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microarray studies reveal a 'differential response' to moderate or severe heat shock of the hrca- and hspr-dependent systems in corynebacterium glutamicum.this article has been retracted at the request of microbiology because identical bands for the 16s rrna probe controls in the northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in journal of bacteriology over a period of 5 years.201323754843
transcriptional analysis of the f0f1 atpase operon of corynebacterium glutamicum atcc 13032 reveals strong induction by alkaline ph.this article has been retracted at the request of microbiology because identical bands for the 16s rrna probe controls in the northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in journal of bacteriology over a period of 5 years.201323754842
direct l-lysine production from cellobiose by corynebacterium glutamicum displaying beta-glucosidase on its cell surface.we constructed beta-glucosidase (bgl)-displaying corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. after screening active bgls, sde1394, which is a bgl from saccharophagus degradans, was successfully displayed on the c. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. the optical density at 600 nm of bgl-displaying c. glutamicum grown on cellobiose as a carbon source reached 23.5 aft ...201323749228
metabolic evolution of corynebacterium glutamicum for increased production of l-ornithine.l-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. the commercial applications mean that efficient biotechnological production of l-ornithine has become increasingly necessary. adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. the evolved strains can be further optimised by metabolic engineering. thus, metabolic evolutio ...201323725060
identification of a gene involved in plasmid structural instability in corynebacterium glutamicum.expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. in order to enhance plasmid stability in recombinant corynebacterium glutamicum, an expression plasmid containing genes of the clostridium acetobutylicum butyryl-coa synthesis operon with high structural instability within wild-type c. glutamicum was employed. from a total of 133 mutants exhibiting disruptions in ...201323703324
a mutational analysis of the active site loop residues in cis-3-chloroacrylic acid dehalogenase.cis-3-chloroacrylic acid dehalogenase (cis-caad) from pseudomonas pavonaceae 170 and a homologue from corynebacterium glutamicum designated cg10062 are 34% identical in sequence (54% similar). the former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. although cg10062 has the six active site residues (pro-1, his-28, arg-70, arg-73, tyr-103, and glu-114) that are critical for cis-caad activity, it s ...201323692140
enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different corynebacterium glutamicum.during l-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (pcx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. to explore the significance of pcx for l-glutamate overproduction, the pyc gene encoding pcx was amplified in corynebacterium glutamicum gdk-9 triggered by biotin limitation and cn1021 triggered by a temperature shock, respectively. in the fed-batch cultures, gdk-9pxmj19pyc exhibited 7.4 % lower l-alanine excretion and no im ...201323690048
pcgr2 copy number depends on the par locus that forms a parc-parb-dna partition complex in corynebacterium glutamicum.to characterize the par system of corynebacterium glutamicum pcgr2 and to manipulate the par components to effectively manipulate plasmid copy number.201323683072
heat shock proteome analysis of wild-type corynebacterium glutamicum atcc 13032 and a spontaneous mutant lacking groel1, a dispensable chaperone. 201323661687
transcriptional analysis of the groes-groel1, groel2, and dnak genes in corynebacterium glutamicum: characterization of heat shock-induced promoters. 201323661686
cord factor (trehalose 6,6'-dimycolate) forms fully stable and non-permeable lipid bilayers required for a functional outer membrane.cord factor (trehalose 6,6'-dimycolate, tdm) is the major lipid in the outer membrane of corynebacteria and mycobacteria. although its role is well recognized in the immune response phenomena, its membrane biophysical properties remained largely unexplored and tdm has often been described as a detergent. we purified the main components of the outer membrane from corynebacterium glutamicum and analyzed their membrane forming properties. in mixture with endogenous cardiolipin, but not alone, the s ...201323643889
isolation of fully synthetic promoters for high-level gene expression in corynebacterium glutamicum.corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. to extend its product spectrum and improve productivity, c. glutamicum needs to undergo further engineering, including the development of applicable promoter system. here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in c. glutamicum. using green fluorescent protein (gfp) as a reporter, highly fl ...201323633298
recombineering in corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation.recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. here, we establish recombineering for corynebacterium glutamicum using rect of prophage rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell ...201323630315
proteome response of corynebacterium glutamicum to high concentration of industrially relevant c₄ and c₅ dicarboxylic acids.more than fifty years of industrial and scientific developments on the amino acid-producer strain corynebacterium glutamicum has generated an extremely huge knowledge highly applicable to the development of new products. despite the production of dicarboxylic acids has already been engineered in c. glutamicum, the effect caused by these acids at competitive industrial levels has not yet been described. thus, aspartic, fumaric, itaconic, malic and succinic acids have been tested on the growth of ...201323624027
