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genr, an iclr-type regulator, activates and represses the transcription of gen genes involved in 3-hydroxybenzoate and gentisate catabolism in corynebacterium glutamicum.the genes required for 3-hydroxybenzoate and gentisate catabolism in corynebacterium glutamicum are closely clustered in three operons. genr, an iclr-type regulator, can activate the transcription of genkh and gendfm operons in response to 3-hydroxybenzoate and gentisate, and it can repress its own expression. footprinting analyses demonstrated that genr bound to four sites with different affinities. two genr-binding sites (dfmn01 and dfmn02) were found to be located between positions --41 and - ...201323354754
direct production of organic acids from starch by cell surface-engineered corynebacterium glutamicum in anaerobic conditions.we produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of corynebacterium glutamicum displaying α-amylase (amya) from streptococcus bovis 148 on the cell surface. notably, reactions performed under anaerobic conditions at 35 and 40°c, which are higher than the optimal growth temperature of 30°c, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate co ...201324342107
whole cell biotransformation for reductive amination reactions.whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. recently, we established an escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. transformation of 2-keto-3-methylvalerate to l-isoleucine by e. coli cells was ...201324406456
characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph bacillus methanolicus.the genome of the facultative ribulose monophosphate (rump) cycle methylotroph bacillus methanolicus encodes two bisphosphatases (glpx), one on the chromosome (glpx(c)) and one on plasmid pbm19 (glpx(p)), which is required for methylotrophy. both enzymes were purified from recombinant escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (fbpases). the fbpase-negative corynebacterium glutamicum δfbp mutant could be phenotypically complemented with glpx(c) and glpx(p) from ...201324013630
evaluation of the food grade expression systems nice and psip for the production of 2,5-diketo-d-gluconic acid reductase from corynebacterium glutamicum.2,5-diketo-d-gluconic acid reductase (2,5-dkg reductase) catalyses the reduction of 2,5-diketo-d-gluconic acid (2,5-dkg) to 2-keto-l-gulonic acid (2-klg), a direct precursor (lactone) of l-ascorbic acid (vitamin c). this reaction is an essential step in the biocatalytic production of the food supplement vitamin c from d-glucose or d-gluconic acid. as 2,5-dkg reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-dkg reductase production that also ...201323356419
heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in escherichia coli.a limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host escherichia coli. in order to systematically investigate the functionality of carotenoid pathway enzymes in e. coli, the pathway genes of carotenogenic microorganisms (brevibacterium linens, corynebacterium glutamicum, rhodobacter sphaeroides, rhodobacter capsulatus, rhodopirellula baltica, and pantoea ananatis) were modified to form synthetic e ...201323144136
systems metabolic engineering of corynebacterium glutamicum for production of the chemical chaperone ectoine.the stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. these rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. there is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally ...201324228689
characterization and molecular mechanism of arop as an aromatic amino acid and histidine transporter in corynebacterium glutamicum.corynebacterium glutamicum is equipped with abundant membrane transporters to adapt to a changing environment. many amino acid transporters have been identified in c. glutamicum, but histidine uptake has not been investigated in detail. here, we identified the aromatic amino acid transporter encoded by arop as a histidine transporter in c. glutamicum by a combination of the growth and histidine uptake features. characterization of histidine uptake showed that arop has a moderate affinity for his ...201324056108
comprehensive discovery and characterization of small rnas in corynebacterium glutamicum atcc 13032.recent discoveries on bacterial transcriptomes gave evidence that small rnas (srnas) have important regulatory roles in prokaryotic cells. modern high-throughput sequencing approaches (rna-seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. whole transcriptome data obtained by rna-seq can be used to detect and characterize all transcript species, including small rnas. here, we describe an rna-seq approach for comprehensive dete ...201324138339
impact of carbon source and variable nitrogen conditions on bacterial biosynthesis of polyhydroxyalkanoates: evidence of an atypical metabolism in bacillus megaterium dsm 509.twenty bacterial strains were examined on their ability to produce polyhydroxyalkanoates (pha) from different carbon sources under rich and depleted nitrogen conditions. preliminary experiments with glucose as sole carbon source allowed to select pha producing bacteria using ftir spectroscopy. they were further tested with eight additional carbon substrates including organic, fatty acids or sugars. pha content and monomer composition of four chosen strains (pseudomonas putida mt-2, bacillus mega ...201323548274
an assay for functional xylose transporters in saccharomyces cerevisiae.it has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. in this study, an assay to screen xylose transporters was established in the saccharomyces cerevisiae strain, which expresses the xylosidase gene of bacillus pumilus intracellularly. the absorbed xylose analog p-nitrophenyl-β-d-xylopyranoside (pnpx) rapidly hydrolyzed to p-nitrophenol (pnp), which displayed a yellow tint when exposed to xylosidase in vivo. the ...201323928049
corynebacterium jeikeium jk0268 constitutes for the 40 amino acid long poracj, which forms a homooligomeric and anion-selective cell wall channel.corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. c. jeikeium k411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. the channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. a gene coding for a 4 ...201324116064
increasing the antibacterial activity of gentamicin in combination with extracted polyphosphate from bacillus megaterium.the aim of this research was production of polyphosphate (poly p) and study on its antibacterial effects.201323332009
