Biotin concentration affects anaplerotic reactions functioning in glutamic acid production in Corynebacterium glutamicum.

The study investigates the effect of biotin concentration on the role of anaplerotic reactions catalysed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) in glutamic acid production by Corynebacterium glutamicum. C. glutamicum requires biotin for its growth, and its glutamic acid production can be induced by the addition of Tween 40 or penicillin or by biotin limitation. The biotin enzyme PC and the non-biotin enzyme PEPC catalyse two anaplerotic reactions to supply oxaloacetic acid to the TCA cycle in C. glutamicum. Therefore, they are crucial for glutamic acid production in this bacterium. In this study, we investigated the contribution of each anaplerotic reaction to Tween 40- and penicillin-induced glutamic acid production using disruptants of PEPC and PC. In the presence of 20 µg l-1 biotin, which is sufficient for growth, the PEPC-catalysed anaplerotic reaction mainly contributed to Tween 40- and penicillin-induced glutamic acid production. However, when increasing biotin concentration 10-fold (i.e. 200 µg l-1), both PC- and PEPC-catalysed reactions could function in glutamic acid production. Western blotting revealed that the amount of biotin-bound PC was reduced by the addition of Tween 40 and penicillin in the presence of 20 µg l-1. However, these induction treatments did not change the amount of biotin-bound PC in the presence of 200 µg l-1 biotin. These results indicate that both anaplerotic reactions are functional during glutamic acid production in C. glutamicum and that biotin concentration mainly affects which anaplerotic reactions function during glutamic acid production.

Integrated metabolome and transcriptome analysis of differences in quality of ripe Lycium barbarum L. fruits harvested at different periods.

BACKGROUND: Wolfberry is well-known for its high nutritional value and medicinal benefits. Due to the continuous ripening nature of Goji berries and the fact that they can be commercially harvested within a few weeks, their phytochemical composition may change during the harvesting process at different periods. RESULT: The involved molecular mechanisms of difference in fruit quality of ripe Lycium barbarum L. harvested at four different periods were investigated by transcriptomic and metabolomics analyses for the first time. According to the results we obtained, it was found that the appearance quality of L. barbarum fruits picked at the beginning of the harvesting season was superior, while the accumulation of sugar substances in L. barbarum fruits picked at the end of the harvesting season was better. At the same time the vitamin C and carotenoids content of wolfberry fruits picked during the summer harvesting season were richer. Ascorbic acid, succinic acid, glutamic acid, and phenolic acids have significant changes in transcription and metabolism levels. Through the network metabolic map, we found that ascorbic acid, glutamic acid, glutamine and related enzyme genes were differentially accumulated and expressed in wolfberry fruits at different harvesting periods. Nevertheless, these metabolites played important roles in the ascorbate-glutathione recycling system. Ascorbic acid, phenolic substances and the ascorbate-glutathione recycling system have antioxidant effects, which makes the L. barbarum fruits harvested in the summer more in line with market demand and health care concepts. CONCLUSION: This study laid the foundation for understanding the molecular regulatory mechanisms of quality differences of ripe wolfberry fruits harvested at different periods, and provides a theoretical basis for enhancing the quality of L. barbarum fruits.

Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient Halomonas elongata.

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

D-arabinose acts as antidepressant by activating the ACSS2-PPARγ/TFEB axis and CRTC1 transcription.

