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non-invasive microbial metabolic activity sensing at single cell level by perfusion of calcein acetoxymethyl ester.phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. we present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. the fluorescent conversi ...201526513257
arar, an l-arabinose-responsive transcriptional regulator in corynebacterium glutamicum atcc 31831, exerts different degrees of repression depending on the location of its binding sites within the three target promoter regions.in corynebacterium glutamicum atcc 31831, a laci-type transcriptional regulator arar, represses the expression of l-arabinose catabolism (arabda), uptake (arae), and the regulator (arar) genes clustered on the chromosome. arar binds to three sites: one (bsb) between the divergent operons (arabda and galm-arar) and two (bse1 and bse2) upstream of arae. l-arabinose acts as an inducer of the arar-mediated regulation. here, we examined the roles of these arar-binding sites in the expression of the a ...201526416832
modular pathway engineering of corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.the glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and ...201526393954
improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step.the biosynthesis of poly(lactic acid) (pla)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme a (coa) and the polyhydroxyalkanoate (pha) synthase-catalyzed polymerization of lactyl-coa. in order to increase polymer productivity, we explored the rate-limiting step in pla-like polymer synthesis based on quantitative metabolite analysis using liq ...201426267112
bi-functional cellulases complexes displayed on the cell surface of corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature.introducing cellulases into corynebacterium glutamicum leads to the direct degradation of lignocellulosic materials for energy sources. in this study, a cellulase complex containing two cellulolytic enzymes, endoglucanase e (cele) and β-glucosidase a (bgla), was established to completely degrade cellulose to glucose. the cellulases complexes were displayed on the cell surface of c. glutamicum by using the mechanosensitive channel (msc) to anchor enzymes in the cytoplasmic membrane. as confirmed ...201425248702
production of l-glutamic acid with corynebacterium glutamicum (ncim 2168) and pseudomonas reptilivora (ncim 2598): a study on immobilization and reusability.l-glutamic acid is one of the major amino acids that is present in a wide variety of foods. it is mainly used as a food additive and flavor enhancer in the form of sodium salt. corynebacterium glutamicum (c. glutamicum) is one of the major organisms widely used for glutamic acid production.201425215180
economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using corynebacterium glutamicum immobilized in porous polyurethane filler.an immobilized fermentation system, using cassava bagasse hydrolysate (cbh) and mixed alkalis, was developed to achieve economical succinic acid production by corynebacterium glutamicum. the c. glutamicum strains were immobilized in porous polyurethane filler (ppf). cbh was used efficiently as a carbon source instead of more expensive glucose. moreover, as a novel method for regulating ph, the easily decomposing nahco3 was replaced by mixed alkalis (naoh and mg(oh)2) for succinic acid production ...201425463799
metabolic engineering of corynebacterium glutamicum for glycolate production.corynebacterium glutamicum - a well-known industrial amino acid producer - has recently been engineered for the production of a variety of new products including diamines, alcohols, carotenoids and organic acids. glycolic acid was shown here not to serve as sole or combined carbon source for c. glutamicum. glycolate affected growth of c. glutamicum only at high concentrations (460mm) and in a comparable manner as other salts (480mm potassium chloride and 490mm sodium chloride). a transcriptome a ...201424486442
modification of chimeric (2s, 3s)-butanediol dehydrogenase based on structural information.a chimeric (2s, 3s)-butanediol dehydrogenase (clbdh) was engineered to have the strict (s)-configuration specificity of the (2s, 3s)-bdh (bslbdh) derived from brevibacterium saccharolyticum as well as the enzymatic stability of the (2r, 3s)-bdh (kpmbdh) from klebsiella pneumonia by swapping the domains of two native bdhs. however, while clbdh possesses the stability, it lacks the specificity. in order to assist in the design a bdh having strict substrate specificity, an x-ray structural analysis ...201425612804
high-level expression in corynebacterium glutamicum of nitrile hydratase from rhodococcus rhodochrous for acrylamide production.the nhhbag gene of rhodococcus rhodochrous m33 that encodes nitrile hydratase (nhase), converting acrylonitrile into acrylamide, was cloned and expressed in corynebacterium glutamicum under the control of an ilvc promoter. the specific enzyme activity in recombinant c. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (dcw). to overexpress the nhase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (ti ...201424493572
biosynthesis of pinene from glucose using metabolically-engineered corynebacterium glutamicum.pinene is a monoterpenes (c10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. herein, we have metabolically-engineered corynebacterium glutamicum, to produce pinene and studied its toxicity in c. glutamicum. geranyl diphosphate synthases (gpps) and pinene synthases (ps), obtained from pinus taeda and abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphat ...201424930112