the lipid ii flippase roda determines morphology and growth in corynebacterium glutamicum.lipid ii flippases play an essential role in cell growth and the maintenance of cell shape in many rod-shaped bacteria. the putative lipid ii flippase roda is an integral membrane protein and member of the seds (shape, elongation, division and sporulation) protein family. in contrast to its homologues in escherichia coli and bacillus subtilis little is known about the role of roda in actinobacteria. in this study, we describe the localization and function of roda in corynebacterium glutamicum, a ...201324118443
metabolic engineering of corynebacterium glutamicum for increasing the production of l-ornithine by increasing nadph availability.the experiments presented here were based on the conclusions of our previous proteomic analysis. increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of spee, which encodes spermidine synthase, improved l-ornithine production by corynebacterium glutamicum. production of l-ornithine requires 2 moles of nadph per mole of l-ornithine. thus, the effect of nadph availability on l-ornithine ...201323836141
development of biotin-prototrophic and -hyperauxotrophic corynebacterium glutamicum strains.to develop the infrastructure for biotin production through naturally biotin-auxotrophic corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. to confer biotin prototrophy on the organism, the cotranscribed biobf genes of escherichia coli were introduced into the c. glutamicum genome, which originally lacked the biof gene. the resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a ...201323709504
engineering of acetate recycling and citrate synthase to improve aerobic succinate production in corynebacterium glutamicum.corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. to address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing c. glutamicum. the acetyl-coa synthetase from bacillus sub ...201323593275
picoliter ndep traps enable time-resolved contactless single bacterial cell analysis in controlled microenvironments.we present a lab-on-a-chip device, the envirostat 2.0, which allows for the first time contactless cultivation of a single bacterial cell by negative dielectrophoresis (ndep) in a precisely controllable microenvironment. stable trapping in perfusing growth medium was achieved by a miniaturization of octupole electrode geometries, matching the dimensions of bacteria. temperature sensitive fluorescent measurements showed that these reductions of microelectrode distances led to reduced joule heatin ...201323223864
formaldehyde degradation in corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase.corynebacterium glutamicum, a gram-positive soil bacterium belonging to the actinomycetes, is able to degrade formaldehyde but the enzyme(s) involved in this detoxification process were not known. acetaldehyde dehydrogenase ald, which is essential for ethanol utilization, and fadh, characterized here as nad-linked mycothiol-dependent formaldehyde dehydrogenase, were shown to be responsible for formaldehyde oxidation since a mutant lacking ald and fadh could not oxidize formaldehyde resulting in ...201324065717
electrophysiological characterization of the mechanosensitive channel msccg in corynebacterium glutamicum.corynebacterium glutamicum msccg, also referred to as ncgl1221, exports glutamate when biotin is limited in the culture medium. msccg is a homolog of escherichia coli mscs, which serves as an osmotic safety valve in e. coli cells. patch-clamp experiments using heterogeneously expressed msccg have shown that msccg is a mechanosensitive channel gated by membrane stretch. although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological ch ...201324047987
a wbla-binding protein, spia, involved in streptomyces oxidative stress response.the streptomyces coelicolor wbla gene is known to play a negative role in both antibiotic biosynthesis and the expression of genes responding to oxidative stress. recently, whca, a wbla ortholog protein, was confirmed to interact with dioxygenase-encoding spia (stress protein interacting with whca) in corynebacterium glutamicum. we describe here the identification of a spia ortholog sco2553 protein (spiasc) that interacts with wbla in s. coelicolor. using heterologous expression in e. coli and i ...201323867703
construction and application of an efficient multiple-gene-deletion system in corynebacterium glutamicum.gene deletion techniques are important for modifying corynebacterium glutamicum, the bacterium for industrial production of amino acids. in this study, a novel multiple-gene-deletion system for c. glutamicum was developed. the system is composed of three plasmids pdtw109, pdtw201 and pdtw202. pdtw109 is a temperature-sensitive vector which harbors a cat gene under the tacm promoter, a cre gene under the tac promoter, an origin orie for replicating in escherichia coli, and another origin rep(ts) ...201323856168
enhancing (l)-isoleucine production by thrabc overexpression combined with alat deletion in corynebacterium glutamicum.l-isoleucine is synthesized from 2-ketobutyrate and pyruvate in corynebacterium glutamicum, and the supplies of these two precursors are important for l-isoleucine synthesis. c. glutamicum yilwδalat with alat gene deletion (encoding alanine aminotransferase, a principal enzyme for l-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrabc genes from escherichia coli (encoding bifunctional aspartate kinase i-homoserine dehydrogenase i, homoserine kinase, ...201323813403
the methylotrophic bacillus methanolicus mga3 possesses two distinct fructose 1,6-bisphosphate aldolases.the thermotolerant gram-positive methylotroph bacillus methanolicus is able to grow with methanol, glucose or mannitol as a sole carbon and energy source. fructose 1,6-bisphosphate aldolase (fba), a key enzyme of glycolysis and gluconeogenesis, is encoded in the genome of b. methanolicus by two putative fba genes, the chromosomally located fba(c) and fba(p) on the naturally occurring plasmid pbm19. their amino acid sequences share 75 % identity and suggest a classification as class ii aldolases. ...201323760818
strain optimization for efficient isobutanol production using corynebacterium glutamicum under oxygen deprivation.microbial production of isobutanol is made difficult by the chemical's high cell toxicity. corynebacterium glutamicum, inherently one of the more isobutanol-tolerant industrial microorganisms, exhibits unprecedented productivity under oxygen deprivation, potentially allowing for high productivity of such toxic chemicals as isobutanol. here, we show that development of c. glutamicum strains proficient in isobutanol production depends not only on modulating the activity of 2-keto acid decarboxylas ...201323737329