oxyr acts as a transcriptional repressor of hydrogen peroxide-inducible antioxidant genes in corynebacterium glutamicum r.oxyr, a lysr-type transcriptional regulator, has been established as a redox-responsive activator of antioxidant genes in bacteria. this study shows that oxyr acts as a transcriptional repressor of kata, dps, ftn and cyda in corynebacterium glutamicum r. kata encodes h2o2-detoxifing enzyme catalase, dps and ftn are implicated in iron homeostasis and cyda encodes a subunit of cytochrome bd oxidase. quantitative rt-pcr analyses revealed that expression of kata and dps, but not of ftn and cyda, was ...201323621709
physiological roles of mycothiol in detoxification and tolerance to multiple poisonous chemicals in corynebacterium glutamicum.mycothiol (msh) plays important roles in maintaining cytosolic redox homeostasis and in adapting to reactive oxygen species in the high-(g + c)-content gram-positive actinobacteria. however, its physiological roles are ill defined compared to glutathione, the functional analog of msh in gram-negative bacteria and most eukaryotes. in this research, we explored the impact of intracellular msh on cellular physiology by using msh-deficient mutants in the model organism corynebacterium glutamicum. we ...201323615850
recovery of high-purity metallic pd from pd(ii)-sorbed biosorbents by incineration.this work reports a direct way to recover metallic palladium with high purity from pd(ii)-sorbed polyethylenimine-modified corynebacterium glutamicum biosorbent using a combined method of biosorption and incineration. this study is focused on the incineration part which affects the purity of recovered pd. the incineration temperature and the amount of pd loaded on the biosorbent were considered as major factors in the incineration process, and their effects were examined. the results showed that ...201323611701
regulatory interaction of the corynebacterium glutamicum whc genes in oxidative stress responses.in this study, we analyzed the regulatory interaction of the corynebacterium glutamicum whc genes that play roles in oxidative stress responses. we found that whce and whca transcription was minimal in the whcb-deleted mutant (δwhcb). however, whcb and whca transcription increased in the δwhce mutant during the log phase, whereas their transcription decreased during the stationary phase. in addition, cells carrying the p180-whcb vector, which showed retarded growth due to uncontrolled whcb overe ...201323608553
development of a secretion system for the production of heterologous proteins in corynebacterium glutamicum using the porin b signal peptide.corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. here, we report the development of a new secretory system for the production of heterologous proteins by using the porin b (porb) signal peptide in c. glutamicum. we examined two different endoxylanases and an antibody fragment (scfv) as model proteins for secretory production. ...201323597779
oxygen supply in disposable shake-flasks: prediction of oxygen transfer rate, oxygen saturation and maximum cell concentration during aerobic growth.dissolved oxygen plays an essential role in aerobic cultivation especially due to its low solubility. under unfavorable conditions of mixing and vessel geometry it can become limiting. this, however, is difficult to predict and thus the right choice for an optimal experimental set-up is challenging. to overcome this, we developed a method which allows a robust prediction of the dissolved oxygen concentration during aerobic growth. this integrates newly established mathematical correlations for t ...201323592306
crystal and solution studies reveal that the transcriptional regulator acnr of corynebacterium glutamicum is regulated by citrate-mg2+ binding to a non-canonical pocket.corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor acnr, assigned by sequence identity to the tetr family. we report the structures of acnr in two crystal forms together with ligan ...201323589369
maltose uptake by the novel abc transport system musefgk2i causes increased expression of ptsg in corynebacterium glutamicum.the gram-positive corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. however, maltose uptake in c. glutamicum has not been investigated. interestingly, the presence of maltose in the medium causes increased expression of ptsg in c. glutamicum by an unknown mechanism, although ...201323543710
kinetic and thermodynamic characterization of lysine production process in brevibacterium lactofermentum.detailed kinetic and thermodynamic parameters for lysine production from brevibacterium lactofermentum are investigated for the first time in this study. production of the essential amino acid, l-lysine, by b. lactofermentum was assessed in a flask and a continuously stirred tank fermentor (22 l). maximum lysine production was achieved after 40 h of growth and at 35 °c. the effect of different nitrogen sources such as nh(4)no(3), (nh(4))(2)so(4), (nh(4))(2)hpo(4), corn steep liquor, nano(3), and ...201323475286
systems metabolic engineering of xylose-utilizing corynebacterium glutamicum for production of 1,5-diaminopentane.the sustainable production of industrial platform chemicals is one of the great challenges facing the biotechnology field. ideally, fermentation feedstocks would rather rely on industrial waste streams than on food-based raw materials. corynebacterium glutamicum was metabolically engineered to produce the bio-nylon precursor 1,5-diaminopentane from the hemicellulose sugar xylose. comparison of a basic diaminopentane producer strain on xylose and glucose feedstocks revealed a 30% reduction in dia ...201323447448
proteome turnover in bacteria: current status for corynebacterium glutamicum and related bacteria.with the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. these dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established 'steady state' proteomics approaches. in this article, ...201323425033
conversion of corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain.in this study a comparative analysis of three corynebacterium glutamicum atcc 13032 respiratory chain mutants lacking either the cytochrome bd branch (δcydab), or the cytochrome bc1-aa3 branch (δqcr), or both branches was performed. the lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1-aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly i ...201323416842
