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significance of arg3, arg54, and tyr58 of l-aspartate α-decarboxylase from corynebacterium glutamicum in the process of self-cleavage.we have elucidated the significance of three key amino acid residues of l-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. these results provide useful fundamental information for engineering l-aspartate α-decarboxylase. l-aspartate α-decarboxylase (adc) from corynebacterium glutamicum, and encoded by pand, was cloned and expressed in escherichia coli and then purified. three amino acid residues were found ...201424104602
succinic acid production from corn cob hydrolysates by genetically engineered corynebacterium glutamicum.corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. in the present work, we constructed a xylose metabolic pathway in c. glutamicum by heterologous expression of the xyla and xylb genes coming from escherichia coli. dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant c. glutamicum nc-2. the results indicated that the available activated charcoal ...201424078255
ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in bacteria and archaea, which contain inositol phospholipid.in eukarya, phosphatidylinositol (pi) is biosynthesized from cdp-diacylglycerol (cdp-dag) and inositol. in archaea and bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. the precursors, inositol 1-phosphate, cdp-archaeol (cdp-aroh), and cdp-dag, form archaetidylinositol phosphate (aip) and phosphatidylinositol phosphate (pip) as intermediates. these intermediates are dephosphorylated to synthesize archaetidylinositol (ai) and pi. to date, the activities of ...201424269814
functional characterization of corynebacterium glutamicum mycothiol s-conjugate amidase.the present study focuses on the genetic and biochemical characterization of mycothiol s-conjugate amidase (mca) of corynebacterium glutamicum. recombinant c. glutamicum mca was heterologously expressed in escherichia coli and purified to apparent homogeneity. the molecular weight of native mca protein determined by gel filtration chromatography was 35 kda, indicating that mca exists as monomers in the purification condition. mca showed amidase activity with mycothiol s-conjugate of monobromobim ...201425514023
engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. it is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. we calculated the fraction of synonymous substitution and the effective number of codons (enc) for nine genes stemming from g ...201425469170
discovery of a bacterial 5-methylcytosine deaminase.5-methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from escherichia coli (coda) will not accept 5-methylcytosine as a substrate. since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. we therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to ...201425384249
metabolic engineering of escherichia coli for the production of riboflavin.riboflavin (vitamin b2), the precursor of the flavin cofactors flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad), is used commercially as an animal feed supplement and food colorant. e. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. thus, the aim of this study was to understand the metabolic capacity of e. coli for the riboflavin production by modification of central me ...201425027702
enhanced acetic acid and succinic acid production under microaerobic conditions by corynebacterium glutamicum harboring escherichia coli transhydrogenase gene pntab.some microorganisms, such as escherichia coli, harbor transhydrogenases that catalyze the interconversion between nadph and nadh. however, such transhydrogenase genes have not been found in the genome of a glutamic acid-producing bacterium corynebacterium glutamicum. in this study, the e. coli transhydrogenase genes udha and pntab were introduced into the c. glutamicum wild-type strain atcc 13032, and the metabolic characteristics of the recombinant strains under aerobic and microaerobic conditi ...201425008167
disruption of pkng enhances production of gamma-aminobutyric acid by corynebacterium glutamicum expressing glutamate decarboxylase.gamma-aminobutyric acid (gaba), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by corynebacterium glutamicum that expresses escherichia coli glutamate decarboxylase (gad) b encoded by gadb. this strain was engineered to produce gaba more efficiently from biomass-derived sugars. to enhance gaba production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pkng (encoding serine/threonine protein kinase ...201424949255
[progress in biosythesis of diaminopentane].air pollution and global warming are increasingly deteriorating. large amounts of polyamides derived from fossil fuel sources are consumed around the world. cadaverine is an important building monomer block of bio-based polyamides, thus biotechnological processes for these polymers possess enormous ecological and economical potential. currently, the engineered strains for biological production of cadaverine are corynebacterium glutamicum and escherichia coli. we review here the latest research p ...201424941739
microbial production of amino acids and derived chemicals: synthetic biology approaches to strain development.amino acids are produced at the multi-million-ton-scale with fermentative production of l-glutamate and l-lysine alone being estimated to amount to more than five million tons in the year 2013. metabolic engineering constantly improves productivities of amino acid producing strains, mainly corynebacterium glutamicum and escherichia coli strains. classical mutagenesis and screening have been accelerated by combination with intracellular metabolite sensing. synthetic biology approaches have allowe ...201424922334
production of the sesquiterpene (+)-valencene by metabolically engineered corynebacterium glutamicum.the sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturally pigmented as it synthesizes the rare fifty carbon atoms (c50) containing carotenoid decaprenoxanthin. since the carotenoid pathway of this gram-positive bacterium has previously b ...201424910970
application of metabolic engineering for the biotechnological production of l-valine.the branched chain amino acid l-valine is an essential nutrient for higher organisms, such as animals and humans. besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l-valine is now emerging into the feed market, and significant increase of sales and world production is expected. in accordance, well-known microbial production bacteria, such as escherichia coli and corynebacterium glutamicum strains, have ...201424816722
synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from corynebacterium glutamicum.β-alanine is mainly produced by chemical methods in current industrial processes. here, pand from corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (adc) was cloned and expressed in escherichia coli bl21(de3). adc c.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. the purified adc c.g was optimal at 55 °c and ph 6 with excellent stability at 16-37 °c and ph 4-7. a ph-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep t ...201424737081
synthetic biology platform of corynebrick vectors for gene expression in corynebacterium glutamicum and its application to xylose utilization.currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as escherichia coli and saccharomyces cerevisiae. in order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called corynebrick, for gene expression in corynebacterium glutamicum as a set of e. coli-c. glutamicum shuttle vectors whose elements are interchangeable with bglbrick standard parts. c. glutamicum is an established ...201424706215
directed evolution and mutagenesis of glutamate decarboxylase from lactobacillus brevis lb85 to broaden the range of its activity toward a near-neutral ph.glutamate decarboxylase (gad) transforms l-glutamate into γ-aminobutyric acid (gaba) with the consumption of a proton. gad derived from lactic acid bacteria exhibits optimum activity at ph 4.0-5.0 and significantly loses activity at near-neutral ph. to broaden the active range of the gad gadb1 from lactobacillus brevis lb85 toward a near-neutral ph, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. for directed evolution of gadb1, a se ...201424910334
production and glucosylation of c50 and c 40 carotenoids by metabolically engineered corynebacterium glutamicum.the yellow-pigmented soil bacterium corynebacterium glutamicum atcc13032 is accumulating the cyclic c50 carotenoid decaprenoxanthin and its glucosides. carotenoid pathway engineering was previously shown to allow for efficient lycopene production. here, engineering of c. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous c50 carotenoids c.p.450 and sarcinaxanthin is described. plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation fr ...201424270893
high-level expression in corynebacterium glutamicum of nitrile hydratase from rhodococcus rhodochrous for acrylamide production.the nhhbag gene of rhodococcus rhodochrous m33 that encodes nitrile hydratase (nhase), converting acrylonitrile into acrylamide, was cloned and expressed in corynebacterium glutamicum under the control of an ilvc promoter. the specific enzyme activity in recombinant c. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (dcw). to overexpress the nhase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (ti ...201424493572
novel and tightly regulated resorcinol and cumate-inducible expression systems for streptomyces and other actinobacteria.inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the laci repressor, and isopropyl β-d-thiogalactopyranoside (iptg). we successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the cymr regulator, the operator sequence (cmt) ...201425012786
hyaluronic acid production with corynebacterium glutamicum: effect of media composition on yield and molecular weight.corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (ha).201424863652
the crystal structures of apo and camp-bound glxr from corynebacterium glutamicum reveal structural and dynamic changes upon camp binding in crp/fnr family transcription factors.the cyclic amp-dependent transcriptional regulator glxr from corynebacterium glutamicum is a member of the super-family of crp/fnr (cyclic amp receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. in c. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the corynebacteriales including mycobacterium tuberculosis ...201425469635
acyl-coa sensing by fasr to adjust fatty acid synthesis in corynebacterium glutamicum.corynebacterium glutamicum, like mycobacterium tuberculosis, is a member of the corynebacteriales, which have linear fatty acids and as branched fatty acids the mycolic acids. we identified accd1 and fasa as key genes of fatty acid synthesis, encoding the β-subunit of the acetyl-coa carboxylase and a type-i fatty acid synthase, respectively, and observed their repression during growth on minimal medium with acetate. we also identified the transcriptional regulator fasr and its binding sites in t ...201425449109
interaction of 2-oxoglutarate dehydrogenase odha with its inhibitor odhi in corynebacterium glutamicum: mutants and a model.pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. in corynebacterium glutamicum and related bacteria like mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein odha catalyzing the conversion of oxoglutarate to succinyl-coenzyme a. this activity is inhibited by the forkhead-associated (fha) domain of the small autoinhibitory protein odhi. here we used a biological screen which enabled us to isola ...201424905147
benzothiazinones mediate killing of corynebacterineae by blocking decaprenyl phosphate recycling involved in cell wall biosynthesis.benzothiazinones (btzs) are a new class of sulfur containing heterocyclic compounds that target dpre1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (dpr) to decaprenyl-phosphoarabinose (dpa) in the corynebacterineae, such as corynebacterium glutamicum and mycobacterium tuberculosis. as a result, btz inhibition leads to inhibition of cell wall arabinan biosynthesis. previous studies have demonstrated the essentiality of dpre1. in contrast, cg-ubia a ribosyltransfera ...201424446451
the concerted action of a positive charge and hydrogen bonds dynamically regulates the pka of the nucleophilic cysteine in the nrdh-redoxin family.nrdh-redoxins shuffle electrons from the nadph pool in the cell to class ib ribonucleotide reductases, which in turn provide the precursors for dna replication and repair. nrdh-redoxins have a cvqc active site motif and belong to the thioredoxin-fold protein family. as for other thioredoxin-fold proteins, the pk(a) of the nucleophilic cysteine of nrdh-redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. recently, the pk(a) value of this cysteine in cory ...201424243781
biosynthesis of pinene from glucose using metabolically-engineered corynebacterium glutamicum.pinene is a monoterpenes (c10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. herein, we have metabolically-engineered corynebacterium glutamicum, to produce pinene and studied its toxicity in c. glutamicum. geranyl diphosphate synthases (gpps) and pinene synthases (ps), obtained from pinus taeda and abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphat ...201424930112
