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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
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
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
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
[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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
[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
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
inactivation of the phosphoglucomutase gene pgm in corynebacterium glutamicum affects cell shape and glycogen metabolism.in corynebacterium glutamicum formation of glc-1-p (α-glucose-1-phosphate) from glc-6-p (glucose-6-phosphate) by α-pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. furthermore, pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-p. we here show that c. glutamicum possesses at least two pgm isoenzymes, the cg2800 (pgm) encoded enzyme contribu ...201323863124
a novel type of n-acetylglutamate synthase is involved in the first step of arginine biosynthesis in corynebacterium glutamicum.arginine biosynthesis in corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by n-acetylglutamate synthase (nags). there are different kinds of known nagss, for example, "classical" arga, bifunctional argj, argo, and s-nags. however, since c. glutamicum possesses a monofunctional argj, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown nags gene.201324138314
c1 metabolism in corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide.methanol is considered an interesting carbon source in "bio-based" microbial production processes. since corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. the c. glutamicum wild type was able to convert (13)c-labeled methanol to (13)co2. analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, ...201324014532
recognition of microbial and mammalian phospholipid antigens by nkt cells with diverse tcrs.cd1d-restricted natural killer t (nkt) cells include two major subgroups. the most widely studied are vα14jα18(+) invariant nkt (inkt) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse t-cell receptor (tcr) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse nkt (dnkt) cells. dnkt cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. he ...201323307809
ornithine cyclodeaminase-based proline production by corynebacterium glutamicum.the soil bacterium corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. glutamate is the precursor of the amino acid proline. proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. the direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. an ornithine o ...201323806148
comprehensive analysis of the corynebacterium glutamicum transcriptome using an improved rnaseq technique.the use of rnaseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. the aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism corynebacterium glutamicum by rnaseq in order to improve its genome annotation and to describe important features for transcription and translation.201324341750
modulation of global low-frequency motions underlies allosteric regulation: demonstration in crp/fnr family transcription factors.allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. there is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. the mechanisms, however, for communicating dynamic fluctuations between sites are debated. we provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. we have generated coarse ...201324058293
complex regulation of the phosphoenolpyruvate carboxykinase gene pck and characterization of its gntr-type regulator iolr as a repressor of myo-inositol utilization genes in corynebacterium glutamicum.dna affinity chromatography with the promoter region of the corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., rama, gntr1, gntr2, and iolr. determination of the phosphoenolpyruvate carboxykinase activity of the δrama, δgntr1 δgntr2, and δiolr deletion mutants indicated that rama represses pck during growth on glucose about 2-fold, whereas gntr1, gntr2, and iolr activate pck expression about 2-fold irres ...201323873914
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
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
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
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
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
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
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
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
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
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
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
[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
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
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
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
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
crystallization and preliminary x-ray crystallographic analysis of the amylomaltase from corynebacterium glutamicum.amylomaltase (am; ec 2.4.1.25) belongs to the 4-α-glucanotransferase group of the α-amylase family. the enzyme can produce cycloamylose or large-ring cyclodextrin through intramolecular transglycosylation or cyclization reactions of α-1,4-glucan. amylomaltase from the mesophilic bacterium corynebacterium glutamicum (cgam) contains extra residues at the n-terminus for which the three-dimensional structure is not yet known. in this study, cgam was overexpressed and purified to homogeneity using de ...201323989149