media optimization of corynebacterium glutamicum for succinate production under oxygen-deprived condition.corynebacterium glutamicum is one of the well-studied industrial strain that is used for the production of nucleotides and amino acids. recently, it has also been studied as a possible producer of organic acids such as succinic acid, based on its ability to produce organic acids under an oxygen deprivation condition. in this study, we conducted the optimization of medium components for improved succinate production from c. glutamicum under an oxygen deprivation condition by plackett-burman desig ...201323412064
chromosome segregation impacts on cell growth and division site selection in corynebacterium glutamicum.spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. in many rod-shaped bacteria, regulatory systems such as the min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. however, regulation of division site selection in bacteria lacking recognizable min and nucleoid occlusion remains less well understood. here, we describe one such rod-shaped organism, corynebacterium glutamicum, which does not always ...201323405112
involvement of regulatory interactions among global regulators glxr, sugr, and rama in expression of rama in corynebacterium glutamicum.the central carbon metabolism genes in corynebacterium glutamicum are under the control of a transcriptional regulatory network composed of several global regulators. it is known that the promoter region of rama, encoding one of these regulators, interacts with its gene product, rama, as well as with the two other regulators, glxr and sugr, in vitro and/or in vivo. although rama has been confirmed to repress its own expression, the roles of glxr and sugr in rama expression have remained unclear. ...201323396909
phosphotransferase system-mediated glucose uptake is repressed in phosphoglucoisomerase-deficient corynebacterium glutamicum strains.corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. amino acid overproduction comes along with a high nadph demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (ppp). in previous studies, the complete redirection of the carbon flux toward the ppp by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of c. glutamicum amino acid production ...201323396334
the salicylate 1,2-dioxygenase as a model for a conventional gentisate 1,2-dioxygenase: crystal structures of the g106a mutant and its adducts with gentisate and salicylate.the salicylate 1,2-dioxygenase (sdo) from the bacterium pseudaminobacter salicylatoxidans bn12 is a versatile gentisate 1,2-dioxygenase (gdo) that converts both gentisate (2,5-dihydroxybenzoate) and various monohydroxylated substrates. several variants of this enzyme were rationally designed based on the previously determined enzyme structure and sequence differences between the sdo and the 'conventional' gdo from corynebacterium glutamicum. this was undertaken in order to define the structural ...201323384287
modification of histidine biosynthesis pathway genes and the impact on production of l-histidine in corynebacterium glutamicum.histidine biosynthesis in corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. c. glutamicum histidine genes are located and transcribed in two unlinked loci, hiseg and hisdcb-orf1-orf2-hisha-impa-hisfi. we constructed plasmid pk18hisdptac to replace the native hisd promoter with the tac promoter, and overexpressed phosphoribosyl-atp-pyrophosphohydrolase, encoded by hi ...201323355034
increasing l-isoleucine production in corynebacterium glutamicum by overexpressing global regulator lrp and two-component export system brnfe.to increase the l-isoleucine production in corynebacterium glutamicum by overexpressing the global regulator lrp and the two-component export system brnfe.201323331988
characterization of shikimate dehydrogenase homologues of corynebacterium glutamicum.the function of three corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsud (cgr_0495), cgr_1216, and aroe (cgr_1677), was investigated. a disruptant of aroe required shikimate for growth, whereas a qsud-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. there was no discernible difference in growth rate between wild-type and a cgr_1216-deficient strain. enzymatic assays showed that aroe both reduced 3-dehydro ...201323306642
corynebacterium glutamicum promoters: a practical approach.transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate 'green waves' in the metabolic pathways leading to the desired metabolites. detailed analyses of corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms ...201323305350
[overexpression of corynebacterium glutamicum nad kinase improves l-isoleucine biosynthesis].nad kinase catalyzes the phosphorylation of coenzyme i [nad(h)] to form coenzyme ii [nadp(h)], and nadph is an important cofactor in l-isoleucine biosynthesis. in order to improve nadph supply, ppnk, the gene encoding nad kinase in corynebacterium glutamicum was cloned and separately expressed in an l-isoleucine synthetic strain, brevibacterium lactofermentum jhi3-156, by an inducible expression vector pdxw-8 and a constitutive expression vector pdxw-9. compared with the control strain jhi3-156/ ...201223289306
adaptive evolution of corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling.corynebacterium glutamicum was adapted in a chemostat for 1,900 h with gradually increasing h2o2 stress to understand the oxidative stress response of an industrial host. after 411 generations of adaptation, c. glutamicum developed the ability to grow under stress of 10 mm h2o2, whereas the wild-type did not. the adapted strain also showed increased stress resistance to diamide and menadione, sds, tween 20, hcl, naoh, and ampicillin. a total of 1,180 genes in the rna-seq transcriptome analysis o ...201323288296
non-invasive online detection of microbial lysine formation in stirred tank bioreactors by using calorespirometry.non-invasive methods for online monitoring of biotechnological processes without compromising the integrity of the reactor system are very important to generate continuous data. even though calorimetry has been used in conventional biochemical analysis for decades, it has not yet been specifically applied for online detection of product formation at technical scale. thus, this article demonstrates a calorespirometric method for online detection of microbial lysine formation in stirred tank biore ...201323280310
