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development of a bacterial biosensor for rapid screening of yeast p-coumaric acid production.transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. we describe the effects of different translation initiation rates on the dynam ...201728532147
comparative analysis of polyspecificity of the endogenous trna synthetase of different expression host towards photocrosslinking amino acids using an in silico approach.photo-induced covalent crosslinking has emerged as the powerful strategy for analyzing and characterizing the protein-protein interaction and mapping protein 3d conformations. in the last decades, a number of photocrosslinking amino acids have been reported but only a few have been efficiently utilized for photocrosslinking purposes. recently, incorporation of diazirine containing photoactivatable analogs such as photo-methionine, photo-leucine, photo-isoleucine and photo-lysine into target prot ...201728641210
enhancing poly-γ-glutamic acid production in bacillus amyloliquefaciens by introducing the glutamate synthesis features from corynebacterium glutamicum.poly-γ-glutamic acid (γ-pga) is a valuable polymer with glutamate as its sole precursor. enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-pga production, especially for those glutamate-independent γ-pga producing strains. corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-pga producin ...201728532451
in vivo roles of fatty acid-biosynthetic enzymes in biosynthesis of biotin and α-lipoic acid in corynebacterium glutamicum.for fatty acid biosynthesis, corynebacterium glutamicum uses two type i fatty acid synthases (fas-i), fasa and fasb, in addition to acetyl-coa carboxylase (acc) consisting of accbc, accd1, and acce. the in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the fas-i pathway. for this study, we used wild-type c. glutamicum ...201728754705
engineering of corynebacterium glutamicum for consolidated conversion of hemicellulosic biomass into xylonic acid.xylonic acid is a promising platform chemical with various applications in the fields of food, pharmaceuticals, and agriculture. however, in the current process, xylonic acid is mainly produced by the conversion of xylose, whose preparation requires substantial cost and time. here, we engineered corynebacterium glutamicum for the consolidated bioconversion of hemicellulosic biomass (xylan) into xylonic acid in a single cultivation. first, for the efficient conversion of xylose to xylonic acid, x ...201728799725
the antituberculosis drug ethambutol selectively blocks apical growth in cmn group bacteria.members of the genus mycobacterium are the most prevalent cause of infectious diseases. mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (magp) is conserved among all cmn group bacteria. the arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. ethambutol (emb), a classical antit ...201728174310
identification of a membrane protein required for lipomannan maturation and lipoarabinomannan synthesis in corynebacterineae.mycobacterium tuberculosis and related corynebacterineae synthesize a family of lipomannans (lm) and lipoarabinomannans (lam) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. here we describe a new membrane protein, highly conserved in all corynebacterineae, that is required for synthesis of full-length lm and lam. deletion of the corynebacterium glutamicum ncgl2760 gene resulted in a complete loss of mature lm/lam and the appeara ...201728167532
heme derived from corynebacterium glutamicum: a potential iron additive for swine and an electron carrier additive for lactic acid bacterial culture.to investigate the potential applications of bacterial heme, aminolevulinic acid synthase (hema) was expressed in a corynebacterium glutamicum ha strain that had been adaptively evolved against oxidative stress. the red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. to investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain ...201728035120
metabolic engineering of corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose.3-hydroxypropionic acid (3-hp) is a promising platform chemical which can be used for the production of various value-added chemicals. in this study,corynebacterium glutamicum was metabolically engineered to efficiently produce 3-hp from glucose and xylose via the glycerol pathway. a functional 3-hp synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from saccharomyces cerevisiae) and 3-hp production (pducdegh from klebsiella pneu ...201727918882
graded response of the multifunctional 2-cysteine peroxiredoxin, cgprx, to increasing levels of hydrogen peroxide in corynebacterium glutamicum.eukaryotic typical 2-cysteine (cys) peroxiredoxins (prxs) are multifunctional proteins subjected to complex regulation and play important roles in oxidative stress resistance, hydrogen peroxide (h2o2) signaling modulation, aging, and cancer, but the information on the biochemical functions and regulation mechanisms of prokaryotic atypical 2-cys prxs is largely lacking.201727324811