production of non-proteinogenic amino acids from α-keto acid precursors with recombinant corynebacterium glutamicum.in the present work, corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. the novel bio-catalytic activity of c. glutamicum was obtained by heterologous expression of the branched chain aminotransferase ilve from escherichia coli. upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (hoa ...201323737264
l-glutamate secretion by the n-terminal domain of the corynebacterium glutamicum ncgl1221 mechanosensitive channel.the corynebacterium glutamicum ncgl1221 mechanosensitive channel mediates l-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. the ncgl1221 protein has an n-terminal domain (1-286 a.a.) homologous to the escherichia coli mscs and a long c-terminal domain (287-533 a.a.) of unknown function. in order to investigate the role of the c-terminal domain in l-glutamate secretion, we constructed a series of c-terminally truncated muta ...201323649271
development of indole-3-acetic acid-producing escherichia coli by functional expression of ipdc, aspc, and iad1.biosynthesis of indole-3-acetic acid (iaa) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspc, indole-3-pyruvic acid decarboxylase encoded by ipdc, and indole-3-acetic acid dehydrogenase encoded by iad1. the ipdc from enterobacter cloacae atcc 13047, aspc from escherichia coli, and iad1 from ustilago maydis were cloned and expressed under the control of the tac and sod promoters in e. coli. according to sds-page and enzyme activity, ipdc and i ...201324043123
ipsa, a novel laci-type regulator, is required for inositol-derived lipid formation in corynebacteria and mycobacteria.the development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. the enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown.201324377418
identification of a mycoloyl transferase selectively involved in o-acylation of polypeptides in corynebacteriales.we have previously described the posttranslational modification of pore-forming small proteins of corynebacterium by mycolic acid, a very-long-chain α-alkyl and β-hydroxy fatty acid. using a combination of chemical analyses and mass spectrometry, we identified the mycoloyl transferase (myt) that catalyzes the transfer of the fatty acid residue to yield o-acylated polypeptides. inactivation of corynomycoloyl transferase c (cg0413 [corynebacterium glutamicum mytc {cgmytc}]), one of the six cgmyt g ...201323852866
identification and characterization of the channel-forming protein in the cell wall of corynebacterium amycolatum.the mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. corynebacterium amycolatum, a pathogenic corynebacterium species, belongs to the corynebacteriaceae family but it lacks corynomycolic acids in i ...201323811360
[expression optimization and characterization of tenebrio molitor antimicrobiol peptides tmamp1m in escherichia coli].to improve the expression level of tmamp1m gene from tenebrio molitor in escherichia coli, we studied the effects of expression level and activity of the fusion protein his-tmamp1m by conditions, such as culture temperature, inducing time and the final concentration of inductor isopropyl beta-d-thiogalactopyranoside (iptg). we analyzed the optimum expression conditions by tricine-sds-page electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. th ...201324063242
expression of nad(h) kinase and glucose-6-phosphate dehydrogenase improve nadph supply and l-isoleucine biosynthesis in corynebacterium glutamicum ssp. lactofermentum.corynebacterium glutamicum is the workhorse for the production of amino acids, including l-isoleucine (ile). during ile biosynthesis, nadph is required as a crucial cofactor. in this study, four nadph-supplying strategies based on nad kinase, nadh kinase, glucose-6-phosphate dehydrogenase, and nad kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on ile biosynthesis were examined. ppnk is a nad kinase of c. glutamicum ssp. lactofermentum jhi3-156 that pre ...201323868449
recent advancements in various steps of ethanol, butanol, and isobutanol productions from woody materials.in this review, the recent advancements and technical challenges associated with the production of ethanol, butanol, and isobutanol via bioconversion routes from celluloses of woody materials are reviewed. physicochemical processes, e.g. steam explosion, seem to be the most viable process for pretreating woody materials. although enzymatic hydrolysis is selective, it is rather a slow process. acid hydrolysis is a relatively fast process with a high yield, but it produces inhibitory compounds of ...201323297056
sample preparation, crystallization and structure solution of hisc from mycobacterium tuberculosis.histidinolphosphate aminotransferase (hisc; rv1600) from mycobacterium tuberculosis was overexpressed in m. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. diffraction-quality crystals suitable for x-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. the crystals belonged to the hexagonal space group p3221, with an unusual high sol ...201323545656
interaction between dahp synthase and chorismate mutase endows new regulation on dahp synthase activity in corynebacterium glutamicum.previous research on corynebacterium glutamicum revealed that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dscg, formerly ds2098) interacts with chorismate mutase (cmcg, formerly cm0819). in this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. our results show that the interaction imparted a new mechanism for regulation of dahp activity: in the absence of cmcg, dscg activity was not regulated by prephenate, whereas in the presence of cm ...201323467831
nrdh-redoxin of mycobacterium tuberculosis and corynebacterium glutamicum dimerizes at high protein concentration and exclusively receives electrons from thioredoxin reductase.nrdh-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. they function as the electron donor for class ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. we solved the x-ray structure of oxidized nrdh-redoxin from corynebacterium glutamicum (cg) at 1.5 å resolution. based on this monomeric structure, we built a homology model of nrdh-redoxin from mycobacterium tuberc ...201323362277
synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.glucuronosyl diacylglycerides (glcagroac2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the corynebacteria. here we describe the complete synthesis of distinct acyl-isoforms of glcagroac2 bearing both acylation patterns of (r)-tuberculostearic acid (c19:0) and palmitic acid (c16:0) and their mass spectral characterization. collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowin ...201323343519