monitoring of population dynamics of corynebacterium glutamicum by multiparameter flow cytometry.phenotypic variation of microbial populations is a well-known phenomenon and may have significant impact on the success of industrial bioprocesses. flow cytometry (fc) and the large repertoire of fluorescent dyes bring the high-throughput analysis of multiple parameters in single bacterial cells into reach. in this study, we evaluated a set of different fluorescent dyes for suitability in fc single cell analysis of the biotechnological platform organism corynebacterium glutamicum. already simple ...201323279937
analysis of in vivo function of predicted isoenzymes:a metabolomic approach.isoenzymes occur in all organisms. often, they are regulated with respect to environmental perturbations to assure metabolic flexibility. in bioinformatics-driven functional genome annotations, the presence of isoenzymes is frequently predicted. it is desirable to verify the functions of putative isoenzymes experimentally for a correct estimation of the metabolic capacities in a given organism. using metabolome analysis, we investigated two knockout mutants of putative shikimate dehydrogenases ( ...201223215805
bio-based production of organic acids with corynebacterium glutamicum.the shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. rational strain design of appropriate microorganisms has ...201323199277
ruthenium recovery from acetic acid waste water through sorption with bacterial biosorbent fibers.a fibrous bacterial biosorbent was developed to bind precious metal-organic complexes in batch and column processes. polyethylenimine (pei)-modified bacterial biosorbent fiber (pbbf) was prepared by spinning corynebacterium glutamicum biomass-chitosan blends, coating them with pei and cross-linking with glutaraldehyde. when an acetic acid waste solution containing 1822.9mg/l ru was used as a model waste solution, ru uptake by the pbbf was 16.5 times higher than that of the commercial ion exchang ...201323196218
influence of sigb inactivation on corynebacterium glutamicum protein secretion.the non-essential corynebacterium glutamicum sigma factor, sigb, modulates global gene expression during the transition from exponential growth to the stationary phase. utilizing a signal peptide derived from c. glutamicum r cgr_0949, a sigb disruption mutant able to secrete 3- to 5-fold more green fluorescence protein (gfp) and α-amylase than the wild type strain was isolated. the signal peptide selectively enabled the mutant to produce greater amounts of both proteins, which were in turn secre ...201323179627
secretory production of an fad cofactor-containing cytosolic enzyme (sorbitol-xylitol oxidase from streptomyces coelicolor) using the twin-arginine translocation (tat) pathway of corynebacterium glutamicum.carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. using the industrial workhorse corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic fad cofactor-containing sorbitol-xylitol oxidase from streptomyces coelicolor was achieved by using the twin-arginine translocation (tat) protein export machinery for protein translocation across the cytoplasmic membrane. our results demonstr ...201323163932
next-generation sequencing-based genome-wide mutation analysis of l-lysine-producing corynebacterium glutamicum atcc 21300 strain.in order to identify single nucleotide polymorphism and insertion/deletion mutations, we performed whole-genome re-sequencing of the enhanced l-lysine-producing corynebacterium glutamicum atcc 21300 strain. in total, 142 single nucleotide polymorphisms and 477 insertion/deletion mutations were identified in the atcc 21300 strain when compared to 3,434 predicted genes of the wild-type c. glutamicum atcc 13032 strain. among them, 110 transitions and 29 transversions of single nucleotide polymorphi ...201223124757
identification of a had superfamily phosphatase, hdpa, involved in 1,3-dihydroxyacetone production during sugar catabolism in corynebacterium glutamicum.corynebacterium glutamicum produces 1,3-dihydroxyacetone (dha) as metabolite of sugar catabolism but the responsible enzyme is yet to be identified. using a transposon mutant library, the gene hdpa (cgr_2128) was shown to encode a haloacid dehalogenase superfamily member that catalyzes dephosphorylation of dihydroxyacetone phosphate to produce dha. inactivation of hdpa led to a drastic decrease in dha production from each of glucose, fructose, and sucrose, indicating that hdpa is the main enzyme ...201223108048
high-resolution detection of dna binding sites of the global transcriptional regulator glxr in corynebacterium glutamicum.the transcriptional regulator glxr has been characterized as a global hub within the gene-regulatory network of corynebacterium glutamicum. chromatin immunoprecipitation with a specific anti-glxr antibody and subsequent high-throughput sequencing (chip-seq) was applied to c. glutamicum to get new in vivo insights into the gene composition of the glxr regulon. in a comparative approach, c. glutamicum cells were grown with either glucose or acetate as the sole carbon source prior to immunoprecipit ...201323103979
metabolic engineering of the purine biosynthetic pathway in corynebacterium glutamicum results in increased intracellular pool sizes of imp and hypoxanthine.purine nucleotides exhibit various functions in cellular metabolism. besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. further, imp and gmp represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. therefore, this work aimed towards the accumulation of imp applying targeted genetic engineering of corynebacterium glutamicum.201223092390
corynebacterium glutamicum csor acts as a transcriptional repressor of two copper/zinc-inducible p(1b)-type atpase operons.the mechanism of regulation of the expression of copa and copb, encoding putative copper-translocating p(1b)-type atpases in corynebacterium glutamicum, was investigated. the levels of copa and copb mrnas were upregulated in response to excess copper as well as excess zinc. disruption of csor, encoding a transcriptional regulator, resulted in constitutive expression of copa and copb. the csor protein bound to the promoter regions of the copa-csor and the cgr_0124-copb-cgr_0126 operon. in vitro d ...201223090582
