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heat shock proteome analysis of wild-type corynebacterium glutamicum atcc 13032 and a spontaneous mutant lacking groel1, a dispensable chaperone. 201323661687
pcgr2 copy number depends on the par locus that forms a parc-parb-dna partition complex in corynebacterium glutamicum.to characterize the par system of corynebacterium glutamicum pcgr2 and to manipulate the par components to effectively manipulate plasmid copy number.201323683072
enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different corynebacterium glutamicum.during l-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (pcx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. to explore the significance of pcx for l-glutamate overproduction, the pyc gene encoding pcx was amplified in corynebacterium glutamicum gdk-9 triggered by biotin limitation and cn1021 triggered by a temperature shock, respectively. in the fed-batch cultures, gdk-9pxmj19pyc exhibited 7.4 % lower l-alanine excretion and no im ...201323690048
a mutational analysis of the active site loop residues in cis-3-chloroacrylic acid dehalogenase.cis-3-chloroacrylic acid dehalogenase (cis-caad) from pseudomonas pavonaceae 170 and a homologue from corynebacterium glutamicum designated cg10062 are 34% identical in sequence (54% similar). the former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. although cg10062 has the six active site residues (pro-1, his-28, arg-70, arg-73, tyr-103, and glu-114) that are critical for cis-caad activity, it s ...201323692140
identification of a gene involved in plasmid structural instability in corynebacterium glutamicum.expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. in order to enhance plasmid stability in recombinant corynebacterium glutamicum, an expression plasmid containing genes of the clostridium acetobutylicum butyryl-coa synthesis operon with high structural instability within wild-type c. glutamicum was employed. from a total of 133 mutants exhibiting disruptions in ...201323703324
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
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
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
metabolic evolution of corynebacterium glutamicum for increased production of l-ornithine.l-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. the commercial applications mean that efficient biotechnological production of l-ornithine has become increasingly necessary. adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. the evolved strains can be further optimised by metabolic engineering. thus, metabolic evolutio ...201323725060
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
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
direct l-lysine production from cellobiose by corynebacterium glutamicum displaying beta-glucosidase on its cell surface.we constructed beta-glucosidase (bgl)-displaying corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. after screening active bgls, sde1394, which is a bgl from saccharophagus degradans, was successfully displayed on the c. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. the optical density at 600 nm of bgl-displaying c. glutamicum grown on cellobiose as a carbon source reached 23.5 aft ...201323749228
transcriptional analysis of the f0f1 atpase operon of corynebacterium glutamicum atcc 13032 reveals strong induction by alkaline ph.this article has been retracted at the request of microbiology because identical bands for the 16s rrna probe controls in the northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in journal of bacteriology over a period of 5 years.201323754842
microarray studies reveal a 'differential response' to moderate or severe heat shock of the hrca- and hspr-dependent systems in corynebacterium glutamicum.this article has been retracted at the request of microbiology because identical bands for the 16s rrna probe controls in the northern blots were reported to correspond to experiments using different strains and experimental conditions in articles published in this journal and in journal of bacteriology over a period of 5 years.201323754843
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
modeling and optimization of glutamic acid production using mixed culture of corynebacterium glutamicum ncim2168 and pseudomonas reptilivora ncim2598.in this study, a hybrid system of response surface methodology followed by genetic algorithm has been adopted to optimize the production medium for l-glutamic acid fermentation with mixed cultures of corynebacterium glutamicum and pseudomonas reptilovora. the optimal combination of media components for maximal production of l-glutamic acid was found to be 49.99 g l(-1) of glucose, 10 g l(-1) of urea, 18.06% (v/v) of salt solution, and 4.99% (v/v) of inoculum size. the experimental glutamic acid ...201323768112
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
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
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
taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. here we present an approach to rapidly generate sets of enzymes overriding this control. it is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. the sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by facs. we randomly mutageni ...201423829416