engineering of corynebacterium glutamicum for minimized carbon loss during utilization of d-xylose containing substrates.biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. the industrially important platform organism corynebacterium glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. however, all currently described c. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of co2, in particular, when aiming f ...201425304460
metabolic engineering of escherichia coli for the production of riboflavin.riboflavin (vitamin b2), the precursor of the flavin cofactors flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad), is used commercially as an animal feed supplement and food colorant. e. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. thus, the aim of this study was to understand the metabolic capacity of e. coli for the riboflavin production by modification of central me ...201425027702
enhanced acetic acid and succinic acid production under microaerobic conditions by corynebacterium glutamicum harboring escherichia coli transhydrogenase gene pntab.some microorganisms, such as escherichia coli, harbor transhydrogenases that catalyze the interconversion between nadph and nadh. however, such transhydrogenase genes have not been found in the genome of a glutamic acid-producing bacterium corynebacterium glutamicum. in this study, the e. coli transhydrogenase genes udha and pntab were introduced into the c. glutamicum wild-type strain atcc 13032, and the metabolic characteristics of the recombinant strains under aerobic and microaerobic conditi ...201425008167
disruption of pkng enhances production of gamma-aminobutyric acid by corynebacterium glutamicum expressing glutamate decarboxylase.gamma-aminobutyric acid (gaba), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by corynebacterium glutamicum that expresses escherichia coli glutamate decarboxylase (gad) b encoded by gadb. this strain was engineered to produce gaba more efficiently from biomass-derived sugars. to enhance gaba production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pkng (encoding serine/threonine protein kinase ...201424949255
[progress in biosythesis of diaminopentane].air pollution and global warming are increasingly deteriorating. large amounts of polyamides derived from fossil fuel sources are consumed around the world. cadaverine is an important building monomer block of bio-based polyamides, thus biotechnological processes for these polymers possess enormous ecological and economical potential. currently, the engineered strains for biological production of cadaverine are corynebacterium glutamicum and escherichia coli. we review here the latest research p ...201424941739
microbial production of amino acids and derived chemicals: synthetic biology approaches to strain development.amino acids are produced at the multi-million-ton-scale with fermentative production of l-glutamate and l-lysine alone being estimated to amount to more than five million tons in the year 2013. metabolic engineering constantly improves productivities of amino acid producing strains, mainly corynebacterium glutamicum and escherichia coli strains. classical mutagenesis and screening have been accelerated by combination with intracellular metabolite sensing. synthetic biology approaches have allowe ...201424922334
production of the sesquiterpene (+)-valencene by metabolically engineered corynebacterium glutamicum.the sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturally pigmented as it synthesizes the rare fifty carbon atoms (c50) containing carotenoid decaprenoxanthin. since the carotenoid pathway of this gram-positive bacterium has previously b ...201424910970
application of metabolic engineering for the biotechnological production of l-valine.the branched chain amino acid l-valine is an essential nutrient for higher organisms, such as animals and humans. besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l-valine is now emerging into the feed market, and significant increase of sales and world production is expected. in accordance, well-known microbial production bacteria, such as escherichia coli and corynebacterium glutamicum strains, have ...201424816722
synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from corynebacterium glutamicum.β-alanine is mainly produced by chemical methods in current industrial processes. here, pand from corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (adc) was cloned and expressed in escherichia coli bl21(de3). adc c.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. the purified adc c.g was optimal at 55 °c and ph 6 with excellent stability at 16-37 °c and ph 4-7. a ph-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep t ...201424737081
synthetic biology platform of corynebrick vectors for gene expression in corynebacterium glutamicum and its application to xylose utilization.currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as escherichia coli and saccharomyces cerevisiae. in order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called corynebrick, for gene expression in corynebacterium glutamicum as a set of e. coli-c. glutamicum shuttle vectors whose elements are interchangeable with bglbrick standard parts. c. glutamicum is an established ...201424706215