sugar transport systems in corynebacterium glutamicum: features and applications to strain development.corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (pts) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. this means that worldwide amino acid production using this organism has depended exclusively on the pts. recently, a better understanding not only of pts-mediated sugar uptake but also of global regulation associat ...201223081775
molecular mechanisms and metabolic engineering of glutamate overproduction in corynebacterium glutamicum.glutamate is a commercially important chemical. it is used as a flavor enhancer and is a major raw material for producing industrially useful chemicals. a coryneform bacterium, corynebacterium glutamicum, was isolated in 1956 by japanese researchers as a glutamate-overproducing bacterium and since then, remarkable progress in glutamate production has been made using this microorganism. currently, the global market for glutamate is over 2.5 million tons per year. glutamate overproduction by c. gl ...201223080255
analysis of corynebacterium glutamicum promoters and their applications.promoters are dna sequences which function as regulatory signals of transcription initiation catalyzed by rna polymerase. since promoters substantially influence levels of gene expression, they have become powerful tools in metabolic engineering. methods for their localization used in corynebacterium glutamicum and techniques for the analysis of their function are described in this review. c. glutamicum promoters can be classified according to the respective σ factors which direct rna polymerase ...201223080252
corynebacterium glutamicum zur acts as a zinc-sensing transcriptional repressor of both zinc-inducible and zinc-repressible genes involved in zinc homeostasis.zur is a zinc-dependent transcriptional repressor of zinc uptake systems in bacteria. in the present study, we examined the role of corynebacterium glutamicum zur in the zinc-inducible expression of two genes: one encoding a cation diffusion facilitator (zrf) and the other a metal-translocating p-type atpase (zra). both genes were shown to be involved in zinc resistance. disruption of the zur gene encoding zur resulted in constitutive expression of zrf and zra mrnas. an electrophoretic mobility ...201223061624
3' untranslated region-dependent degradation of the acea mrna, encoding the glyoxylate cycle enzyme isocitrate lyase, by rnase e/g in corynebacterium glutamicum.we previously reported that the corynebacterium glutamicum rnase e/g encoded by the rneg gene (ncgl2281) is required for the 5' maturation of 5s rrna. in the search for the intracellular target rnas of rnase e/g other than the 5s rrna precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneg knockout mutant cells grown on sodium acetate as the sole carbon source. rifampin chase experiments showed that the half-life of the acea mrna was about ...201223042181
the two-component system chrsa is crucial for haem tolerance and interferes with hrrsa in haem-dependent gene regulation in corynebacterium glutamicum.we recently showed that the two-component system (tcs) hrrsa plays a central role in the control of haem homeostasis in the gram-positive soil bacterium corynebacterium glutamicum. here, we characterized the function of another tcs of this organism, chrsa, which exhibits significant sequence similarity to hrrsa, and provide evidence for cross-regulation of the two systems. in this study, chrsa was shown to be crucial for haem resistance of c. glutamicum by activation of the putative haem-detoxif ...201223038807
the ppm operon is essential for acylation and glycosylation of lipoproteins in corynebacterium glutamicum.due to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (lgt), cleaved off their signal peptides by lipoprotein signal peptidase (lsp) and, in gram-negative bacteria, further triacylated by lipoprotein n-acyl transferase (lnt). the existence of an active apolipoprotein n-acyltransfe ...201223029442
coordinated regulation of gnd, which encodes 6-phosphogluconate dehydrogenase, by the two transcriptional regulators gntr1 and rama in corynebacterium glutamicum.the transcriptional regulation of corynebacterium glutamicum gnd, encoding 6-phosphogluconate dehydrogenase, was investigated. two transcriptional regulators, gntr1 and rama, were isolated by affinity purification using gnd promoter dna. gntr1 was previously identified as a repressor of gluconate utilization genes, including gnd. involvement of rama in gnd expression had not been investigated to date. the level of gnd mrna was barely affected by the single deletion of rama. however, gnd expressi ...201223024346
implication of gluconate kinase activity in l-ornithine biosynthesis in corynebacterium glutamicum.with the purpose of generating a microbial strain for l-ornithine production in corynebacterium glutamicum, genes involved in the central carbon metabolism were inactivated so as to modulate the intracellular level of nadph, and to evaluate their effects on l-ornithine production in c. glutamicum. upon inactivation of the 6-phosphoglucoisomerase gene (pgi) in a c. glutamicum strain, the concomitant increase in intracellular nadph concentrations from 2.55 to 5.75 mmol g⁻¹ (dry cell weight) was ac ...201222987028
a propionate-inducible expression system based on the corynebacterium glutamicum prpd2 promoter and prpr activator and its application for the redirection of amino acid biosynthesis pathways.a novel expression system for corynebacterium glutamicum, based on the transcriptional activator of propionate metabolism genes prpr and its target promoter/operator sequence, was developed and tested. the activator prpr is co-activated by propionate added to the growth medium. in a minimal medium a propionate concentration of only 1 mg l⁻¹ was found to be sufficient for full induction (up to 120-fold) of its native target, the propionate metabolism operon prpdbc2. then, an artificial transcript ...201322982516