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
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
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
expression, crystallization and preliminary crystallographic study of glub from corynebacterium glutamicum.glub is a substrate-binding protein (sbp) which participates in the uptake of glutamic acid in corynebacterium glutamicum, a gram-positive bacterium. it is part of an atp-binding cassette (abc) transporter system. together with the transmembrane proteins gluc and glud and the cytoplasmic protein glua, which couples the hydrolysis of atp to the translocation of glutamate, they form a highly active glutamate-uptake system. as part of efforts to study the amino-acid metabolism, especially the metab ...201323722846
asymmetric chromosome segregation in xanthomonas citri ssp. citri.this study was intended to characterize the chromosome segregation process of xanthomonas citri ssp. citri (xac) by investigating the functionality of the parb factor encoded on its chromosome, and its requirement for cell viability and virulence. using tap tagging we show that parb is expressed in xac. disruption of parb increased the cell doubling time and precluded the ability of xac to colonize the host citrus. moreover, xac mutant cells expressing only truncated forms of parb exhibited the ...201324339434
directed evolution and structural analysis of nadph-dependent acetoacetyl-coa reductase from ralstonia eutropha reveals two mutations responsible for enhanced kinetics.nicotinamide adenine dinucleotide phosphate (nadph)-dependent acetoacetyl-coa reductase (phab) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [p(3hb)], along with β-ketothiolase (phaa) and polyhydroxyalkanoate synthase (phac). in this study, phab from ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone pcr-mediated mutagenesis and a p(3hb) accumulation-based in vivo screening system using escherichia coli. out of approximately twenty thousan ...201323913421
elucidation of a protein-protein interaction network involved in corynebacterium glutamicum cell wall biosynthesis as determined by bacterial two-hybrid analysis.mycobacterium species have a highly complex and unique cell wall that consists of a large macromolecular structure termed the mycolyl-arabinogalactan-peptidoglycan (magp) complex. this complex is essential for growth, survival and virulence of the human pathogen mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. the closely related species corynebacterium glutamicum has proven useful in the study of orthologous m. tuberculosis genes and proteins involved in magp synt ...201425117516
dna cleavage by cgii and ngoavii requires interaction between n- and r-proteins and extensive nucleotide hydrolysis.the stress-sensitive restriction-modification (rm) system cgli from corynebacterium glutamicum and the homologous ngoavii rm system from neisseria gonorrhoeae fa1090 are composed of three genes: a dna methyltransferase (m.cgli and m.ngoavii), a putative restriction endonuclease (r.cgli and r.ngoavii, or r-proteins) and a predicted dead-family helicase/atpase (n.cgli and n.ngoavii or n-proteins). here we report a biochemical characterization of the r- and n-proteins. size-exclusion chromatography ...201425429977
optimization of the ipp precursor supply for the production of lycopene, decaprenoxanthin and astaxanthin by corynebacterium glutamicum.the biotechnologically relevant bacterium corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic c50 carotenoid decaprenoxanthin and its glucosides. the precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (ipp) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (mep) or non-mevalonate pathway. termina ...201425191655
rho and rnase play a central role in fmn riboswitch regulation in corynebacterium glutamicum.riboswitches are rna elements that regulate gene expression in response to their ligand. although these regulations are thought to be performed without any aid of other factors, recent studies suggested the participation of protein factors such as transcriptional termination factor rho and rnase in some riboswitch regulations. however, to what extent these protein factors contribute to the regulation was unclear. here, we studied the regulatory mechanism of the flavin mononucleotide (fmn) ribosw ...201425477389
role of corynebacterium glutamicum spra encoding a serine protease in glxr-mediated global gene regulation.the global regulator glxr of corynebacterium glutamicum is involved in many cellular activities. considering its role, the glxr protein likely interacts with other proteins to obtain, maintain, and control its activity. to isolate proteins interacting with glxr, we used a two-hybrid system with glxr as the bait. subsequently, the partner, a subtilisin-like serine protease, was isolated from a c. glutamicum genomic library. unlike glxr, which showed constitutive expression, the expression of spra ...201424691519
characterization of 3-phosphoglycerate kinase from corynebacterium glutamicum and its impact on amino acid production.corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (pgk) (ec 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bpg) to adp to yield 3-phosphoglycerate (3-pg) and atp in substrate chain phosphorylation.201424593686
peptidoglycan from fermentation by-product triggers defense responses in grapevine.plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. to identify the molecule that triggered this innate immunity, we frac ...201425427192
