As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.
Valine ; Corynebacterium glutamicum/genetics* ; Metabolic Engineering ; Amino Acids, Branched-Chain ; Bioreactors

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. METHODS: The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138⁺ and CD138^- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138⁺ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. RESULTS: Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138⁺ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138⁺ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). CONCLUSIONS MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.
Humans ; Valine-tRNA Ligase ; Multiple Myeloma/genetics* ; Metabolomics ; Amino Acids ; RNA, Transfer

OBJECTIVE: To analyze the difference in the gene expression, amino acid and carnitine levels in the cervical secretions between the endometria of pre-receptive and receptive stages, with an aim to provide clues for identifying new molecular markers for endometrial receptivity. METHODS: Fifty nine infertile women treated at the Department of Reproductive Medicine of Linyi People's Hospital from January 6, 2020 to January 31, 2022 were selected as as the study subjects, which were matched with 3 pairs (6 cases) of infertile women preparing for embryo transfer based on factors such as age, body mass index, and length of infertility. Endometrial tissue samples were collected for gene transcription and expression analysis. Twenty five women who had become pregnant through assisted reproductive technology were selected as the control group, and 28 non-pregnant women receiving ovulation monitoring at the Outpatient Department were enrolled as the case group. Status of endometrial receptivity was determined by ultrasonography. In the former group, endometrial tissues were sampled for sequencing, and GO and KEGG database enrichment analysis of differentially expressed genes was carried out. In the latter group, cervical secretions were collected, and amino acid and carnitine levels were measured by mass spectrometry. Statistical analysis was carried out using rank sum test, t test and chi-square test with SPSS v25.0 software. RESULTS: No difference was found in the clinical data of the patients with regard to age, body mass index, infertility years, AMH, FSH, LH, E2, and type of infertility. Compared with the receptive endometrial tissues, there were 100 significantly up-regulated genes and 191 significantly down-regulated genes in the pre-receptive endometrial tissue, with the most significantly altered ones being HLA-DRB5 and MMP10. The biological processes, molecular functions and pathways enriched by more differentially expressed genes in GO and KEGG were mainly immune regulation, cell adhesion and tryptophan metabolism. Analysis of secretion metabolism also revealed a significant difference in the levels of amino acids and carnitine metabolites between the two groups (P < 0.05), in particular those of Alanine, Valine, 3-hydroxybutyrylcarnitine (C4OH) + malonylcarnitine (C3DC)/captoylcarnitine (C10). CONCLUSION A significant difference has been discovered in the levels of gene transcription and protein expression in the endometrial tissues from the pre-receptive and receptive stages. The levels of amino acids and carnitine, such as Alanine, Valine, 3-hydroxybutyryl carnitine (C4OH)+malonyl carnitine (C3DC)/caproyl carnitine (C10), may be associated with the receptive status of the endometrium, though this need to be verified with larger samples.
Pregnancy ; Humans ; Female ; Infertility, Female/genetics* ; Endometrium/metabolism* ; Amino Acids/metabolism* ; Gene Expression ; Carnitine ; Alanine/metabolism* ; Valine/metabolism*

Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (rFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these rFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated rFXIII-A were characterized. rFXIII-A (V34L) and rFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type rFXIII-A and V35L variant. However, the activated rFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2- plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.
Blood Coagulation Tests ; Catalysis ; Enzyme Activation/genetics ; Escherichia coli/genetics ; Factor XIII/*genetics/*metabolism ; Fibrin/metabolism ; Human ; Immunoblotting ; Leucine/genetics ; Mutagenesis, Site-Directed ; *Polymorphism (Genetics) ; Recombinant Proteins/genetics/metabolism ; Thrombin/metabolism ; Valine/genetics