reactions upstream of glycerate-1,3-bisphosphate drive corynebacterium glutamicum (d)-lactate productivity under oxygen deprivation.we previously demonstrated the simplicity of oxygen-deprived corynebacterium glutamicum to produce d-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous d-ldha gene from lactobacillus delbrueckii. here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production in c. glutamicum. we consequently show that while the reactions catalyzed by glucokin ...201323712891
gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by tlr2.innate immune recognition is the first line of host defense against invading microorganisms. it is a based on the detection, by pattern recognition receptors (prrs), of invariant molecular signatures that are unique to microorganisms. tlr2 is a prr that plays a major role in the detection of gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. these microbe-associated molec ...201324278450
engineering of corynebacterium glutamicum for high-yield l-valine production under oxygen deprivation conditions.we previously demonstrated efficient l-valine production by metabolically engineered corynebacterium glutamicum under oxygen deprivation. to achieve the high productivity, a nadh/nadph cofactor imbalance during the synthesis of l-valine was overcome by engineering nad-preferring mutant acetohydroxy acid isomeroreductase (ahair) and using nad-specific leucine dehydrogenase from lysinibacillus sphaericus. lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene l ...201323241971
enhancement of γ-aminobutyric acid production in recombinant corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from lactobacillus brevis.γ-aminobutyric acid (gaba), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. to establish an effective single-step production system for gaba, a recombinant corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (gad) genes (gadb1 and gadb2) derived from lactobacillus brevis lb85 was constructed. compared with the gaba production of the gadb1 or gadb2 single-expressing strains, gaba production by the gadb1-gadb2 co-expressing st ...201323928903
glycerol as a substrate for aerobic succinate production in minimal medium with corynebacterium glutamicum.corynebacterium glutamicum, an established microbial cell factory for the biotechnological production of amino acids, was recently genetically engineered for aerobic succinate production from glucose in minimal medium. in this work, the corresponding strains were transformed with plasmid pvwex1-glpfkd coding for glycerol utilization genes from escherichia coli. this plasmid had previously been shown to allow growth of c. glutamicum with glycerol as sole carbon source. the resulting strains were ...201322513227
increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-coa in corynebacterium glutamicum.in this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (tca) cycle at the level of succinyl-coa synthetase. the succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-coa to succinate in this aerobic bacterium. the mutant c. glutamicum δsuccd showed a 60% increase in the yield of lys ...201322871505
exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production.dihydrodipicolinate synthase (dhdps, ec 4.2.1.52) catalyzes the first committed reaction of l-lysine biosynthesis in bacteria and plants and is allosterically regulated by l-lysine. in previous studies, dhdpss from different species were proved to have different sensitivity to l-lysine inhibition. in this study, we investigated the key determinants of feedback regulation between two industrially important dhdpss, the l-lysine-sensitive dhdps from escherichia coli and l-lysine-insensitive dhdps f ...201322644522
crude glycerol-based production of amino acids and putrescine by corynebacterium glutamicum.corynebacterium glutamicum possesses genes for glycerol kinase and glycerol-3-phosphate dehydrogenase that were shown to support slow growth with glycerol only when overexpressed from a plasmid. pure glycerol and crude glycerol from biodiesel factories were tested for growth of recombinant strains expressing glpf, glpk and glpd from escherichia coli. some, but not all crude glycerol lots served as good carbon sources. although the inhibitory compound(s) present in these crude glycerol lots remai ...201323562176
enhanced production of l-phenylalanine in corynebacterium glutamicum due to the introduction of escherichia coli wild-type gene aroh.metabolic engineering is a powerful tool which has been widely used for producing valuable products. for improving l-phenylalanine (l-phe) accumulation in corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. the genes involved in the biosynthesis of l-phe were found to be strictly regulated genes by feedback inhibition. as a result, overexpression of the native wild-type genes arof, arog or phea resulted in a slight increase of l-phe. in contra ...201323526182
metabolic engineering of corynebacterium glutamicum to produce gdp-l-fucose from glucose and mannose.wild-type corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (gdp)-l-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. this was done by introducing the gmd and wcag genes of escherichia coli encoding gdp-d-mannose-4,6-dehydratase and gdp-4-keto-6-deoxy-d-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of gdp-l-fuco ...201323404100
glutamate efflux mediated by corynebacterium glutamicum msccg, escherichia coli mscs, and their derivatives.corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, in particular glutamate. the mechanism of glutamate excretion, however, is not yet fully understood. recently, evidence was provided that the ncgl1221 gene product from c. glutamicum atcc 13869, a mscs-type mechanosensitive efflux channel, is responsible for glutamate efflux [1]. the major difference of ncgl1221 and the homologous protein msccg of c. glutamicum atcc 13032 from escherichia coli mscs a ...201323313454
transcriptional control of the f0f1-atp synthase operon of corynebacterium glutamicum: sigmah factor binds to its promoter and regulates its expression at different ph values.corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. its f(0)f(1)-atpase operon (atpbefhagdc) is expressed optimally at ph 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpb gene. expression of this operon is controlled by the sigmah factor. the sigmah gene (sigh) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. a mutant d ...201323298179