histidine biosynthesis, its regulation and biotechnological application in corynebacterium glutamicum.l-histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. for decades l-histidine biosynthesis has been studied mainly in escherichia coli and salmonella typhimurium, revealing fundamental regulatory processes in bacteria. furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium corynebacterium glutamicum, revealing similarities to e. coli and s. typhimurium, a ...201423617600
directed evolution and mutagenesis of glutamate decarboxylase from lactobacillus brevis lb85 to broaden the range of its activity toward a near-neutral ph.glutamate decarboxylase (gad) transforms l-glutamate into γ-aminobutyric acid (gaba) with the consumption of a proton. gad derived from lactic acid bacteria exhibits optimum activity at ph 4.0-5.0 and significantly loses activity at near-neutral ph. to broaden the active range of the gad gadb1 from lactobacillus brevis lb85 toward a near-neutral ph, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. for directed evolution of gadb1, a se ...201424910334
the crystal structures of apo and camp-bound glxr from corynebacterium glutamicum reveal structural and dynamic changes upon camp binding in crp/fnr family transcription factors.the cyclic amp-dependent transcriptional regulator glxr from corynebacterium glutamicum is a member of the super-family of crp/fnr (cyclic amp receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. in c. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the corynebacteriales including mycobacterium tuberculosis ...201425469635
acyl-coa sensing by fasr to adjust fatty acid synthesis in corynebacterium glutamicum.corynebacterium glutamicum, like mycobacterium tuberculosis, is a member of the corynebacteriales, which have linear fatty acids and as branched fatty acids the mycolic acids. we identified accd1 and fasa as key genes of fatty acid synthesis, encoding the β-subunit of the acetyl-coa carboxylase and a type-i fatty acid synthase, respectively, and observed their repression during growth on minimal medium with acetate. we also identified the transcriptional regulator fasr and its binding sites in t ...201425449109
interaction of 2-oxoglutarate dehydrogenase odha with its inhibitor odhi in corynebacterium glutamicum: mutants and a model.pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. in corynebacterium glutamicum and related bacteria like mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein odha catalyzing the conversion of oxoglutarate to succinyl-coenzyme a. this activity is inhibited by the forkhead-associated (fha) domain of the small autoinhibitory protein odhi. here we used a biological screen which enabled us to isola ...201424905147
benzothiazinones mediate killing of corynebacterineae by blocking decaprenyl phosphate recycling involved in cell wall biosynthesis.benzothiazinones (btzs) are a new class of sulfur containing heterocyclic compounds that target dpre1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (dpr) to decaprenyl-phosphoarabinose (dpa) in the corynebacterineae, such as corynebacterium glutamicum and mycobacterium tuberculosis. as a result, btz inhibition leads to inhibition of cell wall arabinan biosynthesis. previous studies have demonstrated the essentiality of dpre1. in contrast, cg-ubia a ribosyltransfera ...201424446451
the concerted action of a positive charge and hydrogen bonds dynamically regulates the pka of the nucleophilic cysteine in the nrdh-redoxin family.nrdh-redoxins shuffle electrons from the nadph pool in the cell to class ib ribonucleotide reductases, which in turn provide the precursors for dna replication and repair. nrdh-redoxins have a cvqc active site motif and belong to the thioredoxin-fold protein family. as for other thioredoxin-fold proteins, the pk(a) of the nucleophilic cysteine of nrdh-redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. recently, the pk(a) value of this cysteine in cory ...201424243781
production and glucosylation of c50 and c 40 carotenoids by metabolically engineered corynebacterium glutamicum.the yellow-pigmented soil bacterium corynebacterium glutamicum atcc13032 is accumulating the cyclic c50 carotenoid decaprenoxanthin and its glucosides. carotenoid pathway engineering was previously shown to allow for efficient lycopene production. here, engineering of c. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous c50 carotenoids c.p.450 and sarcinaxanthin is described. plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation fr ...201424270893
novel and tightly regulated resorcinol and cumate-inducible expression systems for streptomyces and other actinobacteria.inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the laci repressor, and isopropyl β-d-thiogalactopyranoside (iptg). we successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the cymr regulator, the operator sequence (cmt) ...201425012786
hyaluronic acid production with corynebacterium glutamicum: effect of media composition on yield and molecular weight.corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (ha).201424863652
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
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
ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in bacteria and archaea, which contain inositol phospholipid.in eukarya, phosphatidylinositol (pi) is biosynthesized from cdp-diacylglycerol (cdp-dag) and inositol. in archaea and bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. the precursors, inositol 1-phosphate, cdp-archaeol (cdp-aroh), and cdp-dag, form archaetidylinositol phosphate (aip) and phosphatidylinositol phosphate (pip) as intermediates. these intermediates are dephosphorylated to synthesize archaetidylinositol (ai) and pi. to date, the activities of ...201424269814
functional characterization of corynebacterium glutamicum mycothiol s-conjugate amidase.the present study focuses on the genetic and biochemical characterization of mycothiol s-conjugate amidase (mca) of corynebacterium glutamicum. recombinant c. glutamicum mca was heterologously expressed in escherichia coli and purified to apparent homogeneity. the molecular weight of native mca protein determined by gel filtration chromatography was 35 kda, indicating that mca exists as monomers in the purification condition. mca showed amidase activity with mycothiol s-conjugate of monobromobim ...201425514023
engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. it is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. we calculated the fraction of synonymous substitution and the effective number of codons (enc) for nine genes stemming from g ...201425469170
discovery of a bacterial 5-methylcytosine deaminase.5-methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from escherichia coli (coda) will not accept 5-methylcytosine as a substrate. since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. we therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to ...201425384249
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
improving the secretion of cadaverine in corynebacterium glutamicum by cadaverine-lysine antiporter.cadaverine (1,5-pentanediamine, diaminopentane), the desired raw material of bio-polyamides, is an important industrial chemical with a wide range of applications. biosynthesis of cadaverine in corynebacterium glutamicum has been a competitive way in place of petroleum-based chemical synthesis method. to date, the cadaverine exporter has not been found in c. glutamicum. in order to improve cadaverine secretion, the cadaverine-lysine antiporter cadb from escherichia coli was studied in c. glutami ...201424510022
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
coupling bioorthogonal chemistries with artificial metabolism: intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins.in this paper, we present a novel, "single experiment" methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. in particular, we engineered the intracellular biosynthesis of l-azidohomoalanine from o-acetyl-l-homoserine and nan3, and achieved its direct incorporation into recombinant target proteins by aug codon reassignment in a methionine-auxotroph e. coli strain. ...201424434673
heterologous expression of escherichia coli fructose-1,6-bisphosphatase in corynebacterium glutamicum and evaluating the effect on cell growth and l-lysine production.fructose-1,6-bisphosphatase (fbpase), which is mainly used to supply nadph, has an important role in increasing l-lysine production by corynebacterium glutamicum. however, c. glutamicum fbpase is negatively regulated at the metabolic level. strains that overexpressed escherichia coli fructose-1,6-bisphosphatase in c. glutamicum were constructed, and the effects of heterologous fbpase on cell growth and l-lysine production during growth on glucose, fructose, and sucrose were evaluated. the hetero ...201424397720
development and characterization of expression vectors for corynebacterium glutamicum.in an attempt to develop a variety of expression vector systems for corynebacterium glutamicum, six types of promoters, including ptac, psod, psod with a conserved shine-dalgarno (sd) sequence from c. glutamicum, pilvc, pilvc with a conserved sd-1 (pilvc-m1), and pilvc with a conserved sd-2 (pilvc-m2), were cloned into a modified shuttle vector, pcxm48. according to analysis of promoter strength by quantitative reverse transcription pcr, psod and psod-m were superior to tac and ilvc promoters in ...201424169455
aerobic production of succinate from arabinose by metabolically engineered corynebacterium glutamicum.arabinose is considered as an ideal feedstock for the microbial production of value-added chemicals due to its abundance in hemicellulosic wastes. in this study, the arabad operon from escherichia coli was introduced into succinate-producing corynebacterium glutamicum, which enabled aerobic production of succinate using arabinose as sole carbon source. the engineered strain zx1 (pxarabad, peacsaglta) produced 74.4 mm succinate with a yield of 0.58 mol (mol arabinose)(-1), which represented 69.9% ...201424169202
significance of arg3, arg54, and tyr58 of l-aspartate α-decarboxylase from corynebacterium glutamicum in the process of self-cleavage.we have elucidated the significance of three key amino acid residues of l-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. these results provide useful fundamental information for engineering l-aspartate α-decarboxylase. l-aspartate α-decarboxylase (adc) from corynebacterium glutamicum, and encoded by pand, was cloned and expressed in escherichia coli and then purified. three amino acid residues were found ...201424104602