engineering of corynebacterium glutamicum for minimized carbon loss during utilization of d-xylose containing substrates.biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. the industrially important platform organism corynebacterium glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. however, all currently described c. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of co2, in particular, when aiming f ...201425304460
metabolic engineering of corynebacterium glutamicum for glycolate production.corynebacterium glutamicum - a well-known industrial amino acid producer - has recently been engineered for the production of a variety of new products including diamines, alcohols, carotenoids and organic acids. glycolic acid was shown here not to serve as sole or combined carbon source for c. glutamicum. glycolate affected growth of c. glutamicum only at high concentrations (460mm) and in a comparable manner as other salts (480mm potassium chloride and 490mm sodium chloride). a transcriptome a ...201424486442
modification of chimeric (2s, 3s)-butanediol dehydrogenase based on structural information.a chimeric (2s, 3s)-butanediol dehydrogenase (clbdh) was engineered to have the strict (s)-configuration specificity of the (2s, 3s)-bdh (bslbdh) derived from brevibacterium saccharolyticum as well as the enzymatic stability of the (2r, 3s)-bdh (kpmbdh) from klebsiella pneumonia by swapping the domains of two native bdhs. however, while clbdh possesses the stability, it lacks the specificity. in order to assist in the design a bdh having strict substrate specificity, an x-ray structural analysis ...201425612804
corynebacterium glutamicum arnr controls expression of nitrate reductase operon narkghji and nitric oxide (no)-detoxifying enzyme gene hmp in an no-responsive manner.corynebacterium glutamicum arnr is a novel transcriptional regulator that represses expression of the nitrate reductase operon narkghji and the nitric oxide (no)-detoxifying flavohemoglobin gene hmp under aerobic conditions. in a previous study, we showed that arnr-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that arnr senses. in this study, we show that in the absence of nitrate, arnr-mediated repression is maintained under ...201424142248
process inhomogeneity leads to rapid side product turnover in cultivation of corynebacterium glutamicum.corynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. this means the demand for industrial process development is likely to increase. however, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. this leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertai ...201424410842
identification of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.the bacterial genus corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and "cheesy gland" in goats and sheep. thus, identifying new therapeutic targets to treat corynebacteria infections is both medically and economically important. cg2496, a functionally uncharacterized protein from corynebacterium glutamicum, was evaluated using an nmr ligand-affinity screen. a total of 11 compounds from a library of 460 biologically active compounds were shown ...201424403054
role of flavohaemoprotein hmp and nitrate reductase narghji of corynebacterium glutamicum for coping with nitrite and nitrosative stress.the influence of nitrate and nitrite on growth of corynebacterium glutamicum under aerobic conditions in shake flasks was analysed. when dissolved oxygen became limiting at higher cell densities, nitrate was reduced almost stoichiometrically to nitrite by nitrate reductase (narghji). the nitrite concentration also declined slowly, presumably as a result of several reactions including reduction to nitric oxide by a side-activity of nitrate reductase. the flavohaemoglobin gene hmp was most strongl ...201424237595
assessment of robustness against dissolved oxygen/substrate oscillations for c. glutamicum dm1933 in two-compartment bioreactor.corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. l-lysine). production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. oscillations can have an influence on the process p ...201424218302
metabolic engineering of corynebacterium glutamicum for 2-ketoisocaproate production.2-ketoisocaproate (kic) is used as a therapeutic agent, and a kic-producing organism may serve as a platform for products deriving from this 2-keto acid. we engineered corynebacterium glutamicum for the production of kic from glucose by deletion of ltbr and ilve, encoding the transcriptional repressor ltbr and transaminase b, respectively, and additional overexpression of ilvbncd, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. the kic-produc ...201424169948
analysis of sos-induced spontaneous prophage induction in corynebacterium glutamicum at the single-cell level.the genome of the gram-positive soil bacterium corynebacterium glutamicum atcc 13032 contains three integrated prophage elements (cgp1 to -3). recently, it was shown that the large lysogenic prophage cgp3 (∼187 kbp) is excised spontaneously in a small number of cells. in this study, we provide evidence that a spontaneously induced sos response is partly responsible for the observed spontaneous cgp3 induction. whereas previous studies focused mainly on the induction of prophages at the population ...201424163339
deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in corynebacterium glutamicum.allosteric regulation of phosphoenolpyruvate carboxylase (pepc) controls the metabolic flux distribution of anaplerotic pathways. in this study, the feedback inhibition of corynebacterium glutamicum pepc was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. based on rational protein design, six pepc mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. introducing one of the point mutations (n9 ...201424334667
pushing product formation to its limit: metabolic engineering of corynebacterium glutamicum for l-leucine overproduction.using metabolic engineering, an efficient l-leucine production strain of corynebacterium glutamicum was developed. in the wild type of c. glutamicum, the leua-encoded 2-isopropylmalate synthase (ipms) is inhibited by low l-leucine concentrations with a k(i) of 0.4 mm. we identified a feedback-resistant imps variant, which carries two amino acid exchanges (r529h, g532d). the corresponding leua(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, ...201424333966