metabolic engineering of industrial platform microorganisms for biorefinery applications--optimization of substrate spectrum and process robustness by rational and evolutive strategies.bio-based production promises a sustainable route to myriads of chemicals, materials and fuels. with regard to eco-efficiency, its future success strongly depends on a next level of bio-processes using raw materials beyond glucose. such renewables, i.e., polymers, complex substrate mixtures and diluted waste streams, often cannot be metabolized naturally by the producing organisms. this particularly holds for well-known microorganisms from the traditional sugar-based biotechnology, including esc ...201323260271
[effect of overexpressing isocitrate lyase on succinate production in ldh(-1) corynebacterium glutamicum].corynebacterium glutamicum sa001 is a mutant with lactate dehydrogenase (ldha) deletion. in order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene acea coding isocitrate lyase (icl) from escherichia coli k12 into corynebacterium glutamicum sa001 (sa001/pxmj19-acea). after 12 h aerobic induction by adding 0.8 mmol/l of iptg, the recombinant strain was transferred to anaerobic fermentation for 16 ...201324701837
[cost-effective production of protein by using cellulose-binding domain fusion tag in corynebacterium glutamicum].the cbd gene from trichoderma reesei was cloned into the corynebacterium glutamicum secretion expression vector pxmj19-sp, in which green fluorescent protein was inserted to obtain pxmj19-sp-gfp-cbd. after induced by 0.5 mmol/l iptg, gfp-cbd was expressed in corynebacterium glutamicum at high level of 200 mg/l. the gfp-cbd could be purified to high purity with cellulose column. the results indicated cbd can be successfully used in corynebacterium glutamicum expression system and thus offer an ex ...201324010367
design and testing of a synthetic biology framework for genetic engineering of corynebacterium glutamicum.synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. however, most of synthetic biology tools have been developed for escherichia coli. here we provide a platform for rapid engineering of c. glutamicum, a microorganism of great industrial interest. this bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of ...201223134565
model-based analysis of an adaptive evolution experiment with escherichia coli in a pyruvate limited continuous culture with glycerol.: bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. a block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in e. coli. the resulting mutant strain was able to grow on a medium containing glycerol and l ...201223033959
[construction and structural analysis of integrated cellular network of corynebacterium glutamicum].corynebacterium glutamicum is one of the most important traditional industrial microorganisms and receiving more and more attention towards a novel cellular factory due to the recently rapid development in genomics and genetic operation toolboxes for corynebacterium. however, compared to other model organisms such as escherichia coli, there were few studies on its metabolic regulation, especially a genome-scale integrated cellular network model currently missing for corynebacterium, which hinder ...201222916496
construction of in vitro transcription system for corynebacterium glutamicum and its use in the recognition of promoters of different classes.to facilitate transcription studies in corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. this system consists of c. glutamicum rna polymerase (rnap) core (α2, β, β'), a sigma factor and a promoter-carrying dna template, that is specifically recognized by the rnap holoenzyme formed. the rnap core was purified from the c. glutamicum strain with the modified rpoc ge ...201222885668
a high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.we present a novel method for visualizing intracellular metabolite concentrations within single cells of escherichia coli and corynebacterium glutamicum that expedites the screening process of producers. it is based on transcription factors and we used it to isolate new l-lysine producing mutants of c. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (facs). this high-throughput method fills the gap between existing high-throughput methods for mutant ...201222640862
comparative reaction engineering studies for succinic acid production from sucrose by metabolically engineered escherichia coli in fed-batch-operated stirred tank bioreactors.this study presents a comparative reaction engineering analysis of metabolically engineered sucrose-utilizing escherichia coli derived from e. coli k12 mg1655 for the anaerobic production of succinic acid. production capacities of 16 different recombinant strains were evaluated in 48 parallel fed-batch-operated milliliter-scale stirred tank bioreactors (10 ml) with continuous co₂ sparging. the effects of recombinant sucrose-utilization systems (csc-operon or scr-operon), enhancements of anaplero ...201222588847
[a novel bacterial cell-surface display system based on ncgl1221 from corynebacterium glutamicum].to develop a novel escherichia coli cell surface display system by using c-terminally truncated ncgl1221 as the anchoring protein, which greatly enriched or optimized the bacterial displayed systems.201222586995
characterization of the rna polymerase α subunit operon from corynebacterium ammoniagenes.the rpoa gene, which encodes the α subunit of rna polymerase, and the surrounding regions were cloned from corynebacterium ammoniagenes (atcc 6872), a parental strain of an industrial nucleotide producer in korea. this region encodes genes for the following proteins (in order): initiation factor if-1, the ribosomal proteins s13, s11 and s4, the α subunit of rna polymerase and the ribosomal protein l17. transcript mapping via reverse transcription polymerase chain reaction demonstrates that if1, ...201222806862
glutamate is excreted across the cytoplasmic membrane through the ncgl1221 channel of corynebacterium glutamicum by passive diffusion.the ncgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (glu) overproduction by corynebacterium glutamicum, but direct evidence of glu excretion through this channel has not yet been provided. in this study, by electrophysiological methods, we found direct evidence of glu excretion through this channel by passive diffusion. we found that the introduction into phe-producing escherichia coli of mutant ncgl1221 genes that induce glu over ...201222785475
