DNA hypomethylation.Thus,hypomethylation of DNA may be one of the pathogenic mechanisms of AS.

Methylenetrahydrofolate Reductase Gene C677T Polymorphism and Diabetic Retinopathy: a Meta-Analysis / 中国医学科学杂志(英文版)

Objective To investigate the association between the methylenetetrahydrofolate reductase gene C677T (MTHFR C677T) polymorphism and diabetic retinopathy (DR). Methods A total of 6971 subjects including 2707 DR patients and 4264 controls from 23 studies were enrolled in the study. A random-effects model was applied to estimate the overall effects and the stratified effects of the MTHFR C677T polymorphism on the risk of DR, and study quality was also assessed. Results Strong associations were observed between the MTHFR C677T polymorphism and DR. The carries of MTHFR C677T were more likely to be found in the DR group in relative to the healthy control group with odds ratio 1.68, 2.55, and 2.31 respectively in allele contrast model (T vs. C, 95%CI: 1.29-2.18, P<0.001, I 2=78.4%), homozygous model (TT vs. CC, 95%CI: 1.70-3.83, P=0.008, I 2=54.4%) and dominant model (TT+CT vs. CC, 95%CI: 1.62-3.29, P<0.001, I 2=74.7%). This association can also be found in contrast to the Ncd (non-complicated diabetic mellitus) group (allele contrast, OR=1.50, 95%CI: 1.07-2.11, P=0.032, I 2=62.1%; homozygous, OR=2.39, 95%CI: 1.06-5.38, P=0.017, I 2=66.7%; dominant, OR=1.59, 95%CI: 0.97-2.62, P=0.056, I 2=56.5%). For the heterozygous model (CT vs. CC), the association was significant in contrast to the healthy control group (OR=1.46, 95%CI: 1.64-3.69, P=0, I 2=77.3%), while in contrast to the Ncd control group the association was not statistically meaningful (OR=1.38, 95%CI: 0.87-2.18, P=0.131, I 2=43.7%). For the recessive model, 1.92-fold increased risk was found only in contrast to the Ncd control group (95%CI: 1.07-3.43, P=0.064, I 2=55.0%). There was no significant association found in the models in contrast to the DM control group. Conclusion In this meta-analysis, we found an association between the MTHFR C677T polymorphism and DR, especially in contrast to the Ncd control group. Further studies are required to establish more definite relationship.

Association Between Homocysteine Level and Methylenetetrahydrofolate Reductase Gene Polymorphisms in Type 2 Diabetes Accompanied by Dyslipidemia / 中国医学科学杂志(英文版)

Objective To investigate the association between total homocysteine (tHcy) level in plasma and methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genetic polymorphisms in a Chinese Han nationality population with type 2 diabetes mellitus (T2DM) accompanied by dyslipidemia. Methods This case-control study enrolled T2DM patients with dyslipidemia and without dyslipidemia respectively. Sanger dideoxy-mediated chain-termination method was used to detect the gene polymorphisms of MTHFR C677T and A1298C. Plasma tHcy and lipid levels were measured as well. The genotype frequency and allele frequency between the dyslipidemia and non-dyslipidemia groups were compared by using Chi-square test. Plasma tHcy level of T2DM patients who carried the different genotypes was compared by Student's t test. Results Finally, 82 T2DM patients with dyslipidemia and 94 ones without dyslipidemia were included in this study. There was a significant correlation between tHcy level and MTHFR C677T gene polymorphism in T2DM patients (t=2.27, P=0.02). Moreover, the plasma tHcy level in the dyslipidemia patients who carried MTHFR 677 TT genotype was significantly higher than that in those with CT+CC genotype (13.62±6.97 vs. 10.95±3.62 μmol/L, t=2.20, P=0.03); while for patients without dyslipidemia, comparison of the tHcy level between those who carried the above two alleles showed no significantly difference (13.34±6.03 vs. 12.04±5.09 μmol/L, t=1.08, P=0.29). Conclusion MTHFR 677TT genotype might associate with higher tHcy level in T2DM patients with dyslipidemia.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.