platform engineering of corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of l-lysine, l-valine, and 2-ketoisovalerate.exchange of the native corynebacterium glutamicum promoter of the acee gene, encoding the e1p subunit of the pyruvate dehydrogenase complex (pdhc), with mutated dapa promoter variants led to a series of c. glutamicum strains with gradually reduced growth rates and pdhc activities. upon overexpression of the l-valine biosynthetic genes ilvbnce, all strains produced l-valine. among these strains, c. glutamicum acee a16 (pjc4 ilvbnce) showed the highest biomass and product yields, and thus it was f ...201323835179
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
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
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
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 role of the transcriptional regulator lldr (ncgl2814) in glutamate metabolism under biotin-limited conditions in corynebacterium glutamicum.corynebacterium glutamicum is a gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of l-glutamate. lldr (ncgl2814) is known as a repressor for ldha and lldd encoding lactate dehydrogenases. ldha is responsible for production of l-lactate, while lldd is for its assimilation. since l-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, lldr might play a regulatory role in the glutamate metabolism. here for the first ...201323863291
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
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
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
protein s-mycothiolation functions as redox-switch and thiol protection mechanism in corynebacterium glutamicum under hypochlorite stress.protein s-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in firmicutes bacteria. here we used transcriptomics to analyze the naocl stress response in the mycothiol (msh)-producing corynebacterium glutamicum. we further applied thiol-redox proteomics and mass spectrometry (ms) to identify protein s-mycothiolation.201423886307
construction of a prophage-free variant of corynebacterium glutamicum atcc 13032 for use as a platform strain for basic research and industrial biotechnology.the activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. the genome of the industrial amino acid producer corynebacterium glutamicum atcc 13032 contains three prophages (cgp1, cgp2, and cgp3) of so far unknown functionality. several phage genes are regularly expressed, and the large prophage cgp3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. here, we present t ...201323892752
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
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
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
enzyme-substrate complexes of the quinate/shikimate dehydrogenase from corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination.quinate dehydrogenase (qdh) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (nadh) and is involved in the catabolic quinate metabolism required for the degradation of lignin. the enzyme is a member of the family of shikimate/quinate dehydrogenases (sdh/qdh) occurring in bacteria and plants. we characterized the dual-substrate quinate/shikimate dehydrogenase (qsdh) from corynebacterium glutamicum (cglqsdh) kinetically and revealed a clear su ...201323929881
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
effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by corynebacterium glutamicum.biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with tween 40 addition during fermentation. the transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (tp) of glutamate were investigated under the conditions of var ...201423990041
visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level.we describe genetically encoded sensors which transmit elevated cytosolic concentrations of o-acetyl serine (oas) and o-acetyl homoserine (oah)-intermediates of l-cysteine and l-methionine synthesis-into an optical output. the sensor psenoas3 elicits 7.5-fold-increased fluorescence in cultures of a corynebacterium glutamicum strain that excrete l-cysteine. determination of the cytosolic oas concentration revealed an increase to 0.13 mm, whereas the concentration in the reference strain was below ...201323995919
development of fatty acid-producing corynebacterium glutamicum strains.to date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of corynebacterium glutamicum. to develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. this was followed by genome analysis to characterize its genetic background. the selection of spontaneous mutants resistant to the palmitic acid ester surf ...201323995924
beyond growth rate 0.6: what drives corynebacterium glutamicum to higher growth rates in defined medium.in a former study we showed that corynebacterium glutamicum grows much faster in defined cgxii glucose medium when growth was initiated in highly diluted environments [grünberger et al. (2013b) biotechnol bioeng]. here we studied the batch growth of c. glutamicum in cgxii at a comparable low starting biomass concentration of od ≈ 0.005 in more detail. during bioreactor cultivations a bi-phasic growth behavior with changing growth rates was observed. initially the culture grew with μˆ=0.61±0.02 h ...201423996851
[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