succinic acid production from corn cob hydrolysates by genetically engineered corynebacterium glutamicum.corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. in the present work, we constructed a xylose metabolic pathway in c. glutamicum by heterologous expression of the xyla and xylb genes coming from escherichia coli. dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant c. glutamicum nc-2. the results indicated that the available activated charcoal ...201424078255
a novel approach for improving the yield of bacillus subtilis transglutaminase in heterologous strains.the transglutaminase (btg) from bacillus subtilis is considered to be a new type of transglutaminase for the food industry. given that the btg gene only encodes a mature peptide, the expression of btg in heterologous microbial hosts could affect their normal growth due to btg's typical transglutaminase activity which can catalyze cross-linking of proteins in the cells. therefore, we developed a novel approach to suppress btg activity and reduce the toxicity on microbial hosts, thus improving btg ...201424947581
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
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
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
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
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
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
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
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
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
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
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
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
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
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
co₂ /hco₃⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in corynebacterium glutamicum.the exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. while gradients of substrates, ph, or dissolved oxygen are often investigated, oscillating co2/hco3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. hereby, we investigate the metabolic and transcriptional response in corynebacterium glutamicum wild type as well as the impact on l-lysine production ...201425139448
detoxification of furfural in corynebacterium glutamicum under aerobic and anaerobic conditions.the toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. furfural is considered to be one of the most toxic compounds among these inhibitors. here, we describe the detoxification of furfural in corynebacterium glutamicum atcc13032 under both aerobic and anaerobic conditions. under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. the ratio of the ...201425112225
construction of a novel expression system for use in corynebacterium glutamicum.corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. suitable vectors are needed for metabolic engineering in c. glutamicum. most available vectors used in c. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. these vectors, though excellent for laboratory use, are not preferable choices for i ...201425108235
transcriptional response of corynebacterium glutamicum atcc 13032 to hydrogen peroxide stress and characterization of the oxyr regulon.the aerobic soil bacterium corynebacterium glutamicum atcc 13032 has a remarkable natural resistance to hydrogen peroxide. a major player in hydrogen peroxide defense is the lysr type transcriptional regulator oxyr, homologs of which are present in a wide range of bacteria. in this study, the global transcriptional response of c. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome dna microarrays, demonstrating the dynamic reaction of the regulatory networ ...201425107507
metabolic engineering of corynebacterium glutamicum for l-arginine production.l-arginine is an important amino acid for diverse industrial and health product applications. here we report the development of metabolically engineered corynebacterium glutamicum atcc 21831 for the production of l-arginine. random mutagenesis is first performed to increase the tolerance of c. glutamicum to l-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of nadph level, dis ...201425091334
oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.brevibacterium lactofermentum and corynebacterium glutamicum are important biotechnology species of the genus corynebacterium. the single-strand dna annealing protein (ssap)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains b. lactofermentum aj1511 and c. glutamicum atcc13032. when the rpsl gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies p ...201425087479
succinate production from co₂-grown microalgal biomass as carbon source using engineered corynebacterium glutamicum through consolidated bioprocessing.the potential for production of chemicals from microalgal biomass has been considered as an alternative route for co₂ mitigation and establishment of biorefineries. this study presents the development of consolidated bioprocessing for succinate production from microalgal biomass using engineered corynebacterium glutamicum. starch-degrading and succinate-producing c. glutamicum strains produced succinate (0.16 g succinate/g total carbon source) from a mixture of starch and glucose as a model micr ...201425056811
