Encoding Glutamate Dehydrogenase Research Articles

Transcriptomic response to nitrogen availability reveals signatures of adaptive plasticity during tetraploid wheat domestication.

The domestication of crops, coupled with agroecosystem development, is associated with major environmental changes and provides an ideal model of phenotypic plasticity. Here, we examined 32 genotypes of three tetraploid wheat (Triticum turgidum L.) subspecies, wild emmer, emmer, and durum wheat, which are representative of the key stages in the domestication of tetraploid wheat. We developed a pipeline that integrates RNA-Seq data and population genomics to assess gene expression plasticity and identify selection signatures under diverse nitrogen availability conditions. Our analysis revealed differing gene expression responses to nitrogen availability across primary (wild emmer to emmer) and secondary (emmer to durum wheat) domestication. Notably, nitrogen triggered the expression of twice as many genes in durum wheat compared to that in emmer and wild emmer. Unique selection signatures were identified at each stage: primary domestication mainly influenced genes related to biotic interactions, whereas secondary domestication affected genes related to amino acid metabolism, in particular lysine. Selection signatures were found in differentially expressed genes (DEGs), notably those associated with nitrogen metabolism, such as the gene encoding glutamate dehydrogenase (GDH). Overall, our study highlights the pivotal role of nitrogen availability in the domestication and adaptive responses of a major food crop, with varying effects across different traits and growth conditions.

Bioinformatics analysis identifies GLUD1 as a prognostic indicator for clear cell renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a common primary tumor of the kidney and is divided into three major subtypes, of which clear cell renal cell carcinoma (ccRCC) has the highest incidence. Glutamate dehydrogenase 1 (GLUD1) encodes glutamate dehydrogenase 1, which catalyzes the oxidative deamination of glutamate.MethodsWe analyzed TCGA data using R language software and used multiple online databases to explore the relationship of GLUD1 with signaling pathways and drug sensitivity as well as GLUD1 protein expression and methylation.ResultsThe results showed that GLUD1 mRNA expression was reduced in tumor tissues and correlated with the progression of ccRCC. Univariate and multivariate Cox analysis showed that GLUD1 could be used as a prognostic marker for ccRCC. GLUD1 expression in ccRCC was associated with immune cells infiltration and multiple classical signaling pathways. In addition, GLUD1 mRNA expression was related to drug sensitivity.ConclusionsThese findings provide new ideas for finding new prognostic molecular markers and therapeutic targets for ccRCC.

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Salt Stress-Related Mechanisms in Leaves of the Wild Barley Hordeum spontaneum Generated from RNA-Seq Datasets.

This study aims to detect salt stress-related genes and mechanisms of the wild barley Hordeum spontaneum. Among the generated RNA-Seq datasets, several regulated transcripts are influenced by levels of cellular carbon, nitrogen and oxygen. Some of the regulated genes act on photorespiration and ubiquitination processes, as well as promoting plant growth and development under salt stress. One of the genes, encoding alanine:glyoxylate aminotransferase (AGT), participates in signaling transduction and proline biosynthesis, while the gene encoding asparagine synthetase (ASN) influences nitrogen storage and transport in plants under stress. Meanwhile, the gene encoding glutamate dehydrogenase (GDH) promotes shoot and root biomass production as well as nitrate assimilation. The upregulated genes encoding alpha-aminoadipic semialdehyde synthase (AASAS) and small auxin-up RNA 40 (SAUR40) participate in the production of proline and signaling compounds, respectively, while the gene encoding E3 ubiquitin-protein ligase regulates the carbon/nitrogen-nutrient response and pathogen resistance, in addition to some physiological processes under biotic and abiotic stresses via signal transduction. The gene encoding the tetratricopeptide repeat (TPR)-domain suppressor of STIMPY (TSS) negatively regulates the carbon level in the cell. In conclusion, this study sheds light on possible molecular mechanisms underlying salt stress tolerance in wild barley that can be utilized further in genomics-based breeding programs of cultivated species.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

BackgroundItaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy.ResultsIn this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L−1 with a product yield of 81 ± 9 mmol mol−1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L−1 and 116 ± 15 mmol mol−1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L−1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.ConclusionAs shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Multiomics analysis couples mRNA turnover and translational control of glutamine metabolism to the differentiation of the activated CD4+ T cell

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

ODP284 Hyperinsulinism-Hyperammonaemia syndrome: a dive into the rare presentation and diagnostic challenges encountered in adults