high-level secretory production of recombinant single-chain variable fragment (scfv) in corynebacterium glutamicum.we describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scfv) against the anthrax toxin in corynebacterium glutamicum. for efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5' untranslated region (5' utr) sequence, and (5) transcriptional terminator. among all the systems examined, the use of a codon ...201424380967
enhancement of l-ornithine production by disruption of three genes encoding putative oxidoreductases in corynebacterium glutamicum.recently, corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (gdh) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. in this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to gdh of bacillus subtilis. chromosomal in-frame deletion of three genes (ncgl0281, ncgl2582, ...201424402505
construction and application of an expression vector from the new plasmid platc1 of acidithiobacillus caldus.in this study, a recently sequenced 9.8-kb plasmid, platc1, from acidithiobacillus caldus strain sm-1 was characterized and developed into an expression vector. the platc1 backbone carried an oriv, three rep genes, five mob genes, a nic site, and an addiction system. multilocus sequence analysis indicated that platc1 was phylogenetically more related to the incq-like broad host range plasmids than to other incq plasmids. platc1 was able to replicate and reside in gram-negative escherichia coli, ...201424445921
identification and methionine analog tolerance of environmental bacterial isolates selected on methionine analog containing medium.methionine is the first limiting amino acid in poultry feed. currently, methionine supplement is synthesized from an expensive chemical process requiring hazardous chemicals. therefore, the objectives of this study were isolation of methionine producing bacteria from environmental samples and quantification of methionine production in these isolated bacteria. mcgc medium was selected as the isolation medium for methionine-producing bacteria by using corynebacterium glutamicum atcc13032 and esche ...201424502216
engineered coryneform bacteria as a bio-tool for arsenic remediation.despite current remediation efforts, arsenic contamination in water sources is still a major health problem, highlighting the need for new approaches. in this work, strains of the nonpathogenic and highly arsenic-resistant bacterium corynebacterium glutamicum were used as inexpensive tools to accumulate inorganic arsenic, either as arsenate (as(v)) or arsenite (as(iii)) species. the assays made use of "resting cells" from these strains, which were assessed under well-established conditions and c ...201425208910
engineering biotin prototrophic corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.the gram-positive corynebacterium glutamicum is auxotrophic for biotin. besides the biotin uptake system bioymn and the transcriptional regulator bioq, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-coa. heterologous expression of biof from the gram-negative escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of biof together with bioc and bioh from e. coli did not entail biotin proto ...201424486440
analysis of cepa encoding an efflux pump-like protein in corynebacterium glutamicum.a gene encoding a homolog of purine efflux proteins of escherichia coli and bacillus subtilis was identified in the genome of corynebacterium glutamicum and designated as cepa. the gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. to elucidate the function of the gene, we constructed a cepa deletion mutant (δcepa) and a cepa-overexpressing strain and analyzed their physiological characteristics. the c ...201424535744
ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in bacteria and archaea, which contain inositol phospholipid.in eukarya, phosphatidylinositol (pi) is biosynthesized from cdp-diacylglycerol (cdp-dag) and inositol. in archaea and bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. the precursors, inositol 1-phosphate, cdp-archaeol (cdp-aroh), and cdp-dag, form archaetidylinositol phosphate (aip) and phosphatidylinositol phosphate (pip) as intermediates. these intermediates are dephosphorylated to synthesize archaetidylinositol (ai) and pi. to date, the activities of ...201424269814
engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. it is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. we calculated the fraction of synonymous substitution and the effective number of codons (enc) for nine genes stemming from g ...201425469170
discovery of a bacterial 5-methylcytosine deaminase.5-methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from escherichia coli (coda) will not accept 5-methylcytosine as a substrate. since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. we therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to ...201425384249
disruption of pkng enhances production of gamma-aminobutyric acid by corynebacterium glutamicum expressing glutamate decarboxylase.gamma-aminobutyric acid (gaba), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by corynebacterium glutamicum that expresses escherichia coli glutamate decarboxylase (gad) b encoded by gadb. this strain was engineered to produce gaba more efficiently from biomass-derived sugars. to enhance gaba production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pkng (encoding serine/threonine protein kinase ...201424949255