increase in lactate yield by growing corynebacterium glutamicum in a bioelectrochemical reactor.under conditions conductive to growth, corynebacterium glutamicum showed higher lactate yield from glucose (1.62 ± 0.04) in a bioelectrochemical reactor including 0.2 mm of anthraquinone 2,6-disulfonate with the electrode potential regulated at -0.6 v (vs. ag/agcl) than in a non-regulated environment (1.10 ± 0.03), clarifying that low cathodic potential is beneficial for lactate production.201424315531
stimulus analysis of betp activation under in vivo conditions.the secondary active, na(+) coupled glycine betaine carrier betp from corynebacterium glutamicum betp was shown to harbor two different functions, transport catalysis (betaine uptake) and stimulus sensing, as well as activity regulation in response to hyperosmotic stress. by analysis in a reconstituted system, the rise in the cytoplasmic k(+) concentration was identified as a primary stimulus for betp activation. we have now studied regulation of betp in vivo by independent variation of both the ...201424384063
nrdh redoxin enhances resistance to multiple oxidative stresses by acting as a peroxidase cofactor in corynebacterium glutamicum.nrdh redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. although nrdh redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. in this study, we confirmed that the corynebacterium glutamicum nrdh redoxin catalytically reduces the disulfides in the class ib ribonucleotide reductases (rnr), insulin and 5,5'-dithiobis-(2-nitro ...201424375145
reducing lactate secretion by ldha deletion in l-glutamate- producing strain corynebacterium glutamicum gdk-9.l-lactate is one of main byproducts excreted in to the fermentation medium. to improve l-glutamate production and reduce l-lactate accumulation, l-lactate dehydrogenase-encoding gene ldha was knocked out from l-glutamate producing strain corynebacterium glutamicum gdk-9, designated gdk-9δldha. gdk-9δldha produced approximately 10.1% more l-glutamate than the gdk-9, and yielded lower levels of such by-products as α-ketoglutarate, l-lactate and l-alanine. since dissolved oxygen (do) is one of main ...201425763057
economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using corynebacterium glutamicum immobilized in porous polyurethane filler.an immobilized fermentation system, using cassava bagasse hydrolysate (cbh) and mixed alkalis, was developed to achieve economical succinic acid production by corynebacterium glutamicum. the c. glutamicum strains were immobilized in porous polyurethane filler (ppf). cbh was used efficiently as a carbon source instead of more expensive glucose. moreover, as a novel method for regulating ph, the easily decomposing nahco3 was replaced by mixed alkalis (naoh and mg(oh)2) for succinic acid production ...201425463799
bi-functional cellulases complexes displayed on the cell surface of corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature.introducing cellulases into corynebacterium glutamicum leads to the direct degradation of lignocellulosic materials for energy sources. in this study, a cellulase complex containing two cellulolytic enzymes, endoglucanase e (cele) and β-glucosidase a (bgla), was established to completely degrade cellulose to glucose. the cellulases complexes were displayed on the cell surface of c. glutamicum by using the mechanosensitive channel (msc) to anchor enzymes in the cytoplasmic membrane. as confirmed ...201425248702
production of l-glutamic acid with corynebacterium glutamicum (ncim 2168) and pseudomonas reptilivora (ncim 2598): a study on immobilization and reusability.l-glutamic acid is one of the major amino acids that is present in a wide variety of foods. it is mainly used as a food additive and flavor enhancer in the form of sodium salt. corynebacterium glutamicum (c. glutamicum) is one of the major organisms widely used for glutamic acid production.201425215180
idsa is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in corynebacterium glutamicum.corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic c50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. crte was assumed to be the major geranylgeranyl pyrophosphate (ggpp) synthase in carotenogenesis; however, deletion of crte did not abrogate carotenoid synthesis. in silico analysis of the repertoire of prenyltransferases encoded by the c. glutamicum genome revealed two candidate ggpps gen ...201425181035
characterization of a corynebacterium glutamicum dnab mutant that shows temperature-sensitive growth and mini-cell formation.corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. in order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from c. glutamicum wild-type strain atcc 31831, and found that one of them, m45, produced high frequencies of mini-cells with no nucleoids. cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally obs ...201425141796
improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step.the biosynthesis of poly(lactic acid) (pla)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme a (coa) and the polyhydroxyalkanoate (pha) synthase-catalyzed polymerization of lactyl-coa. in order to increase polymer productivity, we explored the rate-limiting step in pla-like polymer synthesis based on quantitative metabolite analysis using liq ...201426267112
co₂ /hco₃⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in corynebacterium glutamicum.the exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. while gradients of substrates, ph, or dissolved oxygen are often investigated, oscillating co2/hco3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. hereby, we investigate the metabolic and transcriptional response in corynebacterium glutamicum wild type as well as the impact on l-lysine production ...201425139448
detoxification of furfural in corynebacterium glutamicum under aerobic and anaerobic conditions.the toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. furfural is considered to be one of the most toxic compounds among these inhibitors. here, we describe the detoxification of furfural in corynebacterium glutamicum atcc13032 under both aerobic and anaerobic conditions. under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. the ratio of the ...201425112225
construction of a novel expression system for use in corynebacterium glutamicum.corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. suitable vectors are needed for metabolic engineering in c. glutamicum. most available vectors used in c. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. these vectors, though excellent for laboratory use, are not preferable choices for i ...201425108235