single-step production of polyhydroxybutyrate from starch by using α-amylase cell-surface displaying system of corynebacterium glutamicum.direct polyhydroxybutyrate (phb) production from starch was for the first time achieved using engineered corynebacterium glutamicum expressing phb biosynthetic genes and displaying α-amylase on its cell surface. the engineered strain accumulated 6.4 wt% phb from starch which was higher than that obtained from glucose (4.9 wt%).201322959444
transcriptional regulation of the operon encoding stress-responsive ecf sigma factor sigh and its anti-sigma factor rsha, and control of its regulatory network in corynebacterium glutamicum.the expression of genes in corynebacterium glutamicum, a gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of rna polymerase, including the stress-responsive ecf-sigma factor sigh. the sigh gene is located in a gene cluster together with the rsha gene, putatively encoding an anti-sigma factor. the aim of this study was to analyze the transcriptional regulation of the sigh and rsha gene cluster and the ef ...201222943411
alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter betp.betaine and na(+) symport has been extensively studied in the osmotically regulated transporter betp from corynebacterium glutamicum, a member of the betaine/choline/carnitine transporter family, which shares the conserved leut-like fold of two inverted structural repeats. betp adjusts its transport activity by sensing the cytoplasmic k(+) concentration as a measure for hyperosmotic stress via the osmosensing carboxy-terminal domain. betp needs to be in a trimeric state for communication between ...201222940865
sialic acid utilization by the soil bacterium corynebacterium glutamicum.the ability to use the sialic acid, n-acetylneuraminic acid, neu5ac, as a nutrient has been characterized in a number of bacteria, most of which are human pathogens that encounter this molecule because of its presence on mucosal surfaces. the soil bacterium corynebacterium glutamicum also has a full complement of genes for sialic acid catabolism, and we demonstrate that it can use neu5ac as a sole source of carbon and energy and isolate mutants with a much reduced growth lag on neu5ac. disruptio ...201222924979
production of l-lysine on different silage juices using genetically engineered corynebacterium glutamicum.corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. the resulting strain c. glutamicum lysc(fbr)dld(psod)pyc(psod)male(psod)fbp(psod)gapx(psod) was based on earlier work (neuner and heinzle, 2011). that mutant carries a point mutation in the aspartokinase (lysc) regulatory subunit gene as well as overexpression of d-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and ...201322898177
beyond growth rate 0.6: corynebacterium glutamicum cultivated in highly diluted environments.fast growth of industrial microorganisms, such as corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. recently, it has been shown that c. glutamicum when grown in a novel picoliter bioreactor (plbr) exhibits a 50% higher growth rate compared to a 1 l batch cultivation [grünberger et al. (2012) lab chip]. we here compare growth of c. glutamicum with glucose as substrate at different scales covering batch cultivations in the ...201322890752
glucosamine as carbon source for amino acid-producing corynebacterium glutamicum.corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. we isolated a spontaneous mutant (m4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. characterisation of the m4 mutant revealed a significantly increased expression of the nagb gene en ...201322854894
isolated microbial single cells and resulting micropopulations grow faster in controlled environments.singularized cells of pichia pastoris, hansenula polymorpha, and corynebacterium glutamicum displayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. this highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the envirostat 2.0 concept.201222820335
significance of the cgl1427 gene encoding cytidylate kinase in microaerobic growth of corynebacterium glutamicum.the cgl1427 gene was previously found to be relevant to the microaerobic growth of corynebacterium glutamicum (ikeda et al. biosci biotechnol biochem 73:2806-2808, 2009). in the present work, cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (cmp kinases) and on the basis of findings that deletion of cgl1427 results in loss of cmp kinase activity. deletion of the cmk gene significantly ...201322810301
postgenomic approaches to using corynebacteria as biocatalysts.corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a factory of primary and secondary metabolites. the versatile capabilities of this organism have long been implemented at the industrial scale to produce an array of amino acids at high yields and conversion rates, thereby enabling the development of an entire industry. the postgenomic era provides a new technological platform not only to further optimize the intrinsic attributes of c. glutamicum whole cells as biocatalys ...201222803796
corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate.here, we show that corynebacterium glutamicum atcc 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. co(2) measurements during bioreactor cultivation and use of (13)c-labelled formate demonstrated that formate is almost completely oxidized to co(2). the deletion of fdhf (cg0618), annotated as formate dehydrogenase (fdh) and located in a cluster of genes conserved in the family corynebacteriaceae, prevented formate utilization. similarly, deletion of fdhd ...201222767548
effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain corynebacterium glutamicum yilw.previous studies have shown that the deletion of brnq from the corynebacterium glutamicum chromosome results in a significant reduction in l-isoleucine uptake rates, while overexpression of brnfe leads to enhanced l-isoleucine export rates. given that net excretion rates would be an important factor for high titers of l-isoleucine accumulation, we have tested the notion that decreased l-isoleucine uptake combined with increased l-isoleucine excretion will further improve high-yield strains that ...201222733295