co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in corynebacterium glutamicum.threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of corynebacterium glutamicum, but their activities are usually feedback-inhibited. in this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain c. glutamicum jhi3-156. sequence analysis showed that there was only a single amino acid substitution (phe383val) in the feedback-resistant threonine dehy ...201222771937
robust production of gamma-amino butyric acid using recombinant corynebacterium glutamicum expressing glutamate decarboxylase from escherichia coli.gamma-amino butyric acid (gaba) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. here, we report a simple and robust system to produce gaba from glucose using the recombinant corynebacterium glutamicum strain gad, which expresses gadb, a glutamate decarboxylase encoded by the gadb gene of escherichia coli w3110. as confirmed by hplc analysis, gaba fermentation by c. glutamicum gad cultured at 30°c in gaba production 1 (gp1) medium containing 50 g/l ...201222759537
development and application of an arabinose-inducible expression system by facilitating inducer uptake in corynebacterium glutamicum.corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. many available inducible expression systems in this species use lac-derived promoters from escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. we developed an arabinose-inducible expression system that contains the l-arabinose regulator arac, the p(bad) promoter from the arabad operon, and the l-arabinose transporter arae, all of which a ...201222685153
improved detection of microbial risk of releasing genetically modified bacteria in soil by using massive sequencing and antibiotic resistance selection.high-throughput 16s rrna gene-targeted pyrosequencing was used with commonly used risk assessment techniques to evaluate the potential microbial risk in soil after inoculating genetically modified (gm) corynebacterium glutamicum. to verify the risk, reference experiments were conducted in parallel using well-defined and frequently used gm escherichia coli and wild-type strains. the viable cell count showed that the number of gm bacteria in the soil was reduced to below the detection limit within ...201222682799
a disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.in the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. during scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. currently, these complex interactions are difficult to investigate. in this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions ...201222511122
the missing link in coenzyme a biosynthesis: panm (formerly yhhk), a yeast gcn5 acetyltransferase homologue triggers aspartate decarboxylase (pand) maturation in salmonella enterica.coenzyme a (coa) is an essential cofactor for all forms of life. the biochemistry underpinning the assembly of coa in escherichia coli and other enterobacteria is well understood, except for the events leading to maturation of the l-aspartate-α-decarboxylase (pand) enzyme that converts pantothenate to β-alanine. pand is synthesized as pro-pand, which undergoes an auto-proteolytic cleavage at residue ser25 to yield the catalytic pyruvoyl moiety of the enzyme. since 1990, it has been known that e. ...201222497218
preliminary investigations of the effect of lipophilic analogues of the active metabolite of isoniazid toward bacterial and plasmodial strains.five lipophilic analogues 1-5 of the active metabolite of the antitubercular drug isoniazid (inh), selected as inhibitors of mycobacterium smegmatis and mycobacterium tuberculosis growth, were evaluated for their activity against corynebacterium glutamicum (lacking in inha activity), escherichia coli (to test mycobacteria selectivity), and plasmodium falciparum (as possible parasite target). compound 3 was the only one that did not inhibit c. glutamicum growth. the poor inha inhibitors 1 and 2 w ...201222405039
improving putrescine production by corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system.corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). previously, we constructed the putrescine-producing strain put1 by deletion of argf, the gene for ornithine transcarbamoylase (otc), and argr, encoding the l-arginine repressor, combined with heterologous expression of the escherichia coli gene for l-ornithine decarboxylase spec. as a consequence of argf deletion, this strain requires supplementation of l-arginine and sh ...201222370950
a synthetic escherichia coli system identifies a conserved origin tethering factor in actinobacteria.in eukaryotic and prokaryotic cells the establishment and maintenance of cell polarity is essential for numerous biological processes. in some bacterial species, the chromosome origins have been identified as molecular markers of cell polarity and polar chromosome anchoring factors have been identified, for example in caulobacter crescentus. although speculated, polar chromosome tethering factors have not been identified for actinobacteria, to date. here, using a minimal synthetic escherichia co ...201222340668
an automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform.high-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and sub ...201223113930
chemometric formulation of bacterial consortium-avs for improved decolorization of resonance-stabilized and heteropolyaromatic dyes.a bacterial consortium-avs, consisting of pseudomonas desmolyticum ncim 2112, kocuria rosea mtcc 1532 and micrococcus glutamicus ncim 2168 was formulated chemometrically, using the mixture design matrix based on the design of experiments methodology. the formulated consortium-avs decolorized acid blue 15 and methylene blue with a higher average decolorization rate, which is more rapid than that of the pure cultures. the uv-vis spectrophotometric, fourier transform infra red spectrophotometric an ...201222940340
mmpl genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.mycolic acids are vital components of the cell wall of the tubercle bacillus mycobacterium tuberculosis and are required for viability and virulence. while mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. we investigated the role of large membrane proteins encoded by mmpl genes in mycolic acid transport in mycobacteria and the related corynebacteria. mmpl3 was found to be essential in mycobacteria and conditional depletion of mmpl3 ...201222520756