metabolic profile of 1,5-diaminopentane producing corynebacterium glutamicum under scale-down conditions: blueprint for robustness to bioreactor inhomogeneities.performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. especially, corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. this study focuses on the identification ...201727641904
protein secretion in corynebacterium glutamicum.corynebacterium glutamicum, a gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. due to several characteristics - its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity - c. glutamicum is also a very favorable host cell for the secretory production of heterolog ...201727737570
mutations in mure, the essential udp-n-acetylmuramoylalanyl-d-glutamate 2,6-diaminopimelate ligase of corynebacterium glutamicum: effect on l-lysine formation and analysis of systemic consequences.to explore systemic effects of mutations in the udp-n-acetylmuramoylalanyl-d-glutamate 2,6-diaminopimelate ligase (mure) of corynebacterium glutamicum, that leads to extracellular l-lysine accumulation by this bacterium.201727783176
identification of the camp phosphodiesterase cpda as novel key player in camp-dependent regulation in corynebacterium glutamicum.the second messenger cyclic amp (camp) plays an important role in the metabolism of corynebacterium glutamicum, as the global transcriptional regulator glxr requires complex formation with camp to become active. whereas a membrane-bound adenylate cyclase, cyab, was shown to be involved in camp synthesis, enzymes catalyzing camp degradation have not been described yet. in this study we identified a class ii camp phosphodiesterase named cpda (cg2761), homologs of which are present in many actinoba ...201727862445
comparative analysis of the corynebacterium glutamicum transcriptome in response to changes in dissolved oxygen levels.the dissolved oxygen (do) level of a culture of corynebacterium glutamicum (c. glutamicum) in a bioreactor has a significant impact on the cellular redox potential and the distribution of energy and metabolites. in this study, to gain a deeper understanding of the effects of do on the metabolism of c. glutamicum, we sought to systematically explore the influence of different do concentrations on genetic regulation and metabolism through transcriptomic analysis. the results revealed that after 20 ...201727844170
functional architecture and global properties of the corynebacterium glutamicum regulatory network: novel insights from a dataset with a high genomic coverage.corynebacterium glutamicum is a gram-positive, anaerobic, rod-shaped soil bacterium able to grow on a diversity of carbon sources like sugars and organic acids. it is a biotechnological relevant organism because of its highly efficient ability to biosynthesize amino acids, such as l-glutamic acid and l-lysine. here, we reconstructed the most complete c. glutamicum regulatory network to date and comprehensively analyzed its global organizational properties, systems-level features and functional a ...201727829123
improved fermentative production of gamma-aminobutyric acid via the putrescine route: systems metabolic engineering for production from glucose, amino sugars, and xylose.gamma-aminobutyric acid (gaba) is a non-protein amino acid widespread in nature. among the various uses of gaba, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. in metabolism, gaba can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. fermentative production of gaba from glucose by recombinant corynebacterium glutamicum has been described via both routes. putrescine-based ...201727800627
the whcd gene of corynebacterium glutamicum plays roles in cell division and envelope formation.in this study, we analysed the whcd gene from corynebacteriumglutamicum, which encodes a homologue of whib, a streptomycescoelicolor gene required for the sporulation of aerial hyphae. deletion of the gene (δwhcd) severely affected cell growth in c. glutamicum. the δwhcd strain exhibited a large filamentous, branched and bud-shaped morphology with multiple septa. the transcription levels of the cell division genes involved in z-ring assembly and septal peptidoglycan synthesis, including ftsz, se ...201727902442
expansion of the substrate range of the gentisate 1,2-dioxygenase from corynebacterium glutamicum for the conversion of monohydroxylated benzoates.the gentisate 1,2-dioxygenases (gdos) from corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). in contrast, the α-proteobacterium pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', sdo) which cleaves gentisate, but also (substituted) salicylate(s). sequence comparisons showed that the sdo belongs to a group of gdos mainly ori ...201727887027