overexpression of nad kinases improves the l-isoleucine biosynthesis in corynebacterium glutamicum ssp. lactofermentum.nadph is the key cofactor in l-isoleucine (ile) biosynthetic pathway. to increase the ile biosynthesis in corynebacterium glutamicum ssp. lactofermentum jhi3-156, nadph supply needs to be enhanced. here nad kinase, the key enzyme for the de novo biosynthesis of nadp(+) and nadph, were cloned and expressed in jhi3-156, and their influences on ile production were analysed. meanwhile, enzyme properties of nad kinase from jhi3-156 (cljppnk) were compared with that from c. glutamicum ssp. lactofermen ...201222664190
a gain-of-function mutation in gating of corynebacterium glutamicum ncgl1221 causes constitutive glutamate secretion.the a-to-v mutation at position 111 (a111v) in the mechanosensitive channel ncgl1221 (msccg) causes constitutive glutamate secretion in corynebacterium glutamicum. patch clamp experiments revealed that ncgl1221 (a111v) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of ncgl1221 leads to the oversecretion of glutamate.201222610427
degradation and assimilation of aromatic compounds by corynebacterium glutamicum: another potential for applications for this bacterium?with the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by corynebacterium glutamicum has been emerging. this review summarizes recent findings on degradation of aromatic compounds by c. glutamicum. among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in c. glutamicum. other important knowledge derived from c. glutamicum would be the discovery of linkages among aromati ...201222588501
a tetracycline inducible expression vector for corynebacterium glutamicum allowing tightly regulable gene expression.here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. using the green fluorescent protein (gfp) as a reporter protein we show that this vector, named pclton1, is characterized by tight repression under non-induced conditions as compared to a conventional iptg inducible expression vector, and that it allows gradua ...201222587824
the development and application of a single-cell biosensor for the detection of l-methionine and branched-chain amino acids.the detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. we have developed a biosensor based on the transcriptional regulator lrp that detects intracellular l-methionine and branched-chain amino acids in corynebacterium glutamicum. in assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. in increasing order, the affinity of lrp for the amino acids is l-valine, l ...201222583745
negative role of wbla in response to oxidative stress in streptomyces coelicolor.in this study, we analyzed the oxidative stress response of wbla (whib-like gene a, sco3579), which was previously shown to be a global antibiotic down-regulator in streptomyces coelicolor. ever since a wbla ortholog named whca in corynebacterium glutamicum was found to play a negative role in the oxidative stress response, s. coelicolor wbla has been proposed to have a similar effect. a wbla-deletion mutant exhibited a less sensitive response to oxidative stress induced by diamide present in so ...201222573149
metabolic engineering and flux analysis of corynebacterium glutamicum for l-serine production.l-serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of l-serine from glucose. in this study, corynebacterium glutamicum atcc 13032 was engineered de novo by blocking and attenuating the conversion of l-serine to pyruvate and glycine, releasing the feedback inhibition by l-serine to 3-phosphoglycerate dehydrogenase (pgdh), in combination with the co-expression of 3-phosphoglycerate kinase (pgk) and feedback-resistant pgdh ...201222566084
(l)-valine production with minimization of by-products' synthesis in corynebacterium glutamicum and brevibacterium flavum.corynebacterium glutamicum atcc13032 and brevibacterium flavum jv16 were engineered for l-valine production by over-expressing ilvebn ( r ) c genes at 31 °c in 72 h fermentation. different strategies were carried out to reduce the by-products' accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. the native promoter of ilva of c. glutamicum was replaced with a weak promoter mpilva (p-ilvam1cg) to reduce the biosynthetic rate of l-isol ...201222552525
purification and structure analysis of mycolic acids in corynebacterium glutamicum.corynebacterium glutamicum is widely used for producing amino acids. mycolic acids, the major components in the cell wall of c. glutamicum might be closely related to the secretion of amino acids. in this study, mycolic acids were extracted from 5 strains of c. glutamicum, including atcc 13032, atcc 13869, atcc 14067, l-isoleucine producing strain iwj-1, and l-valine producing strain vwj-1. structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionizatio ...201222538651
protein turnover quantification in a multilabeling approach: from data calculation to evaluation.liquid chromatography coupled to tandem mass spectrometry in combination with stable-isotope labeling is an established and widely spread method to measure gene expression on the protein level. however, it is often not considered that two opposing processes are responsible for the amount of a protein in a cell--the synthesis as well as the degradation. with this work, we provide an integrative, high-throughput method--from the experimental setup to the bioinformatics analysis--to measure synthes ...201222493176
quantification of proteome dynamics in corynebacterium glutamicum by (15)n-labeling and selected reaction monitoring.selected reaction monitoring allows quantitative measurements of proteins over several orders of magnitude in complex biological samples. here we present a targeted approach for quantification of 19 enzymes from corynebacterium glutamicum applying isotope dilution mass spectrometry coupled to high performance liquid chromatography (idms-lc-ms/ms). investigations of protein dynamics upon growth on acetate and glucose as sole carbon source shows highly stable peptide amounts for enzymes of the cen ...201222476105
global proteome survey of protocatechuate- and glucose-grown corynebacterium glutamicum reveals multiple physiological differences.corynebacterium glutamicum can utilize various monocyclic aromatic carbon sources, including protocatechuate, which is catabolized via the β-ketoadipate pathway. in order to obtain a global survey of occurring physiological adaptations on the proteome level, cytoplasmic and membrane fraction from cells grown on protocatechuate or glucose as sole carbon and energy source were compared. shotgun proteomics and relative protein quantification with metabolic isotope labeling and spectral counting wer ...201222450470