differential arabinan capping of lipoarabinomannan modulates innate immune responses and impacts t helper cell differentiation.toll-like receptors (tlrs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (lm) and lipoarabinomannan (lam). such structures are present in several pathogens, including mycobacterium tuberculosis, being important for the initiation of immune responses. it is well established that the interaction of lm and lam with tlr2 is a process dependent on the structure of the ligands. however, the implications of ...201223144457
the lipoprotein lpqw is essential for the mannosylation of periplasmic glycolipids in corynebacteria.phosphatidylinositol mannosides (pim), lipomannan (lm), and lipoarabinomannan (lam) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen mycobacterium tuberculosis, as well as the related corynebacterineae. we have previously shown that the lipoprotein, lpqw, regulates pim and lm/lam biosynthesis in mycobacteria. here, we provide direct evidence that lpqw regulates the activity of key mannosyltransferases in the periplasmic leaflet of the ce ...201223091062
deletion of manc in corynebacterium glutamicum results in a phospho-myo-inositol mannoside- and lipoglycan-deficient mutant.mannose is an important constituent of the immunomodulatory glycoconjugates of the mycobacterial cell wall: lipoarabinomannan (lam), lipomannan (lm) and the related phospho-myo-inositol mannosides (pims). in mycobacterium tuberculosis and the related bacillus corynebacterium glutamicum, mannose is either imported from the medium or derived from glycolysis, and is subsequently converted into the nucleotide-based sugar donor guanosine diphosphomannose (gdp-mannose). this can be utilized by the gly ...201222539165
growth response of avena sativa in amino-acids-rich soils converted from phenol-contaminated soils by corynebacterium glutamicum.the biodegradation of phenol in laboratory-contaminated soil was investigated using the gram-positive soil bacterium corynebacterium glutamicum. this study showed that the phenol degradation caused by c. glutamicum was greatly enhanced by the addition of 1% yeast extract. from the toxicity test using daphnia magna, the soil did not exhibit any hazardous effects after the phenol was removed using c. glutamicum. additionally, the treatment of the phenolcontaminated soils with c. glutamicum increas ...201222534303
glycerol-3-phosphatase of corynebacterium glutamicum.formation of glycerol as by-product of amino acid production by corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. it was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (gpp) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. gpp was found to be active as a homodimer. the enzyme preferred conditions of neutral ph and requires mg²⁺ or mn²⁺ for i ...201222353596
conservation of structure and mechanism within the transaldolase enzyme family.transaldolase (tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. however, tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. we wondered how this variation is conserved in structure and function. to answer this question we characterised and compared the tals from bacillus subtilis, corynebacterium glutamicum and ...201222212631
extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms.overflow metabolism is well known for yeast, bacteria and mammalian cells. it typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. this phenomenon, also denoted the short-term crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. here we present a comprehensive quantitative and time-dependent analysis of ...201222963408
complete genome sequence, lifestyle, and multi-drug resistance of the human pathogen corynebacterium resistens dsm 45100 isolated from blood samples of a leukemia patient.corynebacterium resistens was initially recovered from human infections and recognized as a new coryneform species that is highly resistant to antimicrobial agents. bacteremia associated with this organism in immunocompromised patients was rapidly fatal as standard minocycline therapies failed. c. resistens dsm 45100 was isolated from a blood culture of samples taken from a patient with acute myelocytic leukemia. the complete genome sequence of c. resistens dsm 45100 was determined by pyrosequen ...201222524407
accelerated pentose utilization by corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine.because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. previous studies have engineered several corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. to improve xylose utilization, different xylose isomerase genes were tested in c. glutamicum. the gene originating from xanthomonas campestris was shown to have the hig ...201223164409
carotenoid biosynthesis and overproduction in corynebacterium glutamicum.corynebacterium glutamicum contains the glycosylated c50 carotenoid decaprenoxanthin as yellow pigment. starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.201222963379
metabolic engineering of corynebacterium glutamicum aimed at alternative carbon sources and new products.corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. however, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. some of thes ...201224688664
reductive whole-cell biotransformation with corynebacterium glutamicum: improvement of nadph generation from glucose by a cyclized pentose phosphate pathway using pfka and gapa deletion mutants.in this study, the potential of corynebacterium glutamicum for reductive whole-cell biotransformation is shown. the nadph-dependent reduction of the prochiral methyl acetoacetate (maa) to the chiral (r)-methyl 3-hydroxybutyrate (mhb) by an alcohol dehydrogenase from lactobacillus brevis (lbadh) was used as model reaction and glucose served as substrate for the regeneration of nadph. since nadph is mainly formed in the oxidative branch of the pentose phosphate pathway (ppp), c. glutamicum was eng ...201222851018
overexpression of genes encoding glycolytic enzymes in corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.we previously reported that corynebacterium glutamicum strain δldhaδppc+alad+gapa, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapa, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (t. jojima, m. fujii, e. mori, m. inui, and h. yukawa, appl. microbiol. biotechnol. 87:159-165, 2010). in this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herei ...201222504802
an in silico platform for the design of heterologous pathways in nonnative metabolite production.microorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. however, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. hence, metabolic network design could benefit grea ...201222578364