CREB-regulated transcription coactivator 1 (CRTC1), a pivotal synaptonuclear messenger, regulates synaptic plasticity and transmission to prevent depression. Despite exhaustive investigations into CRTC1 mRNA reductions in the depressed mice, the regulatory mechanisms governing its transcription remain elusive. Consequently, exploring rapid but non-toxic CRTC1 inducers at the transcriptional level is important for resisting depression. Here, we demonstrate the potential of D-arabinose, a unique monosaccharide prevalent in edible-medicinal plants, to rapidly enter the brain and induce CRTC1 expression, thereby eliciting rapid-acting and persistent antidepressant responses in chronic restrain stress (CRS)-induced depressed mice. Mechanistically, D-arabinose induces the expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and transcription factor EB (TFEB), thereby activating CRTC1 transcription. Notably, we elucidate the pivotal role of the acetyl-CoA synthetase short-chain family member 2 (ACSS2) as an obligatory mediator for PPARγ and TFEB to potentiate CRTC1 transcription. Furthermore, D-arabinose augments ACSS2-dependent CRTC1 transcription by activating AMPK through lysosomal AXIN-LKB1 pathway. Correspondingly, the hippocampal down-regulations of ACSS2, PPARγ or TFEB alone failed to reverse CRTC1 reductions in CRS-exposure mice, ultimately abolishing the anti-depressant efficacy of D-arabinose. In summary, our study unveils a previously unexplored role of D-arabinose in activating the ACSS2-PPARγ/TFEB-CRTC1 axis, presenting it as a promising avenue for the prevention and treatment of depression.

Asparagine: A key metabolic junction in targeted tumor therapy.

Nutrient bioavailability in the tumor microenvironment plays a pivotal role in tumor proliferation and metastasis. Among these nutrients, glutamine is a key substance that promotes tumor growth and proliferation, and its downstream metabolite asparagine is also crucial in tumors. Studies have shown that when glutamine is exhausted, tumor cells can rely on asparagine to sustain their growth. Given the reliance of tumor cell proliferation on asparagine, restricting its bioavailability has emerged as promising strategy in cancer treatment. For instance, the use of asparaginase, an enzyme that depletes asparagine, has been one of the key chemotherapies for acute lymphoblastic leukemia (ALL). However, tumor cells can adapt to asparagine restriction, leading to reduced chemotherapy efficacy, and the mechanisms by which different genetically altered tumors are sensitized or adapted to asparagine restriction vary. We review the sources of asparagine and explore how limiting its bioavailability impacts the progression of specific genetically altered tumors. It is hoped that by targeting the signaling pathways involved in tumor adaptation to asparagine restriction and certain factors within these pathways, the issue of drug resistance can be addressed. Importantly, these strategies offer precise therapeutic approaches for genetically altered cancers.

Succinic acid production by wine yeasts and the influence of GABA and glutamic acid.

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.

A genome-wide association study in autoimmune neurological syndromes with anti-GAD65 autoantibodies.

Autoimmune neurological syndromes (AINS) with autoantibodies against the 65 kDa isoform of the glutamic acid decarboxylase (GAD65) present with limbic encephalitis, including temporal lobe seizures or epilepsy, cerebellitis with ataxia, and stiff-person-syndrome or overlap forms. Anti-GAD65 autoantibodies are also detected in autoimmune diabetes mellitus, which has a strong genetic susceptibility conferred by human leukocyte antigen (HLA) and non-HLA genomic regions. We investigated the genetic predisposition in patients with anti-GAD65 AINS. We performed a genome-wide association study (GWAS) and an association analysis of the HLA region in a large German cohort of 1214 individuals. These included 167 patients with anti-GAD65 AINS, recruited by the German Network for Research on Autoimmune Encephalitis (GENERATE), and 1047 individuals without neurological or endocrine disease as population-based controls. Predictions of protein expression changes based on GWAS findings were further explored and validated in the CSF proteome of a virtually independent cohort of 10 patients with GAD65-AINS and 10 controls. Our GWAS identified 16 genome-wide significant (P < 5 × 10-8) loci for the susceptibility to anti-GAD65 AINS. The top variant, rs2535288 [P = 4.42 × 10-16, odds ratio (OR) = 0.26, 95% confidence interval (CI) = 0.187-0.358], localized to an intergenic segment in the middle of the HLA class I region. The great majority of variants in these loci (>90%) mapped to non-coding regions of the genome. Over 40% of the variants have known regulatory functions on the expression of 48 genes in disease relevant cells and tissues, mainly CD4+ T cells and the cerebral cortex. The annotation of epigenomic marks suggested specificity for neural and immune cells. A network analysis of the implicated protein-coding genes highlighted the role of protein kinase C beta (PRKCB) and identified an enrichment of numerous biological pathways participating in immunity and neural function. Analysis of the classical HLA alleles and haplotypes showed no genome-wide significant associations. The strongest associations were found for the DQA1*03:01-DQB1*03:02-DRB1*04:01HLA haplotype (P = 4.39 × 10-4, OR = 2.5, 95%CI = 1.499-4.157) and DRB1*04:01 allele (P = 8.3 × 10-5, OR = 2.4, 95%CI = 1.548-3.682) identified in our cohort. As predicted, the CSF proteome showed differential levels of five proteins (HLA-A/B, C4A, ATG4D and NEO1) of expression quantitative trait loci genes from our GWAS in the CSF proteome of anti-GAD65 AINS. These findings suggest a strong genetic predisposition with direct functional implications for immunity and neural function in anti-GAD65 AINS, mainly conferred by genomic regions outside the classical HLA alleles.