Abstract Background Hyperinsulinemic-hyperammonemia, a rare form of congenital hyperinsulinemia, can seldom present in adulthood if failed to be diagnosed during infancy, enabling it to be difficult to identify, and predisposing affected individuals to neurological complications. Case A 20-year-old female with intellectual disability, seizure disorder and ornithine transcarbamylase deficiency was admitted for recurrent episodes of asymptomatic hypoglycemia. Her medications included risperidone, fluoxetine, levetiracetam, lacosamide, ferrous sulphate and acetaminophen. Physical examination including vital signs was unremarkable. Relevant labs included a fasting glucose of 46mg/dl [70-99]. Further work up of hypoglycemia revealed an IGF-1 of 215ng/dl [85-350] and AM Cortisol of 6.5mcg/dl [5-25]. Given a low AM cortisol level under stress, an ACTH stimulation test was done with appropriate response of cortisol of 24 following cosyntropin administration, ruling out adrenal insufficiency. The patient continued to have h ypoglycemia noted on several glucometer checks every 4 hours where she remained asymptomatic. She was further evaluated with a mixed meal study where no hypoglycemia was noted. A 72 -hour fast was performed which was terminated based on a venous blood sample glucose reading of 50 mg/dl confirmed after hypoglycemia was noted on a glucometer check. The labs showed elevated C-peptide levels of 4ng/ml [0.4-2], insulin of 4mIU/L [<25] and proinsulin level of 2.3pmol/l [3-20]. Levels of insulin and proinsulin were noted to be inappropriately normal in the setting of hypoglycemia. Beta hydroxybutyrate was low at 0.1mmol/L [<0.5] and glucagon inappropriately normal at 36pg/ml [50-100]. Sulfonylurea, meglitinide and autoimmune antibody screen were negative. CT abdomen and pelvis was negative for pancreatic abnormality. In the absence of evidence for acquired causes of hyperinsulinemic hypoglycemia, congenital cases were investigated. Based on that, further labs were obtained including ammonia levels which was elevated at 177.2 u/dl [15-45]. Accordingly, GLUD-1 gene mutation was sent, given high suspicion for hyperinsulinemic hyperammonemia syndrome, which resulted positive and confirmed the diagnosis. She was started on a protein restricted diet and diazoxide which regulated her blood glucose levels effectively. Discussion Hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism caused by activating mutations of the GLUD 1 gene which encodes Glutamate Dehydrogenase, a key enzyme in the beta cell pathway of amino acid-stimulated insulin secretion. The resultant syndrome is characterized by fasting and protein induced hypoglycemia, elevated serum ammonia levels without typical hyperammonemic symptoms. Management includes effective blood glucose control with an insulin inhibitor such as diazoxide and a protein restricted diet with consideration to predominantly include carbohydrates and fat. Conclusion Hyperinsulinemia hyperammonemia is a rare form of congenital hyperinsulinemia typically diagnosed during infancy and should be considered as a possible differential diagnosis in patients presenting with new onset hypoglycemia, as timely diagnosis and intervention can prevent forthcoming neurological complications. Presentation: No date and time listed

Characterizing the neurological phenotype of the hyperinsulinism hyperammonemia syndrome

BackgroundHyperinsulinism hyperammonemia (HI/HA) syndrome is caused by activating mutations in GLUD1, encoding glutamate dehydrogenase (GDH). Atypical absence seizures and neuropsychological disorders occur at high rates in this form of hyperinsulinism. Dysregulated central nervous system (CNS) glutamate balance, due to GDH overactivity in the brain, has been hypothesized to play a role. This study aimed to describe the neurologic phenotype in HI/HA syndrome and investigate CNS glutamate levels using glutamate weighted chemical exchange saturation transfer magnetic resonance imaging (GluCEST MRI). In this cross-sectional study, 12 subjects with HI/HA syndrome had plasma ammonia measurement, self- or parent-completed neurocognitive assessments, electroencephalogram (EEG), and GluCEST MRI at 7 T performed. GluCEST MRI measures were compared to a historic reference population of 10 healthy adults.ResultsSubjects were five males and seven females with median age of 25.5 years. Seventy-five percent of subjects reported a history of neurodevelopmental problems and 42% had neurocognitive assessment scores outside the normal range. Fifty percent had interictal EEG findings of generalized, irregular spike and wave discharges. Higher variability in hippocampal GluCEST asymmetry (p = 0.002), and in peak hippocampal GluCEST values (p = 0.008), was observed in HI/HA subjects (n = 9 with interpretable MRI) compared to the healthy reference population (n = 10).ConclusionsThe high prevalence of abnormal neurocognitive assessment scores and interictal EEG findings observed highlights the importance of longitudinal neuropsychological assessment for individuals with HI/HA syndrome. Our findings demonstrate the potential application of GluCEST to investigate persistent knowledge gaps in the mechanisms underlying the unique neurophenotype of this disorder.