OBJECTIVE: To investigate the relationship between serum brain-derived neurotrophic factor, concentrations and BDNF gene Val66Met polymorphism and amnestic mild cognitive impairment (aMCI), and neuropsychological characteristics. METHODS: Ninety-nine aMCI patients and 99 matched normal controls were recruited for the study. Multi-dimension neuropsychologic tests were used to extensively assess the cognitive function, an enzyme-linked immunosorbent assay was applied to measure serum BDNF concentrations, and polymerase chain reaction-restriction fragment length polymorphism was used to analyse BDNF gene Val66Met polymorphism in the subjects. RESULTS: The scores of neuropsychologic tests in aMCI patients were significantly lower than those in the normal controls (all P<0.001), with the largest impairment on delayed recall of the auditory verbal memory test (AVMT) which reflect verbal episodic memory. The serum concentrations of BDNF in aMCI patients (median: 4.37 microg/L) were significantly lower than those of the normal controls (median: 4.98 microg/L) (z=-2.449, P=0.014). There was positive correlation between the serum concentrations of BDNF and the scores on delayed recall of AVMT (r=0.264, P=0.008). No significant differences were found for the genotype and allele distribution of BDNF Val66Met polymorphism between aMCI patients and the normal controls. BDNF Val66Met polymorphism was not associated with serum BDNF concentrations and cognitive assessment scores in aMCI patients (P>0.05). CONCLUSION aMCI is characterized by episodic memory impairment. Decreased BDNF concentrations may play a role in the pathophysiology of aMCI, and BDNF gene Val66Met polymorphism may not be an important genetic factor in susceptibility to aMCI.
Aged ; Aged, 80 and over ; Alzheimer Disease ; genetics ; Amnesia ; blood ; genetics ; Brain-Derived Neurotrophic Factor ; blood ; genetics ; Cognition Disorders ; blood ; genetics ; Female ; Humans ; Male ; Methionine ; genetics ; Polymorphism, Genetic ; genetics ; Valine ; genetics

In industrial fermentation of amino acids the cells are often forced to synthesize the biochemicals excessive of their physiological needs. The knowledge of metabolic networks and their regulation relevant usually come from biochemical research, especially from enzymology, not from engineering study. To enrich the knowledge of metabolic sub-network of L-valine syntheses for higher production of L-valine, Corynebacterium glutamicum AS1.495 and its genetic derivatives AA361, AAT231, AATV341 were used for metabolic flux analysis. AS1.495 is a leucine auxotrophic (Leu-), and the three derivatives carry additional mutations. AA361 contains D-aspartic acid-beta-hydroxamate supersensitive marker (Leu-, L-AAHss), AAT231 (Leu-, L-AAHss, 2-TAr) is D-aspartic acid-beta-hydroxamate supersensitive and 2-thiazole alanine resistant, and AAT341 (Leu-, L-AAHss, 2-TAr, Vd-) is a D-aspartic acid-beta-hydroxamate supersensitive, 2-thiazole alanine resistant and valine-decompose-ability imperfect (Vd-). The concentrations of extra-cellular metabolites were determined under sub-steady-state of the batch culture. The metabolic flux distribution maps of the four strains were obtained, compared and analyzed. Our analysis showed that the flux ratio of EMP and HMP from the glucose-6-phosphate had increased from 0.205 in the parental strain AS1.495 to 0.321 in the multiple-mutation strain AATV341; the flux ratio of L-valine synthesis branch and the rest branches from the pyruvate node increased from 0.188 in AS1.495 to 3.29 in AATV341; the flux of lactic acid synthesis branch decreased from 11.1 in AS1.495 to 1.16 in AATV341; the flux of L-valine synthesis branch increased from 5.37 in AS1.495 to 37.3 in AATV341; and the productivity of L-valine correspondently increased from 4 g/L in AS1.495 to 24.5 g/L in AATV341. These results indicate that the introduction of analog supersensitive marker L-AAH55 and/or analog resistant marker 2-TAr skew the metabolic flux towards the formation of L-valine. This study revealed the usefulness of the metabolic flux analysis as a tool for verification of existing production strains. The analysis may play an important role in helping us b to rationally re-design metabolism for further improvement of fermentation process.
Corynebacterium glutamicum ; classification ; genetics ; metabolism ; Fermentation ; Glucose-6-Phosphate Isomerase ; metabolism ; Industrial Microbiology ; methods ; Mutation ; Pyruvic Acid ; metabolism ; Valine ; analysis ; biosynthesis