Temporal lobe epilepsy with GAD antibodies: neurons killed by T cells not by complement membrane attack complex.

Temporal lobe epilepsy (TLE) is one of the syndromes linked to antibodies against glutamic acid decarboxylase (GAD). It has been questioned whether 'limbic encephalitis with GAD antibodies' is a meaningful diagnostic entity. The immunopathogenesis of GAD-TLE has remained enigmatic. Improvement of immunological treatability is an urgent clinical concern. We retrospectively assessed the clinical, MRI and CSF course as well as brain tissue of 15 adult patients with GAD-TLE who underwent temporal lobe surgery. Brain tissue was studied by means of immunohistochemistry, multiplex fluorescent microscopy and transcriptomic analysis for inflammatory mediators and neuronal degeneration. In 10 patients, there was a period of mediotemporal swelling and T2 signal increase; in nine cases this occurred within the first 6 years after symptom onset. This resulted in unilateral or bilateral hippocampal sclerosis; three cases developed hippocampal sclerosis within the first 2 years. All CSF studies done within the first year (n = 6) revealed intrathecal synthesis of immunoglobulin G. Temporal lobe surgeries were done after a median disease duration of 9 years (range 3 weeks to 60 years). Only two patients became seizure-free. Brain parenchyma collected during surgery in the first 6 years revealed high numbers of plasma cells but no signs of antibody-mediated tissue damage. Even more dense was the infiltration by CD8+ cytotoxic T lymphocytes (CTLs) that were seen to locally proliferate. Further, a portion of these cells revealed an antigen-specific resident memory T cell phenotype. Finally, CTLs with cytotoxic granzyme B+ granules were also seen in microglial nodules and attached to neurons, suggesting a CTL-mediated destruction of these cells. With longer disease duration, the density of all lymphocytes decreased. Whole transcriptome analysis in early/active cases (but not in late/inactive stages) revealed 'T cell immunity' and 'Regulation of immune processes' as the largest overrepresented clusters. To a lesser extent, pathways associated with B cells and neuronal degeneration also showed increased representation. Surgically treated patients with GAD-TLE go through an early active inflammatory, 'encephalitic' stage (≤6 years) with CTL-mediated, antigen-driven neuronal loss and antibody-producing plasma cells but without signs of complement-mediated cell death. Subsequently, patients enter an apparently immunologically inactive or low-active stage with ongoing seizures, probably caused by the structural damage to the temporal lobe. 'Limbic encephalitis' with GAD antibodies should be subsumed under GAD-TLE. The early tissue damage explains why immunotherapy does not usually lead to freedom from seizures.

A novel chitosan-glutamic acid membrane for multi-pollutant amputation: Investigational and RSM optimizations.