Transcriptome Analysis and RNA Interference Reveal GhGDH2 Regulating Cotton Resistance to Verticillium Wilt by JA and SA Signaling Pathways.

Verticillium wilt, caused by Verticillium dahliae, is one of the most damaging and widespread soil-borne cotton diseases. The molecular mechanisms underlying the cotton defense against V. dahliae remain largely elusive. Here, we compared the transcriptional differences between Upland cotton cultivars: one highly resistant (HR; Shidalukang 1) and one highly susceptible (HS; Junmian 1). This was done at multiple time points after V. dahliae inoculation, which identified 2010 and 1275 differentially expressed genes (DEGs) in HR and HS, respectively. Plant hormone signal transduction-related genes were enriched in HR, whereas genes related to lignin biosynthesis were enriched in both HR and HS. Weighted gene co-expression network analysis (WGCNA) using the 2868 non-redundant genes differentially expressed between the V. dahliae infected and uninfected samples in HR or HS identified 10 different gene network modules and 22 hub genes with a potential role in regulating cotton defense against V. dahliae infection. GhGDH2, encoding glutamate dehydrogenase (GDH), was selected for functional characterization. Suppressing the expression level of GhGDH2 by virus-induced gene silencing (VIGS) in HS led to inhibition of the salicylic acid (SA) biosynthesis/signaling pathways and activation of the jasmonic acid (JA) biosynthesis/signaling pathways, which resulted in an increase of 42.1% JA content and a reduction of 78.9% SA content in cotton roots, and consequently enhanced V. dahliae resistance. Our finding provides new insights on the molecular mechanisms of cotton resistance to V. dahliae infection and candidate genes for breeding V. dahliae resistance cotton cultivars by genetic modification.

Increasing Agmatine Production in Escherichia coli through Metabolic Engineering.

In this study, to obtain higher agmatine yields using the previously developed E. coli strain AUX4 (JM109 ΔspeC ΔspeF ΔspeB ΔargR), the genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), transhydrogenase (pntAB), and biosynthetic arginine decarboxylase (speA) were sequentially overexpressed by replacing their native promoters with the heterologous strong trp, core-trc, or 5Ptacs promoters to generate the plasmid-free E. coli strain AUX11. The fermentation results obtained using a 3-L bioreactor showed that AUX11 produced 2.93 g L-1 agmatine with the yield of 0.29 g agmatine g-1 glucose in the batch fermentation, and the fed-batch fermentation of AUX11 allowed the production of 40.43 g L-1 agmatine with the productivity of 1.26 g L-1 h-1 agmatine. The results showed that the engineered E. coli strain AUX11 can be used for the industrial fermentative production of agmatine.

Familial case of congenital hyperinsulinism associated with mutation in the GLUD1 gene

Congenital hyperinsulinism (CHI) is a rare hereditary disease characterized by hypoglycemia in children during the first year of life. Late diagnosis and inadequate therapy may result in severe neurological complications. Mutations in the GLUD1 gene that encodes glutamate dehydrogenase are one of the causes of CHI. This form of CHI is characterized by protein-induced hyperinsulinemic hypoglycemia and hyperammonemia in patients. Diagnosing can be challenging because hyperinsulinemic hypoglycemia cannot be detected using the conventional fasting glucose test. Extensive examination including the protein load test is needed to refine diagnosis. We report a familial case where the mother and two daughters were diagnosed with CHI and had a mutation in the catalytic domain of the GLUD1 gene. The clinical presentation, the laboratory data, the outcome of therapy, and the dynamic follow up data for the patients are presented.