enhanced glucose consumption and organic acid production by engineered corynebacterium glutamicum based on analysis of a pfkb1 deletion mutant.in the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of corynebacterium glutamicum deficient in pfkb1, which encodes fructose-1-phosphate kinase. after being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly l-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. this considerably higher fermen ...201727881414
microbial production of amino acid-related compounds.corynebacterium glutamicum is the workhorse of the production of proteinogenic amino acids used in food and feed biotechnology. after more than 50 years of safe amino acid production, c. glutamicum has recently also been engineered for the production of amino acid-derived compounds, which find various applications, e.g., as synthons for the chemical industry in several markets including the polymer market. the amino acid-derived compounds such as non-proteinogenic ω-amino acids, α,ω-diamines, an ...201727872963
boosting anaplerotic reactions by pyruvate kinase gene deletion and phosphoenolpyruvate carboxylase desensitization for glutamic acid and lysine production in corynebacterium glutamicum.in the 1980s, shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by corynebacterium glutamicum: (1) low-activity-level citrate synthase (cs(l)), (2) phosphoenolpyruvate carboxylase (pepc) resistant to feedback inhibition by aspartic acid (pepc(r)), and (3) pyruvate kinase (pyk) deficiency. here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant dna techniques. ...201727872961
a novel synthetic pathway enables microbial production of polyphenols independent from the endogenous aromatic amino acid metabolism.numerous plant polyphenols have potential applications as pharmaceuticals or nutraceuticals. stilbenes and flavonoids as most abundant polyphenols are synthesized from phenylpropanoids, which are exclusively derived from aromatic amino acids in nature. several microorganisms were engineered for the synthesis of biotechnologically interesting plant polyphenols; however, low activity of heterologous ammonia lyases, linking endogenous microbial aromatic amino acid biosynthesis to phenylpropanoid sy ...201727936616
glutamate fermentation-2: mechanism of l-glutamate overproduction in corynebacterium glutamicum.the nonpathogenic coryneform bacterium, corynebacterium glutamicum, was isolated as an l-glutamate-overproducing microorganism by japanese researchers and is currently utilized in various amino acid fermentation processes. l-glutamate production by c. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and β-lactam antibiotics. these treatments affect the cell surface structures of c. glutamicum. after the discovery of c. glutamicum, many researchers have i ...201727913829
discovery and history of amino acid fermentation.there has been a strong demand in japan and east asia for l-glutamic acid as a seasoning since monosodium glutamate was found to present umami taste in 1907. the discovery of glutamate fermentation by corynebacterium glutamicum in 1956 enabled abundant and low-cost production of the amino acid, creating a large market. the discovery also prompted researchers to develop fermentative production processes for other l-amino acids, such as lysine. currently, the amino acid fermentation industry is so ...201727909736
the three-component system esrisr regulates a cell envelope stress response in corynebacterium glutamicum.when the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. here, we show that the two-component system esrsr regulates a cell envelope stress response in the actinobacterium corynebacterium glutamicum. the sensor kinase esrs possesses an amino-terminal phage shock protein c (pspc) domain, a property that sets esrsr apart from all other two-component systems characterized so far. an ...201728922502
functional comparison of methionine sulphoxide reductase a and b in corynebacterium glutamicum.methionine sulphoxide reductases (msr) are able to reduce methionine sulfoxide to methionine and protect bacteria against reactive oxygen species (ros). many organisms express both methionine sulphoxide reductase a (msra), specific for methionine-s-sulfoxide and methionine sulphoxide reductase b (msrb), active against methionine-r-sulfoxide. corynebacterium glutamicum expresses msra, the function of which has been well defined; however, the function of msrb has not been studied. whether msrb and ...201728904252
effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor, odhi, involved in glutamate production in corynebacterium glutamicum.in corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (odh) complex is negatively regulated by the unphosphorylated form of odhi protein, which is critical for l-glutamate overproduction. we examined the potential impact of protein acylation at lysine (k)-132 of odhi in c. glutamicum atcc13032. the k132e succinylation-mimic mutation reduced the ability of odhi to bind odha, the catalytic subunit of the odh complex, which reduced the inhibition of odh activity. in vitro ...201728899215