The novel glutamic acid crosslinked chitosan membrane (CsG) was fabricated and tested for its adsorption capabilities for the removal of multiple pollutants like Cr (VI), cyanide, fluoride and diclofenac sodium from wastewater. This fabricated CsG membrane was characterized by various techniques like FT-IR, SEM, EDX and XRD, BET to assess its structural, compositional and morphological properties. The working parameters studied by batch experiments were solution pH, CsG dose, contact time, pollutant concentration and solution temperature. The CsG membrane exhibited maximum adsorption capacity of 410.7 mg/g, 310.2 mg/g, 14.3 mg/g, 132.7 mg/g for Cr (VI), cyanide, fluoride and diclofenac respectively. The validation of the operational parameters was performed by Response Surface Methodology (RSM). The experimental data fitted well with Langmuir isotherm model and followed pseudo second order kinetics for all the four targeted contaminants. The spontaneity of the process was checked by thermodynamics studies. The high partition coefficients of 7669 L/kg Cr(VI), 23,309 L/kg (CN-), 649 L/kg (F-) and 2613 L/kg (DFC) are the indicators of excellent attractive interaction between CsG membrane and target toxicants. The CsG membrane showed efficient regenerative adsorption properties up to 5 adsorption-desorption cycles. Overall, the developed novel CsG membrane promised as an effective material for the removal of multiple number of pollutants from water.

Multifunctional property of N,N-bis (carboxymethyl) glutamic acid modified biomass material: adsorption and degradation removals of cationic dyes in wastewater.

As a common type of pollutants in industrial wastewater, cationic dyes have attracted great attentions. Using biodegradable N,N-di (carboxymethyl) glutamic acid (GLDA) as ligand and corn stalk (CS) as matrix, a novel and green biomass modified material GLDA-CS was successfully prepared. The multifunctional property of GLDA-CS for removing methylene blue (MB), malachite green (MG) and alkaline red 46 (R-46) from wastewater was evaluated. The dyes were removed by the electrostatic adsorption based on the cationic adsorption properties of GLDA-CS. The removal rates of MB, MG and R-46 can quickly reach 90.4%, 96.8% and 94.8% in short time. especially for MG and R-46 even only 20 min. The adsorption capacities of the dyes still remain more than 86.5% of the initial values after 5 cycles. In a heterogeneous system, the dyes were removed by Fenton-like degradation based on the metal chelating property of GLDA-CS. 100% degradation rates of the dyes can be achieved in 35 min under the acidic region. Even if at pH 7, degradation rates are 44.1%, 47.1% and 56.6% higher than those under the conventional homogeneous system, and the degradation rate remained at 83.7% after 5 cycles. Regardless of the adsorption or degradation, GLDA-CS shows strong anti-anion interference ability. The potential mechanisms of adsorption and degradation for the dyes by GLDA-CS were deduced by quantization calculation. It is concluded that the adsorption removal of the dyes by GLDA-CS follows MG > R-46 > MB, and mainly depends on the electrostatic interaction between -COOH in GLDA-CS and -N- in the dye molecules. Based on the degradation mechanism of Fenton-like reaction, the possible active sites of the dyes attacked by free radicals and their possible degradation intermediates were predicted by the calculations of Fukui function.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

MiR-375: it could be a general biomarker of metabolic changes and inflammation in type 1 diabetes patients and their siblings.

PURPOSE: Type 1 diabetes (T1D) is a chronic autoimmune illness that results in loss of pancreatic beta cells and insulin insufficiency. MicroRNAs (miRNAs) are linked to immune system functions contributing to the pathophysiology of T1D, miRNA-375 is significantly expressed in the human pancreas and its circulatory levels might correspond to beta cell alterations. Pancreatic islet cell antibodies (ICA) and Glutamic acid decarboxylase antibodies (GADA) have roles in autoimmune pathogenesis and are predictive markers of T1D. The aim of this work was to detect serum level changes of miRNA-375, ICA, and GADA in T1D patients, and their siblings compared to healthy controls and correlate them with T1D biochemical parameters. METHODS: The study included 66 T1D patients (32 males and 34 females; age range 3-18 years), 22 patients' siblings (13 males and 9 females; age range 4-17 years), and 23 healthy controls (7 males and 16 females; age range 4-17 years). MiRNA-375 levels were measured using quantitative reverse transcription polymerase chain reaction (RT-qPCR), while ICA and GADA levels were measured using enzyme-linked immunosorbent assay (ELISA). Data analysis was done utilizing SPSS-17 software. RESULTS: MiR-375 levels were downregulated in T1D patients and further decreased in their siblings when compared to healthy controls. Furthermore, miR-375 exhibited inverse correlations with HbA1c levels but no correlations with Total Insulin Dose, disease duration, or autoantibodies (GADA & ICA). CONCLUSION: Our study indicates that miR-375 is significantly downregulated in children with T1D and their siblings, suggesting its potential role as a biomarker for beta-cell function and glycemic control.