transcriptional response of corynebacterium glutamicum atcc 13032 to hydrogen peroxide stress and characterization of the oxyr regulon.the aerobic soil bacterium corynebacterium glutamicum atcc 13032 has a remarkable natural resistance to hydrogen peroxide. a major player in hydrogen peroxide defense is the lysr type transcriptional regulator oxyr, homologs of which are present in a wide range of bacteria. in this study, the global transcriptional response of c. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome dna microarrays, demonstrating the dynamic reaction of the regulatory networ ...201425107507
metabolic engineering of corynebacterium glutamicum for l-arginine production.l-arginine is an important amino acid for diverse industrial and health product applications. here we report the development of metabolically engineered corynebacterium glutamicum atcc 21831 for the production of l-arginine. random mutagenesis is first performed to increase the tolerance of c. glutamicum to l-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of nadph level, dis ...201425091334
oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.brevibacterium lactofermentum and corynebacterium glutamicum are important biotechnology species of the genus corynebacterium. the single-strand dna annealing protein (ssap)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains b. lactofermentum aj1511 and c. glutamicum atcc13032. when the rpsl gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies p ...201425087479
succinate production from co₂-grown microalgal biomass as carbon source using engineered corynebacterium glutamicum through consolidated bioprocessing.the potential for production of chemicals from microalgal biomass has been considered as an alternative route for co₂ mitigation and establishment of biorefineries. this study presents the development of consolidated bioprocessing for succinate production from microalgal biomass using engineered corynebacterium glutamicum. starch-degrading and succinate-producing c. glutamicum strains produced succinate (0.16 g succinate/g total carbon source) from a mixture of starch and glucose as a model micr ...201425056811
lactate production as representative of the fermentation potential of corynebacterium glutamicum 2262 in a one-step process.the fermentative properties of thermo-sensitive strain corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. in particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. in both processes, lactate was produced in significant amount, 27 g/l in batch culture, and up to 55.8 g/l in fed-batch culture, but the specific production rate in the fed-batch cul ...201425036691
genome-wide analysis of the role of global transcriptional regulator gntr1 in corynebacterium glutamicum.the transcriptional regulator gntr1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in corynebacterium glutamicum. gluconate lowers the dna binding affinity of gntr1, which is probably the mechanism of gluconate-dependent induction of these genes. in addition, gntr1 positively regulates ptsg, a gene encoding a major glucose transporter, and pck, a gene encoding phosphoenolpyruvate carboxykinase. here, we searched for the new target of gntr1 on a genome-wide scale b ...201424982307
rapid assessment of oxygen transfer impact for corynebacterium glutamicum.oxygen supply is crucial in industrial application of microbial systems, such as corynebacterium glutamicum, but oxygen transfer is often neglected in early strain characterizations, typically done under aerobic conditions. in this work, a new procedure for oxygen transfer screening is presented, assessing the impact of maximum oxygen transfer conditions (otrmax) within microtiter plate-based cultivation for enhanced throughput. oxygen-dependent growth and productivity were characterized for c. ...201424981020
a de novo nadph generation pathway for improving lysine production of corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. in this work, a de novo nadph generation pathway is proposed by altering the coenzyme specificity of a native nad-dependent glyceraldehyde 3-phosphate dehydrogenase (gapdh) to nadp, which consequently has the potential to produce additional nadph in the glycolytic pathway. specifically, the coenzyme specificity of gapdh of corynebacterium glutami ...201424953302
artificial oxidative stress-tolerant corynebacterium glutamicum.we have reported a transcription profile of an adapted corynebacterium glutamicum that showed enhanced oxidative stress resistance. to construct an artificial oxidative stress-resistant strain, gene clusters in the β-ketoadipate pathway, which were up-regulated in the adapted strain, were artificially expressed in the wild-type c. glutamicum. the wild-type strain was unable to grow under 2 mm h2o2 containing minimal medium, while the strains expressing pca gene clusters restored growth under the ...201424949252
rnd transporters protect corynebacterium glutamicum from antibiotics by assembling the outer membrane.corynebacterium-mycobacterium-nocardia (cmn) group are the causative agents of a broad spectrum of diseases in humans. a distinctive feature of these gram-positive bacteria is the presence of an outer membrane of unique structure and composition. recently, resistance-nodulation-division (rnd) transporters (nicknamed mmpls, mycobacterial membrane protein large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. in this study, ...201424942069
transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.the gram-positive bacterium corynebacterium glutamicum belongs to the order corynebacteriales and is used as a producer of amino acids at industrial scales. due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. applying the high-resolution technique of transcriptome sequencing (rna-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the c. glutamicum transcriptome. ...201424910972
expanding the laccase-toolbox: a laccase from corynebacterium glutamicum with phenol coupling and cuprous oxidase activity.laccases are oxidases with potential for application in biotechnology. up to now only fungal laccases have been applied in technical processes, although bacterial laccases are generally easier to handle and more stable at alkaline ph values and elevated temperatures. to increase the toolbox of bacterial laccases and to broaden our knowledge about them, new enzymes have to be characterized. within this study, we describe the new bacterial laccase cgl1 from corynebacterium glutamicum. cgl1 was fou ...201424910971