the role of corynebacterium glutamicum spia gene in whca-mediated oxidative stress gene regulation.the corynebacterium glutamicum whca protein, which inhibits the expression of oxidative stress response genes, is known to interact with the spia protein. in this study, we constructed and analyzed spia mutant cells with the goal of better understanding the function of the spia gene. a c. glutamicum strain overexpressing the spia gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. expression of the spia and whca genes was repressed by oxidant diamide, indi ...201222443283
improved l-lysine production with corynebacterium glutamicum and systemic insight into citrate synthase flux and activity.we here developed a series of corynebacterium glutamicum strains with gradual decreased specific citrate synthase (cs) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on l-lysine formation and growth. we achieved an intended gradual l-lysine yield increase and recognized and overcame further new limitations in the l-lysine biosynthesis pathway to result in a strain with the highest yield repor ...201222392073
toward homosuccinate fermentation: metabolic engineering of corynebacterium glutamicum for anaerobic production of succinate from glucose and formate.previous studies have demonstrated the capability of corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. in this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. to eliminate acetate formation, a derivative of the type strain atcc 13032 (strain bol-1), which lacked all known pathways for acetate and lactate synthesis (δcat δpqo δpta- ...201222389371
gntr-type transcriptional regulator pckr negatively regulates the expression of phosphoenolpyruvate carboxykinase in corynebacterium glutamicum.the pck (cg3169) gene of corynebacterium glutamicum encodes a phosphoenolpyruvate carboxykinase (pepck). here, a candidate transcriptional regulator that binds to the promoter region of pck was detected using a dna affinity purification approach. an isolated protein was identified to be pckr (cg0196), a gntr family transcriptional regulator which consists of 253 amino acids with a mass of 27 kda as measured by peptide mass fingerprinting. the results of electrophoretic mobility shift assays veri ...201222366416
disruption of genes for the enhanced biosynthesis of α-ketoglutarate in corynebacterium glutamicum.the development of microbial strains for the enhanced production of α-ketoglutarate (α-kg) was investigated using a strain of corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-kg biosynthetic pathway. the pathways competing with the biosynthesis of α-kg were blocked by knocking out acea (encoding isocitrate lyase, icl), gdh (encoding glutamate dehydrogenase, l-gludh), and gltb (encoding glutamate synthase or glutamate-2-oxoglutarate aminotra ...201222356563
ndnr is an nad-responsive transcriptional repressor of the ndnr operon involved in nad de novo biosynthesis in corynebacterium glutamicum.the corynebacterium glutamicum ndnr gene, which is chromosomally located in a gene cluster involved in nad de novo biosynthesis, negatively regulates expression of the cluster genes, i.e. nada, nadc, nads and ndnr itself. although ndnr encodes a member of the recently identified nrtr family of transcriptional regulators, whether or not the ndnr protein directly regulates these nad biosynthesis genes remains to be verified. here, two ndnr binding sites in the promoter region of the ndnr-nada-nadc ...201222301909
characterization of the biotin uptake system encoded by the biotin-inducible bioymn operon of corynebacterium glutamicum.the amino acid-producing gram-positive corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-coa is still functional. it possesses accbc, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. comparative genome analyses suggested that the putative transport system bioymn encoded by cg2147, cg2148 and cg2149 might be involved in bio ...201222243621
the gene encoding the alternative thymidylate synthase thyx is regulated by sigma factor sigb in corynebacterium glutamicum atcc 13032.both thya and thyx proteins catalyze the transfer of the methyl group from methylenetetrahydrofolate (ch(2) h(4) -folate) to dump, forming dtmp. to estimate the relative steady state expression levels of thya and thyx, western blot analysis was performed using thya or thyx antiserum on total protein from the wild-type, δthyx, and thyx-complemented strains of corynebacterium glutamicum. the level of thya decreased gradually during the stationary growth phase but that of thyx was maintained steadi ...201222224900
error propagation analysis for quantitative intracellular metabolomics.model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. for quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. the total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. here, we present a framework for the quantification of intr ...201224957773
glutamate overproduction in corynebacterium glutamicum triggered by a decrease in the level of a complex comprising dtsr and a biotin-containing subunit.glutamate overproduction in corynebacterium glutamicum is induced by tween 40, biotin-limitation, or sublethal amounts of penicillin. disruption of the dtsr gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. we report here that overexpression of dtsr inhibits the induction of glutamate overproduction. in contrast, the level of dtsr in the wild type strain was found to decrease in the pr ...199927380236
nadph oxidase system as a superoxide-generating cyanide-resistant pathway in the respiratory chain of corynebacterium glutamicum.the respiratory chain of corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. the membranes of c. glutamicum had nadh, succinate, lactate, and nadph oxidase activities, and menaquinone, and cytochromes a 598, b 562(558), and c 550 as respiratory components. the nadh, succinate, lactate, and nadph oxidase systems, all of which were more cyanide-resistant than n,n,n',n'-tetramethyl-p-phenylene diamine oxidase activity (cytoch ...199827385451
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