CARACTERIZAÇÃO IN SILICO DOS GENES ENVOLVIDOS NO METABOLISMO DO NITROGÊNIO EM MILHO (Zea mays)

O N é o nutriente mineral requerido pelas plantas em maior quantidade e frequentemente limita o crescimento e produtividade. A partir de uma análise in silico foram identificados 26 genes envolvidos na assimilação do nitrogênio: quatro genes que codificam a enzima nitrato redutase (ZmNR), oito nitrato redutase de transporte (ZmNRT), uma nitrito redutase (ZmNRi), uma nitrito de transporte (ZmNRiT), seis glutamina sintetase (ZmGS), quatro glutamato sintase (ZmGOGAT) e duas glutamato desidrogenase (ZmGDH). A árvore filogenética foi construída onde foi possível observar a formação de cinco grupos distintos de acordo com as funções. A análise da estrutura dos genes mostrou que o número de íntrons variou de 0 a 32. A análise dos domínios conservados mostrou que a maioria dos genes identificados possuem o domínios específicos a função que desempenham na rota de assimilação do N em milho. Além disso, esses genes apresentaram padrões de expressão diferenciais em tecidos e órgãos. Os dados gerados neste trabalho forneceram subsídios para selecionar genes-candidatos para futuras análises funcionais a serem utilizados nos programas de melhoramento de milho.

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyzes the reductive amination of 2-oxoglutarate with ammonium and the oxidative deamination of glutamate. In some plants species, GDH is a hexamer and can be separated into seven isoenzymes that are composed of two distinct subunits: α and β. The large number of isoenzymes is the reason why GDH functions are still being intensively researched and widely studied. Until recently, the β subunit of GDH in triticale, a common Polish cereal, was thought to be encoded by the TsGDH1 gene, which can undergo posttranslational modifications and form a heterohexameric enzyme. Here, we report the cloning and molecular characterization of a second glutamate dehydrogenase gene—TsGDH2 (encoding α subunits). The TsGDH2 cDNA contains a 1236-bp open reading frame encoding a 411-amino-acid polypeptide with a calculated molecular mass of 44.5 kDa. To clarify the role of TsGDH2 in triticale, we used triticale GDH α-subunit cDNA to generate transgenic A. thaliana lines with increased and decreased GDH activity via alternation of α-subunit levels.

Cloning and characterization of the glutamate dehydrogenase gene in Streptococcus bovis.

Streptococcus bovis, an etiologic agent of rumen acidosis in cattle, is a rumen bacterium that can grow in a chemically defined medium containing ammonia as a sole source of nitrogen. To understand its ability to assimilate inorganic ammonia, we focused on the function of glutamate dehydrogenase. In order to identify the gene encoding this enzyme, we first amplified an internal region of the gene by using degenerate primers corresponding to hexameric family I and NAD(P)+ binding motifs. Subsequently, inverse PCR was used to identify the whole gene, comprising an open reading frame of 1350bp that encodes 449 amino acid residues that appear to have the substrate binding site of glutamate dehydrogenase observed in other organisms. Upon introduction of a recombinant plasmid harboring the gene into an Escherichia coli glutamate auxotroph lacking glutamate dehydrogenase and glutamate synthase, the transformants gained the ability to grow on minimal medium without glutamate supplementation. When cell extracts of the transformant were resolved by blue native polyacrylamide gel electrophoresis followed by activity staining, a single protein band appeared that corresponded to the size of S. bovis glutamate dehydrogenase. Based on these results, we concluded that the gene obtained encodes glutamate dehydrogenase in S. bovis.

Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress.

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.

Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering

A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15g/L. The γ-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.

Characterization of natural leaf senescence in tobacco (Nicotiana tabacum) plants grown in vitro.

Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.

Intractable Absence Seizures in Hyperinsulinism-Hyperammonemia Syndrome

A girl with intractable absence seizures and facial myoclonia at age 7 years was eventually diagnosed with hyperinsulinism-hyperammonemia syndrome because of hypoglycemia, hyperinsulinism, hyperammonemia, and the results of an oral l-leucine loading test. Her seizures occurred even during periods of relatively normal blood glucose levels, and were completely suppressed by diazoxide treatment only. Her diagnosis of hyperinsulinism-hyperammonemia syndrome was confirmed by a loss of sensitivity of glutamate dehydrogenase for guanosine 5'-triphosphate. Genetic studies identified the I444M mutation in the GLUD1 gene, which encodes glutamate dehydrogenase. This case illustrates the complex relationship between seizures and hypoglycemia in hyperinsulinism-hyperammonemia syndrome that can create diagnostic difficulties. The possibility of hyperinsulinism-hyperammonemia syndrome should be considered in patients with refractory absence seizures with myoclonia.

High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

BackgroundBacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.ResultsCompared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874.ConclusionsWe demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.