lactate production as representative of the fermentation potential of corynebacterium glutamicum 2262 in a one-step process.the fermentative properties of thermo-sensitive strain corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. in particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. in both processes, lactate was produced in significant amount, 27 g/l in batch culture, and up to 55.8 g/l in fed-batch culture, but the specific production rate in the fed-batch cul ...201425036691
genome-wide analysis of the role of global transcriptional regulator gntr1 in corynebacterium glutamicum.the transcriptional regulator gntr1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in corynebacterium glutamicum. gluconate lowers the dna binding affinity of gntr1, which is probably the mechanism of gluconate-dependent induction of these genes. in addition, gntr1 positively regulates ptsg, a gene encoding a major glucose transporter, and pck, a gene encoding phosphoenolpyruvate carboxykinase. here, we searched for the new target of gntr1 on a genome-wide scale b ...201424982307
rapid assessment of oxygen transfer impact for corynebacterium glutamicum.oxygen supply is crucial in industrial application of microbial systems, such as corynebacterium glutamicum, but oxygen transfer is often neglected in early strain characterizations, typically done under aerobic conditions. in this work, a new procedure for oxygen transfer screening is presented, assessing the impact of maximum oxygen transfer conditions (otrmax) within microtiter plate-based cultivation for enhanced throughput. oxygen-dependent growth and productivity were characterized for c. ...201424981020
a de novo nadph generation pathway for improving lysine production of corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. in this work, a de novo nadph generation pathway is proposed by altering the coenzyme specificity of a native nad-dependent glyceraldehyde 3-phosphate dehydrogenase (gapdh) to nadp, which consequently has the potential to produce additional nadph in the glycolytic pathway. specifically, the coenzyme specificity of gapdh of corynebacterium glutami ...201424953302
artificial oxidative stress-tolerant corynebacterium glutamicum.we have reported a transcription profile of an adapted corynebacterium glutamicum that showed enhanced oxidative stress resistance. to construct an artificial oxidative stress-resistant strain, gene clusters in the β-ketoadipate pathway, which were up-regulated in the adapted strain, were artificially expressed in the wild-type c. glutamicum. the wild-type strain was unable to grow under 2 mm h2o2 containing minimal medium, while the strains expressing pca gene clusters restored growth under the ...201424949252
rnd transporters protect corynebacterium glutamicum from antibiotics by assembling the outer membrane.corynebacterium-mycobacterium-nocardia (cmn) group are the causative agents of a broad spectrum of diseases in humans. a distinctive feature of these gram-positive bacteria is the presence of an outer membrane of unique structure and composition. recently, resistance-nodulation-division (rnd) transporters (nicknamed mmpls, mycobacterial membrane protein large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. in this study, ...201424942069
transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.the gram-positive bacterium corynebacterium glutamicum belongs to the order corynebacteriales and is used as a producer of amino acids at industrial scales. due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. applying the high-resolution technique of transcriptome sequencing (rna-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the c. glutamicum transcriptome. ...201424910972
expanding the laccase-toolbox: a laccase from corynebacterium glutamicum with phenol coupling and cuprous oxidase activity.laccases are oxidases with potential for application in biotechnology. up to now only fungal laccases have been applied in technical processes, although bacterial laccases are generally easier to handle and more stable at alkaline ph values and elevated temperatures. to increase the toolbox of bacterial laccases and to broaden our knowledge about them, new enzymes have to be characterized. within this study, we describe the new bacterial laccase cgl1 from corynebacterium glutamicum. cgl1 was fou ...201424910971
engineered cytidine triphosphate synthetase with reduced product inhibition.cytidine triphosphate (ctp) synthetase (ctps) (ec number 6.3.4.2) is a key enzyme involved in de novo synthesis of ctp. it catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. in this study, a novel ctps from corynebacterium glutamicum atcc 13032 (cgctps) was cloned, expressed and characterized. a series of mutagenesis in its n-terminal ammonia ligase (alase) domain was performed in order to reduce ctp product inhibition. all single mutation varian ...201424902851
metabolic engineering corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway.the experiments presented here were based on the conclusions of our previous results. in order to avoid introduction of expression plasmid and to balance the nadh/nad ratio, the nadh biosynthetic enzyme, i.e., nad-dependent glyceraldehyde-3-phosphate dehydrogenase (gadph), was replaced by nadp-dependent gadph, which was used to biosynthesize nadph rather than nadh. the results indicated that the nadh/nad ratio significantly decreased, and glucose consumption and l-lysine production drastically i ...201424879631