engineered cytidine triphosphate synthetase with reduced product inhibition.cytidine triphosphate (ctp) synthetase (ctps) (ec number 6.3.4.2) is a key enzyme involved in de novo synthesis of ctp. it catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. in this study, a novel ctps from corynebacterium glutamicum atcc 13032 (cgctps) was cloned, expressed and characterized. a series of mutagenesis in its n-terminal ammonia ligase (alase) domain was performed in order to reduce ctp product inhibition. all single mutation varian ...201424902851
metabolic engineering corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway.the experiments presented here were based on the conclusions of our previous results. in order to avoid introduction of expression plasmid and to balance the nadh/nad ratio, the nadh biosynthetic enzyme, i.e., nad-dependent glyceraldehyde-3-phosphate dehydrogenase (gadph), was replaced by nadp-dependent gadph, which was used to biosynthesize nadph rather than nadh. the results indicated that the nadh/nad ratio significantly decreased, and glucose consumption and l-lysine production drastically i ...201424879631
corynebacterium glutamicum sdha encoding succinate dehydrogenase subunit a plays a role in cysr-mediated sulfur metabolism.the corynebacterium glutamicum cysr protein plays a critical regulatory role in sulfur metabolism. in this study, we isolated a protein interacting with cysr by employing a two-hybrid system. subsequent analysis identified the gene as sdha annotated to encode succinate dehydrogenase flavoprotein subunit a, a krebs cycle enzyme. deletion of the gene (δsdha) severely affected cell growth and final cell yield, particularly in complex media. in addition, the δsdha mutant strain was unable to use ace ...201424866946
investigation of ptsg gene in response to xylose utilization in corynebacterium glutamicum.corynebacterium glutamicum strains nc-2 were able to grow on xylose as sole carbon sources in our previous work. nevertheless, it exhibited the major shortcoming that the xylose consumption was repressed in the presence of glucose. so far, regarding c. glutamicum, there are a number of reports on ptsg gene, the glucose-specific transporter, involved in glucose metabolism. recently, we found ptsg had influence on xylose utilization and investigated the ptsg gene in response to xylose utilization ...201424859809
effect of cofactor folate on the growth of corynebacterium glutamicum syps-062 and l-serine accumulation.the direct fermentative production of l-serine from sugar has attracted increasing attention. corynebacterium glutamicum syps-062 can directly convert sugar to l-serine. in this study, the effects of exogenous and endogenous regulation of cofactor folate on c. glutamicum syps-062 growth and l-serine accumulation were investigated. for exogenous regulation, the inhibitor (sulfamethoxazole) or precursor (p-aminobenzoate) of folate biosynthesis was added to the medium, respectively. for endogenous ...201424859773
from zero to hero - production of bio-based nylon from renewable resources using engineered corynebacterium glutamicum.polyamides are important industrial polymers. currently, they are produced exclusively from petrochemical monomers. herein, we report the production of a novel bio-nylon, pa5.10 through an integration of biological and chemical approaches. first, systems metabolic engineering of corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. in this way, a hyper-producer, with a high diaminopentane yield of 41% ...201424831706
involvement of the global regulator glxr in 3-hydroxybenzoate and gentisate utilization by corynebacterium glutamicum.corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. an iclr-type regulator genr has been characterized to activate the transcription of gendfm and genkh operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. on the other hand, glxr, a global regulator of the cyclic amp (camp) receptor protein-fumarate nitrate reductase regulator (crp-fnr) type, was also ...201424795375
phosphatase activity of the histidine kinases ensures pathway specificity of the chrsa and hrrsa two-component systems in corynebacterium glutamicum.the majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. the gram-positive soil bacterium corynebacterium glutamicum contains two homologous two-component systems (tcs) involved in the haem-dependent regulation of gene expression. whereas the hrrsa system is crucial for utilization of haem as an alternative iron source, chrsa is required to cope with high toxic haem levels. in this study, we analyse ...201424779520
pupylated proteins in corynebacterium glutamicum revealed by mudpit analysis.in a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. however, not all actinobacteria possessing the pup protein also contain a proteasome. in this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism corynebacterium glutamicum. a defined pup deletion mutant of c. glutamicum atcc 13032 grew aerobically as the parent strain in standard glucose min ...201424737727
enhancing corynebacterium glutamicum robustness by over-expressing a gene, msha, for mycothiol glycosyltransferase.over-expression of the gene, msha, coding for mycothiol glycosyl transferase improved the robustness of corynebacterium glutamicum to various stresses. intracellular mycothiol (msh) content was increased by 114 % in wt(pxmj19-msha) compared to wt(pxmj19). survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to wt(pxmj19) under stress by h2o2 (40 mm), methylglyoxal (5.8 mm), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), cd(2+) (0.01 mm), mn(2+) ...201424737070
double mutation of cell wall proteins cspb and pbp1a increases secretion of the antibody fab fragment from corynebacterium glutamicum.among other advantages, recombinant antibody-binding fragments (fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length iggs. although production of recombinant fab using microbial expression systems has been reported, yields of active fab have not been satisfactory. we recently developed the corynebacterium glutamicum protein expression system (corynex®) and demonstrated improved yield and purity for some applications, alth ...201424731213
the laci-type transcriptional regulator arar acts as an l-arabinose-responsive repressor of l-arabinose utilization genes in corynebacterium glutamicum atcc 31831.the corynebacterium glutamicum atcc 31831 arabda operon consists of three l-arabinose catabolic genes, upstream of which the galm, arar, and arae genes are located in opposite orientation. arar encodes a laci-type transcriptional regulator that negatively regulates the l-arabinose-inducible expression of arabda and arae (encoding an l-arabinose transporter), through a mechanism that has yet to be identified. here we show that the arar protein binds in vitro to three sites: one upstream of arabda ...201424706742