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
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
improvement of cell growth and l-lysine production by genetically modified corynebacterium glutamicum during growth on molasses.fructose-1,6-bisphosphatase (fbpase) and fructokinase (scrk) have important roles in regenerating glucose-6-phosphate in the pentose phosphate pathway (ppp), and thus increasing l-lysine production. this article focuses on the development of l-lysine high-producing strains by heterologous expression of fbpase gene fbp and scrk gene scrk in c. glutamicum lysc (fbr) with molasses as the sole carbon source. heterologous expression of fbp and scrk lead to a decrease of residual sugar in fermentation ...201324029876
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
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
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
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
[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
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
functional analysis of tetr-family regulator amtrsav in streptomyces avermitilis.in actinomycetes, two main regulators, the ompr-like glnr and the tetr-type amtr, have been identified as the central regulators for nitrogen metabolism. glnr-mediated regulation was previously identified in different actinomycetes except for members of the genus corynebacterium, in which amtr plays a predominant role in nitrogen metabolism. interestingly, some actinomycetes (e.g. streptomyces avermitilis) harbour both glnr- and amtr-homologous genes in the chromosome. thus, it will be interesti ...201324068239
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
nmr localization of the o-mycoloylation on porh, a channel forming peptide from corynebacterium glutamicum.porh and pora are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of corynebacterium glutamicum. specific post-translational modifications on pora and porh are required for the formation of a functional ion channel. the assignment of porh proton nmr chemical shifts in dmso, allowed identifying unambiguously the exact position of the porh o-mycoloylation on ser 56 side chain. this was further confirmed by site directed mutagenesis and mass spectrome ...201324100136
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
current knowledge on mycolic acids in corynebacterium glutamicum and their relevance for biotechnological processes.corynebacterium glutamicum is the world's largest producer of glutamate and lysine. industrial glutamate overproduction is induced by empirical processes, such as biotin limitation, supplementation with specific surfactants or addition of sublethal concentration of certain antibiotics to the culture media. although gram-positive bacteria, c. glutamicum and related bacterial species and genera contain, in addition to the plasma membrane, an outer permeability membrane similar to that of gram-nega ...201324113823
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
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
a structural and dynamic investigation of the inhibition of catalase by nitric oxide.determining the chemical and structural modifications occurring within a protein during fundamental processes such as ligand or substrate binding is essential to building up a complete picture of biological function. currently, significant unanswered questions relate to the way in which protein structural dynamics fit within the structure-function relationship and to the functional role, if any, of bound water molecules in the active site. addressing these questions requires a multidisciplinary ...201324121528
improved succinate production in corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system.a dual route for anaerobic succinate production was engineered into corynebacterium glutamicum. the glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. the engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). further overexpression of succinate exporter, suce, increased succinate yield to 1.43 mol (mol glucose)(-1). metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineeri ...201424129953
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
comprehensive discovery and characterization of small rnas in corynebacterium glutamicum atcc 13032.recent discoveries on bacterial transcriptomes gave evidence that small rnas (srnas) have important regulatory roles in prokaryotic cells. modern high-throughput sequencing approaches (rna-seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. whole transcriptome data obtained by rna-seq can be used to detect and characterize all transcript species, including small rnas. here, we describe an rna-seq approach for comprehensive dete ...201324138339
impact of different co2/hco3- levels on metabolism and regulation in corynebacterium glutamicum.we investigated the growth kinetics and transcriptional responses of corynebacterium glutamicum in environments with low (pco2<40 mbar) and high (pco2 ≥ 300 mbar) co2/hco3(-) levels compared to standard conditions. when cultivated at high co2/hco3(-)-levels, c. glutamicum showed increased (63%) biomass to substrate yields during the initial growth phase. other kinetic parameters such as growth rate (μ), specific glucose consumption rate (qs), and selected enzymatic activities of anaplerotic reac ...201324140290