developing genome-reduced pseudomonas chlororaphis strains for the production of secondary metabolites.the current chassis organisms or various types of cell factories have considerable advantages and disadvantages. therefore, it is necessary to develop various chassis for an efficient production of different bioproducts from renewable resources. in this context, synthetic biology offers unique potentialities to produce value-added products of interests. microbial genome reduction and modification are important strategies for constructing cellular chassis and cell factories. many genome-reduced s ...201728893188
amino acids production focusing on fermentation technologies - a review.amino acids are attractive and promising biochemicals with market capacity requirements constantly increasing. their applicability ranges from animal feed additives, flavour enhancers and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. this review gives an overview of the processes applied for amino acids production and points out the main advantages and disadvantages of each. due to the advances made in the genetic engineering techniques, the biotechnologica ...201728888551
synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms.genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). however, engineering microbial workhorses for biomanufacturing of commodity compounds remains challenging. first, microbes cannot afford burdens with both overexpression of multiple enzymes and metabolite drainage for product synthesis. second, synthetic circuits and introduced heterologous pathways are not yet as "robust and reliable" as native pathways due to hosts' ...201728884354
[biocatalytic access to β-alanine by a two-enzyme cascade synthesis].enzymatic synthesis is an important way to produce β-alanine, but the biological method is expensive generally because of the high price of substrate. in this paper, a two-step enzymatic cascade system was developed, combining l-aspartase from escherichia coli dh5α and l-aspartate α-decarboxylase from corynebacterium glutamicum. this system catalyzes fumarate and ammonia to β-alanine. the optimal ratio of aspa and pand was 1:80 (w/w), and the concentration of aspa was 10 μg/ml, at 37 ℃ and ph 7. ...201728876041
identification and functional characterization of small alarmone synthetases in corynebacterium glutamicum.the hyperphosphorylated guanosine derivatives ppgpp and pppgpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. although it was assumed that both, (p)ppgpp synthesis and hydrolysis, are catalyzed by one bifunctional rsh-protein in the actinobacterial model organism corynebacterium glutamicum atcc 13032, two putative short alarmone synthetases (sass) were identified by bioinformatic analyses. the predicted sequences of ...201728871248
enhanced biosynthesis of hyaluronic acid using engineered corynebacterium glutamicum via metabolic pathway regulation.hyaluronic acid (ha) is a polysaccharide used in many industries such as medicine, surgery, cosmetics and food. to avoid potential pathogenicity caused by its native producer, streptococcus, efforts have been made to create a recombinant host for ha production. in this work, a gras (generally recognized as safe) strain, corynebacterium glutamicum, was engineered for enhanced biosynthesis of ha via metabolic pathway regulation. five enzymes (hasa-hase) involved in the ha biosynthetic pathway were ...201728869338
cgii cleaves dna using a mechanism distinct from other atp-dependent restriction endonucleases.the restriction endonuclease cgli from corynebacterium glutamicum recognizes an asymmetric 5'-gccgc-3' site and cleaves the dna 7 and 6/7 nucleotides downstream on the top and bottom dna strands, respectively, in an ntp-hydrolysis dependent reaction. cgli is composed of two different proteins: an endonuclease (r.cgli) and a dead-family helicase-like atpase (h.cgli). these subunits form a heterotetrameric complex with r2h2 stoichiometry. however, the r2h2·cgli complex has only one nuclease active ...201728854738
structure, function, and regulation of enzymes involved in amino acid metabolism of bacteria and archaea.amino acids are essential components in all organisms because they are building blocks of proteins. they are also produced industrially and used for various purposes. for example, l-glutamate is used as the component of "umami" taste and lysine has been used as livestock feed. recently, many kinds of amino acids have attracted attention as biological regulators and are used for a healthy life. thus, to clarify the mechanism of how amino acids are biosynthesized and how they work as biological re ...201728840778