chorismate-dependent transcriptional regulation of quinate/shikimate utilization genes by lysr-type transcriptional regulator qsur in corynebacterium glutamicum: carbon flow control at metabolic branch point.the qsu operon of corynebacterium glutamicum comprises four genes (qsuabcd) that underpin the microorganism's quinate/shikimate utilization pathways. the genes encode enzymes that catalyse reactions at the metabolic branch point between the biosynthesis route for synthesis of aromatic compounds and the catabolic route for degradation of quinate and shikimate for energy production. a qsur gene located immediately upstream of qsua encodes a protein (qsur) which activates the operon in the presence ...201424674055
analysis of putative protomer crosstalk in the trimeric transporter betp: the heterotrimer approach.the homotrimeric, secondary active betaine carrier betp from corynebacterium glutamicum is a model system for stress-regulated transport in bacteria. its activity responds to hyperosmotic stress and it harbors two different functions, transport catalysis (betaine uptake) and stimulus sensing, resp. activity regulation. structural information from 2d and 3d crystals as well as functional analysis of monomerized betp suggested the presence of conformational crosstalk between the individual protome ...201424637177
recent progress in development of synthetic biology platforms and metabolic engineering of corynebacterium glutamicum.the paradigm of synthetic biology has been evolving, along with relevant engineering, to achieve designed bio-systems. synthetic biology has reached the point where it is possible to develop microbial strains to produce desired chemicals. recent advances in this field have promoted metabolic engineering of corynebacterium glutamicum as an amino-acid producer for use in intelligent microbial-cell factories. here, we review recent advances that address c. glutamicum as a potential model organism f ...201424632177
a method for gene amplification and simultaneous deletion in corynebacterium glutamicum genome without any genetic markers.a method for the simultaneous replacement of a given gene by a target gene, leaving no genetic markers, has been developed. the method is based on insertional inactivation and double-crossover homologous recombination. with this method, the lysc(t311i), fbp and ddh genes were inserted into corynebacterium glutamicum genome, and the pck, alat and avta genes were deleted. mobilizable plasmids with lysc(t311i), fbp and ddh cassettes and two homologous arms on the ends of pck, alat and avta were con ...201424613758
carbon flux analysis by 13c nuclear magnetic resonance to determine the effect of co2 on anaerobic succinate production by corynebacterium glutamicum.wild-type corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. we investigated the effect of co2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. the presence of co2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in a ...201424610842
the physiological role of riboflavin transporter and involvement of fmn-riboswitch in its gene expression in corynebacterium glutamicum.riboflavin is a precursor of flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad), which work as cofactors of numerous enzymes. understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. in this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expressio ...201424531272
construction and application of novel feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases by engineering the n-terminal domain for l-phenylalanine synthesis.3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp synthase) encoded by arof is the first enzyme of the shikimate pathway. in the present study, an arof variant with a deficiency in residue ile11 (named arof*) was shown to be insensitive to l-tyrosine. according to three-dimensional structure analysis, nine arof variants were constructed with truncation of different n-terminal fragments, and overexpression of the variants arof(δ(1-9)) , arof(δ(1-10)) , arof(δ(1-12)) and, in particular, a ...201424517515
high-throughput screening of a corynebacterium glutamicum mutant library on genomic and metabolic level.due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. this strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. of these platforms, metabolic profiling has the strongest correlation with the phenotype. we previously published a high-throughput metabolic profiling method for c. glutamicum as well as the ...201424504095
the pyruvate dehydrogenase complex of corynebacterium glutamicum: an attractive target for metabolic engineering.the pyruvate dehydrogenase complex (pdhc) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-coa and co2. since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. this review summarizes the current knowledge and achievements on metabolic engineering approaches to ...201424486441
genetic characterization of 4-cresol catabolism in corynebacterium glutamicum.corynebacterium glutamicum uses 4-cresol as sole carbon source for growth. protocatechuate 3,4-dioxygenase activity had been detected when c. glutamicum was grown with 4-cresol. in this work, we found that 4-cresol was catabolized via 4-hydroxybenzoate and protocatechuate as intermediate metabolites, and a genetic cluster called cre (designated for 4-cresol, creabcdefghir, tagged as ncgl0521-ncgl0531 in ncbi) was identified. the cre gene cluster comprises of 11 genes, and six of them were experi ...201424480572
application of a genetically encoded biosensor for live cell imaging of l-valine production in pyruvate dehydrogenase complex-deficient corynebacterium glutamicum strains.the majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. in this study, we have applied the recently developed lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered l-valine producing corynebacterium glutamicum strains based on the p ...201424465669
effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by corynebacterium glutamicum.biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with tween 40 addition during fermentation. the transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (tp) of glutamate were investigated under the conditions of var ...201423990041
improved succinate production in corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system.a dual route for anaerobic succinate production was engineered into corynebacterium glutamicum. the glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. the engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). further overexpression of succinate exporter, suce, increased succinate yield to 1.43 mol (mol glucose)(-1). metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineeri ...201424129953