corynebacterium glutamicum sdha encoding succinate dehydrogenase subunit a plays a role in cysr-mediated sulfur metabolism.the corynebacterium glutamicum cysr protein plays a critical regulatory role in sulfur metabolism. in this study, we isolated a protein interacting with cysr by employing a two-hybrid system. subsequent analysis identified the gene as sdha annotated to encode succinate dehydrogenase flavoprotein subunit a, a krebs cycle enzyme. deletion of the gene (δsdha) severely affected cell growth and final cell yield, particularly in complex media. in addition, the δsdha mutant strain was unable to use ace ...201424866946
investigation of ptsg gene in response to xylose utilization in corynebacterium glutamicum.corynebacterium glutamicum strains nc-2 were able to grow on xylose as sole carbon sources in our previous work. nevertheless, it exhibited the major shortcoming that the xylose consumption was repressed in the presence of glucose. so far, regarding c. glutamicum, there are a number of reports on ptsg gene, the glucose-specific transporter, involved in glucose metabolism. recently, we found ptsg had influence on xylose utilization and investigated the ptsg gene in response to xylose utilization ...201424859809
effect of cofactor folate on the growth of corynebacterium glutamicum syps-062 and l-serine accumulation.the direct fermentative production of l-serine from sugar has attracted increasing attention. corynebacterium glutamicum syps-062 can directly convert sugar to l-serine. in this study, the effects of exogenous and endogenous regulation of cofactor folate on c. glutamicum syps-062 growth and l-serine accumulation were investigated. for exogenous regulation, the inhibitor (sulfamethoxazole) or precursor (p-aminobenzoate) of folate biosynthesis was added to the medium, respectively. for endogenous ...201424859773
from zero to hero - production of bio-based nylon from renewable resources using engineered corynebacterium glutamicum.polyamides are important industrial polymers. currently, they are produced exclusively from petrochemical monomers. herein, we report the production of a novel bio-nylon, pa5.10 through an integration of biological and chemical approaches. first, systems metabolic engineering of corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. in this way, a hyper-producer, with a high diaminopentane yield of 41% ...201424831706
involvement of the global regulator glxr in 3-hydroxybenzoate and gentisate utilization by corynebacterium glutamicum.corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. an iclr-type regulator genr has been characterized to activate the transcription of gendfm and genkh operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. on the other hand, glxr, a global regulator of the cyclic amp (camp) receptor protein-fumarate nitrate reductase regulator (crp-fnr) type, was also ...201424795375
phosphatase activity of the histidine kinases ensures pathway specificity of the chrsa and hrrsa two-component systems in corynebacterium glutamicum.the majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. the gram-positive soil bacterium corynebacterium glutamicum contains two homologous two-component systems (tcs) involved in the haem-dependent regulation of gene expression. whereas the hrrsa system is crucial for utilization of haem as an alternative iron source, chrsa is required to cope with high toxic haem levels. in this study, we analyse ...201424779520
pupylated proteins in corynebacterium glutamicum revealed by mudpit analysis.in a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. however, not all actinobacteria possessing the pup protein also contain a proteasome. in this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism corynebacterium glutamicum. a defined pup deletion mutant of c. glutamicum atcc 13032 grew aerobically as the parent strain in standard glucose min ...201424737727
enhancing corynebacterium glutamicum robustness by over-expressing a gene, msha, for mycothiol glycosyltransferase.over-expression of the gene, msha, coding for mycothiol glycosyl transferase improved the robustness of corynebacterium glutamicum to various stresses. intracellular mycothiol (msh) content was increased by 114 % in wt(pxmj19-msha) compared to wt(pxmj19). survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to wt(pxmj19) under stress by h2o2 (40 mm), methylglyoxal (5.8 mm), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), cd(2+) (0.01 mm), mn(2+) ...201424737070
double mutation of cell wall proteins cspb and pbp1a increases secretion of the antibody fab fragment from corynebacterium glutamicum.among other advantages, recombinant antibody-binding fragments (fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length iggs. although production of recombinant fab using microbial expression systems has been reported, yields of active fab have not been satisfactory. we recently developed the corynebacterium glutamicum protein expression system (corynex®) and demonstrated improved yield and purity for some applications, alth ...201424731213