From lab bench to hope: a review of gene therapies in clinical trials for Parkinson's disease and challenges.

Parkinson's disease (PD) is a chronic neurological disorder that is identified by a characteristic combination of symptoms such as bradykinesia, resting tremor, rigidity, and postural instability. It is the second most common neurodegenerative disease after Alzheimer's disease and is characterized by the progressive loss of dopamine-producing neurons in the brain. Currently, available treatments for PD are symptomatic and do not prevent the disease pathology. There is growing interest in developing disease-modifying therapy that can reduce disease progression and improve patients' quality of life. One of the promising therapeutic approaches under evaluation is gene therapy utilizing a viral vector, adeno-associated virus (AAV), to deliver transgene of interest into the central nervous system (CNS). Preclinical studies in small animals and nonhuman primates model of PD have shown promising results utilizing the gene therapy that express glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), aromatic L-amino acid decarboxylase (AADC), and glutamic acid decarboxylase (GAD). This study provides a comprehensive review of the current state of the above-mentioned gene therapies in various phases of clinical trials for PD treatment. We have highlighted the rationale for the gene-therapy approach and the findings from the preclinical and nonhuman primates studies, evaluating the therapeutic effect, dose safety, and tolerability. The challenges associated with gene therapy for heterogeneous neurodegenerative diseases, such as PD, have also been described. In conclusion, the review identifies the ongoing promising gene therapy approaches in clinical trials and provides hope for patients with PD.

Production and molecular weight variation of poly-γ-glutamic acid using a recombinant Bacillus subtilis with various Pgs-component ratios.

Poly-γ-glutamic acid (PGA) has been of interest as a sustainable biopolymer in industrial applications. PGA biosynthesis in Bacillus subtilis is catalyzed by a transmembrane protein complex comprising PgsB, PgsC, and PgsA. To determine the Pgs component responsible for PGA overproduction, we constructed recombinants in which the promoter of the host-derived pgs gene was replaced with another host-derived gene promoter. These recombinants were then transformed using high-copy-number plasmids with various pgs-gene combinations to enhance Pgs component in different ratios. Subsequently, PGA production was investigated in batch cultures with l-glutamate supplemented medium. The recombinant strain enhanced with pgsB alone significantly overproduced PGA (maximum production 35.8 g/L) than either the pgsC- or pgsA-enhanced strain. The molecular weight of the PGA produced with the pgsB-enhanced strain was also greater than that for the pgsC- or pgsA-enhanced strain (approximately 10-fold). Hence, PgsB enhancement alone contributes to PGA overproduction with increased molecular weight.

Expression of an engineered salt-inducible proline biosynthetic operon in a glutamic acid over-producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata Glutamic acid Over-Producing, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

Glutamic-acid grafted hyaluronic acid inhibits inflammatory factors via fibroblast and skin model tests.

BACKGROUND: Excessive inflammation may cause tissue damage and disrupt the function of the skin barrier. Hyaluronic acid (HA), an endogenous component, was found to regulate multiple inflammatory factors for skin health. This work aims to further enhance its efficacy by grafting amino acid onto its molecule. METHODS: Glutamic acid (Glu) was selected as the ligand to react with low-molecular-weight HA. Fibroblast tests and a 3D skin model were used to investigate the anti-inflammation efficacy of HA-Glu. RESULTS: For IL-1α, IL-6 and TNF-α, the grafted compound presents stronger inhibition ability versus native HA. Moreover, HA-Glu could promote the repair of damaged skin by improving the compactness of the stratum corneum and increasing the thickness of the living cell layer. CONCLUSION: The application of HA-Glu compound in skin care formulas would be effective to alleviate inflammation-induced skin symptoms and skin aging.

Differences in Brain Metabolite Profiles Between Normothermia and Hypothermia.