significance of arg3, arg54, and tyr58 of l-aspartate α-decarboxylase from corynebacterium glutamicum in the process of self-cleavage.we have elucidated the significance of three key amino acid residues of l-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. these results provide useful fundamental information for engineering l-aspartate α-decarboxylase. l-aspartate α-decarboxylase (adc) from corynebacterium glutamicum, and encoded by pand, was cloned and expressed in escherichia coli and then purified. three amino acid residues were found ...201424104602
functional characterization of corynebacterium glutamicum mycothiol s-conjugate amidase.the present study focuses on the genetic and biochemical characterization of mycothiol s-conjugate amidase (mca) of corynebacterium glutamicum. recombinant c. glutamicum mca was heterologously expressed in escherichia coli and purified to apparent homogeneity. the molecular weight of native mca protein determined by gel filtration chromatography was 35 kda, indicating that mca exists as monomers in the purification condition. mca showed amidase activity with mycothiol s-conjugate of monobromobim ...201425514023
biosynthesis of trans-4-hydroxyproline by recombinant strains of corynebacterium glutamicum and escherichia coli.trans-4-hydroxy-l-proline (trans-hyp), one of the hydroxyproline (hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. although there are some natural biosynthetic pathways of trans-hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. until now the production of trans-hyp is mainly from the acid hydrolysis of collagen. due to the increasing environmental concerns on those severe chemical processes and compli ...201424885047
l-citrulline production by metabolically engineered corynebacterium glutamicum from glucose and alternative carbon sources.l-citrulline plays an important role in human health and nutrition and is an intermediate of the l-arginine biosynthetic pathway. l-citrulline is a by-product of l-arginine production by corynebacterium glutamicum. in this study, c. glutamicum was engineered for overproduction of l-citrulline as major product without l-arginine being produced as by-product. to this end, l-arginine biosynthesis was derepressed by deletion of the arginine repressor gene argr and conversion of l-citrulline towards ...201426267114
a chromosomally encoded t7 rna polymerase-dependent gene expression system for corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.corynebacterium glutamicum has become a favourite model organism in white biotechnology. nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous rna polymerase. in this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (iptg)-inducible t7 expression system in the prophage-free strain c. glutamicum mb001. for this purpose, part of the de3 region of escherichia coli bl21(de3 ...201425488698
metabolic engineering of pseudomonas sp. strain vlb120 as platform biocatalyst for the production of isobutyric acid and other secondary metabolites.over the recent years the production of ehrlich pathway derived chemicals was shown in a variety of hosts such as escherichia coli, corynebacterium glutamicum, and yeast. exemplarily the production of isobutyric acid was demonstrated in escherichia coli with remarkable titers and yields. however, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. we aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and othe ...201424397404
cell division in corynebacterineae.bacterial cells must coordinate a number of events during the cell cycle. spatio-temporal regulation of bacterial cytokinesis is indispensable for the production of viable, genetically identical offspring. in many rod-shaped bacteria, precise midcell assembly of the division machinery relies on inhibitory systems such as min and noc. in rod-shaped actinobacteria, for example corynebacterium glutamicum and mycobacterium tuberculosis, the divisome assembles in the proximity of the midcell region, ...201424782835
synthetic biology platform of corynebrick vectors for gene expression in corynebacterium glutamicum and its application to xylose utilization.currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as escherichia coli and saccharomyces cerevisiae. in order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called corynebrick, for gene expression in corynebacterium glutamicum as a set of e. coli-c. glutamicum shuttle vectors whose elements are interchangeable with bglbrick standard parts. c. glutamicum is an established ...201424706215
engineering of corynebacterium glutamicum for growth and l-lysine and lycopene production from n-acetyl-glucosamine.sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. the workhorse of industrial amino acid production corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. utilization of the chitin-derived aminosugar n-acetyl-glucosamine (glcnac) for both cultivation and production with c. glutamicum has hitherto not been investigated. albeit this organism harbors the enzymes n-acetylglucosamine-6-phosphatedeacetyl ...201424668244
synthetic promoter libraries for corynebacterium glutamicum.the ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. here, we demonstrate that a previously published easy and fast pcr-based method for modulating gene expression in lactic acid bacteria is also applicable to corynebacterium glutamicum. we constructed constitutive promoter libraries based on various combinations of a previously reported c. glutamicum -10 consensus sequence (gngnta(c/t)aatgg) and the escherichia coli -35 consensus, either wit ...201424458563
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