We investigated the association between superoxide dismutase (SOD) Ala16Val polymorphism and the levels of oxidized LDL lipoprotein-C (ox-LDL-C) in two age-different Greek cohorts. Four hundred fifteen middle-aged (n=147 females: 43.2+/-13 years, n=268 males: 43.3+/-14 years) Caucasian Greek subjects consisted the middle aged cohort. One hundred seventy five elderly (n=88 females: 79.9+/-4 years; n=87 males: 80.6+/-4 years) were selected from the elderly cohort. Genotype data were obtained for all of them. Multiple linear regression analysis, stratified by gender and adjusted for age, smoking habits and body mass index as covariates, showed higher ox-LDL-C levels for the middle aged men with the Val/Val genotype, compared to the other allele (Ala/Ala and Ala/Val) carriers (65.9+/-25.7 vs. 55.7+/-20.5 mg/dl; standardized beta coefficient=0.192, P=0.012). On the contrary, elderly women with the Val/Val genotype occurred with lower ox-LDL-C levels compared to the Ala/Ala or Ala/Val genotype (74.2+/-22.1 vs. 86.5+/-26.6 mg/dl; standardized beta coefficient= -0.269, P=0.015). The same trend was also recorded in elderly men, however without reaching statistical significance (standardized b coefficient= -0.187, P=0.077). Moreover, elderly men and women with the Ala/Ala or Ala/Val genotype presented higher triglycerides levels compared to Val/Val (women: 145.2+/-68.7 vs. 114.3+/-34.3 mg/dl, P= 0.027; men: 147.8+/-72.4 vs. 103.7 +/-38.0 mg/dl, P=0.002). Additionally, middle aged men with the Val/Val genotype had higher HDL-C levels compared to the Ala allele carriers. The results suggest that SOD Ala16Val polymorphism is an age-dependent modulator of ox-LDL-C levels in middle-aged men and elderly women.
Adult ; Aged ; Aged, 80 and over ; Aging/*genetics ; Alanine/*genetics ; Female ; Genotype ; Humans ; Lipoproteins, LDL/*metabolism ; Male ; Middle Aged ; Polymorphism, Single Nucleotide/*genetics ; Regression Analysis ; Sex Characteristics ; Superoxide Dismutase/*genetics ; Valine/*genetics

OBJECTIVETo investigate the relationship between efficacy of Chaihu Taoren Decoction (CTD) and the polymorphism of valine-alanine missense mutation of 9th codan (Val9Ala, T1183C) of superoxide dismutase (SOD) in patients with tardive dyskinesia (TD).

METHODSSeverity of TD was assessed by abnormal involuntary movement scale (AIMS), and the psychologic symptoms were rated by the positive and negative symptoms scale (PANSS). The sample size consisted of 119 patients with TD assigned to the TD group, 129 patients of chronic schizophrenia with the general condition matched strictly with that of the enrolled TD patients assigned to the non-TD group, and 148 healthy persons assigned to the normal group. The gene distribution rate of Val9Ala gene was analyzed using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis, and the therapeutic effect of CTD on 36 patients with TD was observed after 12 weeks treatment.

RESULTSThere was no significant difference in genotype and allelic gene frequency of SOD Val9Ala among the TD, non-TD and normal groups (P > 0.05). Comparison of the AIMS score in TD patients with various Val9Ala genotypes showed that the difference of AIMS scores in patients with TT and CT genotype was not significant (P > 0.05), but CTD did show a better efficacy in TD patients with CT heterozygote than in those with TT homozygote (P < 0.05).

CONCLUSIONThe CTD could effectively relieve the symptoms of TD, its efficacy might be related with the genotype of SOD, and 9Ala is considered to be a protective factor for the susceptibility to TD.

Adolescent ; Adult ; Capsules ; Drugs, Chinese Herbal ; therapeutic use ; Dyskinesia, Drug-Induced ; drug therapy ; etiology ; genetics ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Polymorphism, Genetic ; genetics ; Superoxide Dismutase ; genetics ; Valine ; genetics ; Young Adult

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 investigate the effects of ox-LDL, ACEI and ARB on expressions and activities of TF and TFPI in VSMC.

METHODS(1) Rabbit VSMC was cultured by explant-attached method in vitro. (2) The effects of ox-LDL and valsartan on TF and TFPI expressions were analyzed by immunohistochemistry and immunofluorescence. Laser scanning confocal microscopy were applied to analyze the effects of ox-LDL and valsartan on TF expression. The effects of ox-LDL, valsartan and captopril on TF and TFPI antigen expressions were analyzed by ELISA. Chromogenic substrate method was used to determine the effects of ox-LDL, valsartan and captopril on TF activity. The effects of ox-LDL and valsartan on TF mRNA expression were analyzed by RT-PCR.

RESULTS(1) ox-LDL could upregulate TF antigen, activity and TF expression at mRNA level and downregulate TFPI antigen. (2) Valsartan and captopril could reduce TF antigen and activity in VSMC treated by ox-LDL, and valsartan reduce it in a dose-dependent manner. Valsartan could also attenuate TF expression at mRNA level in VSMC treated by ox-LDL. (3) Using ELISA, valsartan and captopril could also enhance TFPI antigen in VSMC treated by ox-LDL.

CONCLUSIONOur study showed upregulated TF and downregulated TFPI expression and activity by ox-LDL and these effects could be reversed by ACEI and ARB indicating a new insight on the antiatherosclerotic effects of ACEI and ARB.

Animals ; Captopril ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Male ; Proton-Translocating ATPases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Rabbits ; Tetrazoles ; pharmacology ; Thromboplastin ; biosynthesis ; genetics ; Valine ; analogs & derivatives ; pharmacology ; Valsartan