the laci-type transcriptional regulator arar acts as an l-arabinose-responsive repressor of l-arabinose utilization genes in corynebacterium glutamicum atcc 31831.the corynebacterium glutamicum atcc 31831 arabda operon consists of three l-arabinose catabolic genes, upstream of which the galm, arar, and arae genes are located in opposite orientation. arar encodes a laci-type transcriptional regulator that negatively regulates the l-arabinose-inducible expression of arabda and arae (encoding an l-arabinose transporter), through a mechanism that has yet to be identified. here we show that the arar protein binds in vitro to three sites: one upstream of arabda ...201424706742
chorismate-dependent transcriptional regulation of quinate/shikimate utilization genes by lysr-type transcriptional regulator qsur in corynebacterium glutamicum: carbon flow control at metabolic branch point.the qsu operon of corynebacterium glutamicum comprises four genes (qsuabcd) that underpin the microorganism's quinate/shikimate utilization pathways. the genes encode enzymes that catalyse reactions at the metabolic branch point between the biosynthesis route for synthesis of aromatic compounds and the catabolic route for degradation of quinate and shikimate for energy production. a qsur gene located immediately upstream of qsua encodes a protein (qsur) which activates the operon in the presence ...201424674055
analysis of putative protomer crosstalk in the trimeric transporter betp: the heterotrimer approach.the homotrimeric, secondary active betaine carrier betp from corynebacterium glutamicum is a model system for stress-regulated transport in bacteria. its activity responds to hyperosmotic stress and it harbors two different functions, transport catalysis (betaine uptake) and stimulus sensing, resp. activity regulation. structural information from 2d and 3d crystals as well as functional analysis of monomerized betp suggested the presence of conformational crosstalk between the individual protome ...201424637177
recent progress in development of synthetic biology platforms and metabolic engineering of corynebacterium glutamicum.the paradigm of synthetic biology has been evolving, along with relevant engineering, to achieve designed bio-systems. synthetic biology has reached the point where it is possible to develop microbial strains to produce desired chemicals. recent advances in this field have promoted metabolic engineering of corynebacterium glutamicum as an amino-acid producer for use in intelligent microbial-cell factories. here, we review recent advances that address c. glutamicum as a potential model organism f ...201424632177
a method for gene amplification and simultaneous deletion in corynebacterium glutamicum genome without any genetic markers.a method for the simultaneous replacement of a given gene by a target gene, leaving no genetic markers, has been developed. the method is based on insertional inactivation and double-crossover homologous recombination. with this method, the lysc(t311i), fbp and ddh genes were inserted into corynebacterium glutamicum genome, and the pck, alat and avta genes were deleted. mobilizable plasmids with lysc(t311i), fbp and ddh cassettes and two homologous arms on the ends of pck, alat and avta were con ...201424613758
carbon flux analysis by 13c nuclear magnetic resonance to determine the effect of co2 on anaerobic succinate production by corynebacterium glutamicum.wild-type corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. we investigated the effect of co2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. the presence of co2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in a ...201424610842
the physiological role of riboflavin transporter and involvement of fmn-riboswitch in its gene expression in corynebacterium glutamicum.riboflavin is a precursor of flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad), which work as cofactors of numerous enzymes. understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. in this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expressio ...201424531272
construction and application of novel feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases by engineering the n-terminal domain for l-phenylalanine synthesis.3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp synthase) encoded by arof is the first enzyme of the shikimate pathway. in the present study, an arof variant with a deficiency in residue ile11 (named arof*) was shown to be insensitive to l-tyrosine. according to three-dimensional structure analysis, nine arof variants were constructed with truncation of different n-terminal fragments, and overexpression of the variants arof(δ(1-9)) , arof(δ(1-10)) , arof(δ(1-12)) and, in particular, a ...201424517515
high-throughput screening of a corynebacterium glutamicum mutant library on genomic and metabolic level.due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. this strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. of these platforms, metabolic profiling has the strongest correlation with the phenotype. we previously published a high-throughput metabolic profiling method for c. glutamicum as well as the ...201424504095
the pyruvate dehydrogenase complex of corynebacterium glutamicum: an attractive target for metabolic engineering.the pyruvate dehydrogenase complex (pdhc) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-coa and co2. since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. this review summarizes the current knowledge and achievements on metabolic engineering approaches to ...201424486441
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