corynebacterium glutamicum arnr controls expression of nitrate reductase operon narkghji and nitric oxide (no)-detoxifying enzyme gene hmp in an no-responsive manner.corynebacterium glutamicum arnr is a novel transcriptional regulator that represses expression of the nitrate reductase operon narkghji and the nitric oxide (no)-detoxifying flavohemoglobin gene hmp under aerobic conditions. in a previous study, we showed that arnr-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that arnr senses. in this study, we show that in the absence of nitrate, arnr-mediated repression is maintained under ...201424142248
elucidation of the regulation of ethanol catabolic genes and ptsg using a glxr and adenylate cyclase gene (cyab) deletion mutants of corynebacterium glutamicum atcc 13032.the cyclic amp receptor protein (crp) homolog, glxr, controls the expression of several genes involved in the regulation of diverse physiological processes in corynebacterium glutamicum. in silico analysis has revealed the presence of glxr binding sites upstream of genes adha, ald, and ptsg, encoding glucose-specific phosphotransferase system protein, alcohol dehydrogenase (adh), and acetaldehyde dehydrogenase (aldh), respectively. however, the involvement of the glxr-camp complex on the express ...201324150494
analysis of sos-induced spontaneous prophage induction in corynebacterium glutamicum at the single-cell level.the genome of the gram-positive soil bacterium corynebacterium glutamicum atcc 13032 contains three integrated prophage elements (cgp1 to -3). recently, it was shown that the large lysogenic prophage cgp3 (∼187 kbp) is excised spontaneously in a small number of cells. in this study, we provide evidence that a spontaneously induced sos response is partly responsible for the observed spontaneous cgp3 induction. whereas previous studies focused mainly on the induction of prophages at the population ...201424163339
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
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
metabolic engineering of corynebacterium glutamicum for 2-ketoisocaproate production.2-ketoisocaproate (kic) is used as a therapeutic agent, and a kic-producing organism may serve as a platform for products deriving from this 2-keto acid. we engineered corynebacterium glutamicum for the production of kic from glucose by deletion of ltbr and ilve, encoding the transcriptional repressor ltbr and transaminase b, respectively, and additional overexpression of ilvbncd, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. the kic-produc ...201424169948
assessment of robustness against dissolved oxygen/substrate oscillations for c. glutamicum dm1933 in two-compartment bioreactor.corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. l-lysine). production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. oscillations can have an influence on the process p ...201424218302
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
role of flavohaemoprotein hmp and nitrate reductase narghji of corynebacterium glutamicum for coping with nitrite and nitrosative stress.the influence of nitrate and nitrite on growth of corynebacterium glutamicum under aerobic conditions in shake flasks was analysed. when dissolved oxygen became limiting at higher cell densities, nitrate was reduced almost stoichiometrically to nitrite by nitrate reductase (narghji). the nitrite concentration also declined slowly, presumably as a result of several reactions including reduction to nitric oxide by a side-activity of nitrate reductase. the flavohaemoglobin gene hmp was most strongl ...201424237595
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
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
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
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
exploring signal transduction in heteromultimeric protein based on energy dissipation model.dynamic intersubunit interactions are key elements in the regulation of many biological systems. a better understanding of how subunits interact with each other and how their interactions are related to dynamic protein structure is a fundamental task in biology. in this paper, a heteromultimeric allosteric protein, corynebacterium glutamicum aspartokinase, is used as a model system to explore the signal transduction involved in intersubunit interactions and allosteric communication with an empha ...201524279729
increase in lactate yield by growing corynebacterium glutamicum in a bioelectrochemical reactor.under conditions conductive to growth, corynebacterium glutamicum showed higher lactate yield from glucose (1.62 ± 0.04) in a bioelectrochemical reactor including 0.2 mm of anthraquinone 2,6-disulfonate with the electrode potential regulated at -0.6 v (vs. ag/agcl) than in a non-regulated environment (1.10 ± 0.03), clarifying that low cathodic potential is beneficial for lactate production.201424315531
pushing product formation to its limit: metabolic engineering of corynebacterium glutamicum for l-leucine overproduction.using metabolic engineering, an efficient l-leucine production strain of corynebacterium glutamicum was developed. in the wild type of c. glutamicum, the leua-encoded 2-isopropylmalate synthase (ipms) is inhibited by low l-leucine concentrations with a k(i) of 0.4 mm. we identified a feedback-resistant imps variant, which carries two amino acid exchanges (r529h, g532d). the corresponding leua(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, ...201424333966
deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in corynebacterium glutamicum.allosteric regulation of phosphoenolpyruvate carboxylase (pepc) controls the metabolic flux distribution of anaplerotic pathways. in this study, the feedback inhibition of corynebacterium glutamicum pepc was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. based on rational protein design, six pepc mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. introducing one of the point mutations (n9 ...201424334667
microfluidic picoliter bioreactor for microbial single-cell analysis: fabrication, system setup, and operation.in this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (plbr) is described in detail. the plbr can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. the device features continuous media flow enabling constant environm ...201324336165
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
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
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
rational design of allosteric regulation of homoserine dehydrogenase by a nonnatural inhibitor l-lysine.allosteric proteins, which can sense different signals, are interesting biological parts for synthetic biology. in particular, the design of an artificial allosteric enzyme to sense an unnatural signal is both challenging and highly desired, for example, for a precise and dynamical control of fluxes of growth-essential but byproduct pathways in metabolic engineering of industrial microorganisms. in this work, we used homoserine dehydrogenase (hsdh) of corynebacterium glutamicum, which is natural ...201524344690
nrdh redoxin enhances resistance to multiple oxidative stresses by acting as a peroxidase cofactor in corynebacterium glutamicum.nrdh redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. although nrdh redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. in this study, we confirmed that the corynebacterium glutamicum nrdh redoxin catalytically reduces the disulfides in the class ib ribonucleotide reductases (rnr), insulin and 5,5'-dithiobis-(2-nitro ...201424375145
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
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
stimulus analysis of betp activation under in vivo conditions.the secondary active, na(+) coupled glycine betaine carrier betp from corynebacterium glutamicum betp was shown to harbor two different functions, transport catalysis (betaine uptake) and stimulus sensing, as well as activity regulation in response to hyperosmotic stress. by analysis in a reconstituted system, the rise in the cytoplasmic k(+) concentration was identified as a primary stimulus for betp activation. we have now studied regulation of betp in vivo by independent variation of both the ...201424384063
next-generation sequencing-based transcriptome analysis of l-lysine-producing corynebacterium glutamicum atcc 21300 strain.in the present study, 151 genes showed a significant change in their expression levels in corynebacterium glutamicum atcc 21300 compared with those of c. glutamicum atcc 13032. of these 151 genes, 56 genes (2%) were up-regulated and 95 genes (3%) were down-regulated. rna sequencing analysis also revealed that 11 genes, involved in the l-lysine biosynthetic pathway of c. glutamicum, were up- or down-regulated compared with those of c. glutamicum atcc 13032. of the 151 genes, 10 genes were identif ...201324385368
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
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
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 of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.the bacterial genus corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and "cheesy gland" in goats and sheep. thus, identifying new therapeutic targets to treat corynebacteria infections is both medically and economically important. cg2496, a functionally uncharacterized protein from corynebacterium glutamicum, was evaluated using an nmr ligand-affinity screen. a total of 11 compounds from a library of 460 biologically active compounds were shown ...201424403054
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
process inhomogeneity leads to rapid side product turnover in cultivation of corynebacterium glutamicum.corynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. this means the demand for industrial process development is likely to increase. however, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. this leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertai ...201424410842
lysine overproducing corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states.a homoserine auxotroph strain of corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. industrially lysine is produced from c. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. ample threonine in the medium supports growth and inhibits lysine production (phenotype-i) and its complete absence leads to inhibition o ...201324432142
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
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
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
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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