application of den refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from corynebacterium glutamicum.phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. here, the process of determining and refining the structure of cgl1109, a putative succinyl-diaminopimelate desuccinylase from corynebacterium glutamicum, at ∼3 å resolution is described using a combination of homology modeling with modeller, molecular-replacement phasing with phaser, deformable elastic network (den) refi ...201222505259
identification and characterization of γ-aminobutyric acid uptake system gabpcg (ncgl0464) in corynebacterium glutamicum.corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (gaba). in this study, a c. glutamicum gaba-specific transporter (gabp(cg)) encoded by ncgl0464 was identified and characterized. gabp(cg) plays a major role in gaba uptake and is essential to c. glutamicum growing on gaba. gaba uptake by gabp(cg) was weakly competed by l-as ...201222307305
transcriptional cross-regulation between gram-negative and gram-positive bacteria, demonstrated using argp-argo of escherichia coli and lysg-lyse of corynebacterium glutamicum.the protein-gene pairs argp-argo of escherichia coli and lysg-lyse of corynebacterium glutamicum are orthologous, with the first member of each pair being a lysr-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. whereas lyse is an exporter of arginine (arg) and lysine (lys) whose expression is induced by arg, lys, or histidine (his), argo exports arg alone, and its expression is activated by arg but not lys or his. we have now reconstituted in e. ...201222904281
destabilized eyfp variants for dynamic gene expression studies in corynebacterium glutamicum.fluorescent reporter proteins are widely used for the non-invasive monitoring of gene expression patterns, but dynamic measurements are hampered by the extremely high stability of gfp and homologue proteins. in this study, we used ssra-mediated peptide tagging for the construction of unstable variants of the gfp derivative eyfp (enhanced yellow fluorescent protein) and applied those for transient gene expression analysis in the industrial platform organism corynebacterium glutamicum.201222938655
the tetr-type transcriptional repressor rolr from corynebacterium glutamicum regulates resorcinol catabolism by binding to a unique operator, rolo.the rol (designated for resorcinol) gene cluster rolrhmd is involved in resorcinol catabolism in corynebacterium glutamicum, and rolr is the tetr-type regulator. in this study, we investigated how rolr regulated the transcription of the rol genes in c. glutamicum. the transcription start sites and promoters of rolr and rolhmd were identified. quantitative reverse transcription-pcr and promoter activity analysis indicated that rolr negatively regulated the transcription of rolhmd and of its own g ...201222706057
biochemical and molecular characterization of the gentisate transporter genk in corynebacterium glutamicum.gentisate (2,5-dihydroxybenzoate) is a key ring-cleavage substrate involved in various aromatic compounds degradation. corynebacterium glutamicum atcc13032 is capable of growing on gentisate and genk was proposed to encode a transporter involved in this utilization by its disruption in the restriction-deficient mutant res167. its biochemical characterization by uptake assay using [(14)c]-labeled gentisate has not been previously reported.201222808015
two-component signal transduction in corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets.in bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. in the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target ...201222539022
characterization of oxyr as a negative transcriptional regulator that represses catalase production in corynebacterium diphtheriae.corynebacterium diphtheriae and corynebacterium glutamicum each have one gene (cat) encoding catalase. in-frame δcat mutants of c. diphtheriae and c. glutamicum were hyper-sensitive to growth inhibition and killing by h(2)o(2). in c. diphtheriae c7(β), both catalase activity and cat transcription decreased ~2-fold during transition from exponential growth to early stationary phase. prototypic oxyr in escherichia coli senses oxidative stress and it activates katg transcription and catalase produc ...201222438866
phenylacetic acid catabolism and its transcriptional regulation in corynebacterium glutamicum.the industrially important organism corynebacterium glutamicum has been characterized in recent years for its robust ability to assimilate aromatic compounds. in this study, c. glutamicum strain as 1.542 was investigated for its ability to catabolize phenylacetic acid (paa). the paa genes were identified; they are organized as a continuous paa gene cluster. the type strain of c. glutamicum, atcc 13032, is not able to catabolize paa, but the recombinant strain atcc 13032/pec-k18mob2::paa gained t ...201222685150
altered large-ring cyclodextrin product profile due to a mutation at tyr-172 in the amylomaltase of corynebacterium glutamicum.corynebacterium glutamicum amylomaltase (cgam) catalyzes the formation of large-ring cyclodextrins (lr-cds) with a degree of polymerization of 19 and higher. the cloned cgam gene was ligated into the pet-17b vector and used to transform escherichia coli bl21(de3). site-directed mutagenesis of tyr-172 in cgam to alanine (y172a) was performed to determine its role in the control of lr-cd production. both the recombinant wild-type (wt) and y172a enzymes were purified to apparent homogeneity and cha ...201222865069
regulation of the malic enzyme gene male by the transcriptional regulator malr in corynebacterium glutamicum.corynebacterium glutamicum is a gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. here, we investigated the transcriptional control of the male gene encoding malic enzyme (male) in c. glutamicum atcc13032, which is known to involve the nitrogen regulator amtr. gel shift experiments using purified regulators rama and ramb revealed binding of these regulators to the male promoter. in dna-affinity purification experiments a hitherto uncharacteriz ...201222261175
genome sequence of corynebacterium glutamicum atcc 14067, which provides insight into amino acid biosynthesis in coryneform bacteria.we report the genome sequence of corynebacterium glutamicum atcc 14067 (once named brevibacterium flavum), which is useful for taxonomy research and further molecular breeding in amino acid production. preliminary comparison with those of the reported coryneform strains revealed some notable differences that might be related to the difficulties in molecular manipulation.201222247536
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