BACKGROUND: This study evaluated the difference in brain metabolite profiles between normothermia and hypothermia reaching 25°C in humans in vivo. METHODS: Thirteen patients who underwent thoracic aorta surgery under moderate hypothermia were prospectively enrolled. Plasma samples were collected simultaneously from the arteries and veins to estimate metabolite uptake or release. Targeted metabolomics based on liquid chromatographic mass spectrometry and direct flow injection were performed, and changes in the profiles of respective metabolites from normothermia to hypothermia were compared. The ratios of metabolite concentrations in venous blood samples to those in arterial blood samples (V/A ratios) were calculated, and log2 transformation of the ratios [log2(V/A)] was performed for comparison between the temperature groups. RESULTS: Targeted metabolomics were performed for 140 metabolites, including 20 amino acids, 13 biogenic amines, 10 acylcarnitines, 82 glycerophospholipids, 14 sphingomyelins, and 1 hexose. Of the 140 metabolites analyzed, 137 metabolites were released from the brain in normothermia, and the release of 132 of these 137 metabolites was decreased in hypothermia. Two metabolites (dopamine and hexose) showed constant release from the brain in hypothermia, and 3 metabolites (2 glycophospholipids and 1 sphingomyelin) showed conversion from release to uptake in hypothermia. Glutamic acid demonstrated a distinct brain metabolism in that it was taken up by the brain in normothermia, and the uptake was increased in hypothermia. CONCLUSION: Targeted metabolomics demonstrated various degrees of changes in the release of metabolites by the hypothermic brain. The release of most metabolites was decreased in hypothermia, whereas glutamic acid showed a distinct brain metabolism.

Hybrid substrate-based pH autobuffering GABA fermentation by Levilactobacillus brevis CD0817.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (L-glutamic acid or L-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium L-glutamate plus acidic L-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved L-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10 L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery L-glutamic acid (750 g/L), monosodium L-glutamate (34 g/L [0.2 mol/L]), glucose (5 g/L), yeast extract (35 g/L), MnSO4·H2O (50 mg/L [0.3 mmol/L]), and Tween 80 (1.0 g/L). The appropriate fermentation temperature was 30 °C. The fermenter trial results revealed that GABA was slowly synthesized from 0-4 h, rapidly synthesized until 32 h, and finally reached 353.1 ± 8.3 g/L at 48 h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA fermentations.

Effects of N, N-bis (carboxymethyl)-L-glutamic acid and polyaspartic acid on the phytoremediation of cadmium in contaminated soil at the presence of pyrene: Biochemical properties and transcriptome analysis.

Chelator-assisted phytoremediation is an efficacious method for promoting the removal efficiency of heavy metals (HMs). The effects of N, N-bis(carboxymethyl)-L-glutamic acid (GLDA) and polyaspartic acid (PASP) on Cd uptake and pyrene removal by Solanum nigrum L. (S. nigrum) were compared in this study. Using GLDA or PASP, the removal efficiency of pyrene was over 98%. And PASP observably raised the accumulation and transport of Cd by S. nigrum compared with GLDA. Meanwhile, both GLDA and PASP markedly increased soil dehydrogenase activities (DHA) and microbial activities. DHA and microbial activities in the PASP treatment group were 1.05 and 1.06 folds of those in the GLDA treatment group, respectively. Transcriptome analysis revealed that 1206 and 1684 differentially expressed genes (DEGs) were recognized in the GLDA treatment group and PASP treatment group, respectively. Most of the DEGs found in the PASP treatment group were involved in the metabolism of carbohydrates, the biosynthesis of brassinosteroid and flavonoid, and they were up-regulated. The DEGs related to Cd transport were screened, and ABCG3, ABCC4, ABCG9 and Nramp5 were found to be relevant with the reduction of Cd stress in S. nigrum by PASP. Furthermore, with PASP treated, transcription factors (TFs) related to HMs such as WRKY, bHLH, AP2/ERF, MYB were down-regulated, while more MYB and bZIP TFs were up-regulated. These TFs associated with plant stress resistance would work together to induce oxidative stress. The above results indicated that PASP was more conducive for phytoremediation of Cd-pyrene co-contaminated soil than GLDA.