beyond growth rate 0.6: what drives corynebacterium glutamicum to higher growth rates in defined medium.in a former study we showed that corynebacterium glutamicum grows much faster in defined cgxii glucose medium when growth was initiated in highly diluted environments [grünberger et al. (2013b) biotechnol bioeng]. here we studied the batch growth of c. glutamicum in cgxii at a comparable low starting biomass concentration of od ≈ 0.005 in more detail. during bioreactor cultivations a bi-phasic growth behavior with changing growth rates was observed. initially the culture grew with μˆ=0.61±0.02 h ...201423996851
taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. here we present an approach to rapidly generate sets of enzymes overriding this control. it is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. the sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by facs. we randomly mutageni ...201423829416
protein s-mycothiolation functions as redox-switch and thiol protection mechanism in corynebacterium glutamicum under hypochlorite stress.protein s-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in firmicutes bacteria. here we used transcriptomics to analyze the naocl stress response in the mycothiol (msh)-producing corynebacterium glutamicum. we further applied thiol-redox proteomics and mass spectrometry (ms) to identify protein s-mycothiolation.201423886307
effect of tween 40 and dtsr1 on l-arginine overproduction in corynebacterium crenatum.l-glutamate is an important precursor in the l-arginine (l-arg) biosynthetic pathway. various methods, including polyoxyethylene sorbitan monopalmitate (tween 40) addition and dtsr1 disruption, have been widely used to induce l-glutamate overproduction in corynebacterium glutamicum. in this study, a novel strategy for l-arg overproduction through tween 40 trigger and δdtsr1 mutant were proposed in corynebacterium crenatum.201526264811
regulation of the expression of de novo pyrimidine biosynthesis genes in corynebacterium glutamicum.expression of pyrimidine de novo biosynthesis is downregulated by an exogenous uracil in many bacteria. in this study, we show that a putative binding motif sequence of pyrr is required for uracil-mediated repression of pyrr-lacz translational fusion. however, the uracil response was still observed in the strain with the pyrr gene deleted, implying the existence of a uracil response factor other than pyrr which also acts through the pyrr binding loop region. deletion of rho, encoding the transcr ...201526260458
exploring the role of sigma factor gene expression on production by corynebacterium glutamicum: sigma factor h and fmn as example.bacteria are known to cope with environmental changes by using alternative sigma factors binding to rna polymerase core enzyme. sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. in this study, the effect of overexpressing each annotated sigma factor gene in corynebacterium glutamicum wt was assayed using an iptg inducible plasmid system and different iptg concentrations. it was revealed that growth was severely decreased whe ...201526257719
trna-dependent alanylation of diacylglycerol and phosphatidylglycerol in corynebacterium glutamicum.aminoacyl-phosphatidylglycerol synthases (aapgss) are membrane proteins that utilize aminoacylated trnas to modify membrane lipids with amino acids. aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aapgss utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (pg) and cardiolipin. here we ...201526235234
live cell imaging of sos and prophage dynamics in isogenic bacterial populations.almost all bacterial genomes contain dna of viral origin, including functional prophages or degenerated phage elements. a frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (spi) even in the absence of an external stimulus. in this study, we have analyzed spi of the large, degenerated prophage cgp3 (187 kbp), which is integrated into the genome of the gram-positive corynebacterium glutamicum atcc 13032. time-lapse fluorescence microscopy of fluorescent report ...201526235130
engineering microbial cell factories: metabolic engineering of corynebacterium glutamicum with a focus on non-natural products.corynebacterium glutamicum is the workhorse of biotechnological amino acid production. for more than 50 years amino acid producing strains of this actinomycete have been improved by classical breeding, metabolic engineering and systems and synthetic biology approaches. this review focusses mainly on recent developments on c. glutamicum strain development for non-natural products. recently, metabolite sensors have accelerated classical strain breeding. synthetic pathways for access to alternative ...201526216246
co-production of s-adenosyl-l-methionine and l-isoleucine in corynebacterium glutamicum.in this study, production of s-adenosyl-l-methionine in corynebacterium glutamicum was investigated by overexpressing genes metk and vgb. compared with vector control, overexpression of metk alone in c. glutamicum atcc13032 and iwj001 increased sam production 5.11 and 11.65 times, respectively; while overexpression of metk and vgb in c. glutamicum atcc13032 and iwj001 increased sam production 5.83 and 14.95 times, respectively. further studies on iwj001/pdxw-8-metk-vgb showed that the limiting f ...201526215341
thermal and solvent stress cross-tolerance conferred to corynebacterium glutamicum by adaptive laboratory evolution.reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. here, we report on adaptive laboratory evolution (ale) of the amino acid-producing bacterium corynebacterium glutamicum under thermal stress. after 65 days of serial passage of the transgenic strain gly3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were ...201525595768
copper homeostasis-related genes in three separate transcriptional units regulated by csor in corynebacterium glutamicum.in corynebacterium glutamicum r, csor acts as a transcriptional repressor not only of the cognate copa-csor operon but also of the copz1-copb-cgr_0126 operon. it is predicted that copa and copb encode p-type atpases for copper efflux and copz1 encodes a metallochaperone. here, a csor-binding motif was found upstream of another copz-like gene, copz2, and the in vitro binding of the csor protein to its promoter was confirmed. the monocistronic copz2 transcript was upregulated by excess copper in a ...201525592736
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