ph fluctuations imperil the robustness of c. glutamicum to short term oxygen limitation.the presence of complex gradients for, e.g., nutrients, oxygen or ph in industrial scale fed batch processes are a major challenge for process performance. to consider such impact of scale-up during laboratory scale process development, scale-down bioreactor simulation, i.e. mimicking inhomogeneous conditions, became the method of choice. however, most scale-down studies simulate combined inhomogeneities of more than one parameter, so that the impact of the individual parameters remains unclear. ...201728837821
chemical shift assignments of polyketide cyclase_like protein cgl2373 from corynebacterium glutamicum.protein cgl2373 from corynebacterium glutamicum, which is 155 amino acids long and 17.7 kda, is a member of the polyketide_cyc2 family. as a potential polyketide cyclase, it may play an important role in the biosynthesis of aromatic polyketides that are the source of many bioactive molecules. here we report the complete (1)h, (13)c and (15)n chemical shift assignments of cgl2373, which lays a foundation for further structural and functional research.201728825188
polymerization dynamics of the prophage-encoded actin-like protein alpc is influenced by the dna-binding adapter alpa.the corynebacterium glutamicum atcc 13032 prophage cgp3 encodes an actin-like protein, alpc that was shown to be involved in viral dna transport and efficient viral dna replication. alpc binds to an adapter, alpa that in turn binds to specific dna sequences, termed alps sites. thus, the alpac system is similar to the known plasmid segregation system parmrs. so far it is unclear how the alpacs system mediates dna transport and, whether alpa and alpc functionally interact. we show here that alpa m ...201728824563
corynebacterium glutamicum chassis c1*: building and testing a novel platform host for synthetic biology and industrial biotechnology.targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. recently, we created a library of 26 genome-reduced strains of corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 ...201728803482
recombineering using recet in corynebacterium glutamicum atcc14067 via a self-excisable cassette.gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain corynebacterium glutamicum (c. glutamicum) atcc14067, which is used for amino acid production. here, we report an effective and sequential deletion method for c. glutamicum atcc14067 using the exonuclease-recombinase pair rece + rect (recet) for recombineering via a designed self-excisable linear double-strand dna (dsdna) cas ...201728801604
physiological roles of sigma factor sigd in corynebacterium glutamicum.sigma factors are one of the components of rna polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of sigd in transcriptional regulation in c. glutamicum has been mostly unknown.201728701150
development of a single-cell glxr-based camp biosensor for corynebacterium glutamicum.cyclic adenosine monophosphate (camp) plays a regulatory role as second messenger in many species. in the industrial model organism corynebacterium glutamicum, camp acts as effector of the global transcriptional regulator glxr, a homolog of enterobacterial crp. the camp-glxr complex activates or represses the expression of about 200 target genes. cyab, a membrane-bound class iii adenylate cyclase, synthesizes camp from atp, but another yet unknown camp-forming enzyme is likely present in c. glut ...201728698098
a new genome-scale metabolic model of corynebacterium glutamicum and its application.corynebacterium glutamicum is an important platform organism for industrial biotechnology to produce amino acids, organic acids, bioplastic monomers, and biofuels. the metabolic flexibility, broad substrate spectrum, and fermentative robustness of c. glutamicum make this organism an ideal cell factory to manufacture desired products. with increases in gene function, transport system, and metabolic profile information under certain conditions, developing a comprehensive genome-scale metabolic mod ...201728680478
structural insights into substrate specificity of cystathionine γ-synthase from corynebacterium glutamicum.cystathionine γ-synthase (metb) condenses o-acetyl-l-homoserine (oahs) or o-succinyl-l-homoserine (oshs) with cysteine to produce cystathionine. to investigate the molecular mechanisms and substrate specificity of metb from corynebacterium glutamicum (cgmetb), we determined its crystal structure at 1.5 å resolution. the pyridoxal phosphate cofactor is covalently bound to lys204 via a schiff base linkage in the deep cavity. superposition with the structure of metb from nicotiana tabacum in comple ...201728675039
assignment of sigma factors of rna polymerase to promoters in corynebacterium glutamicum.corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. the c. glutamicum genome encodes seven sigma subunits (factors) of rna polymerase: the primary sigma factor siga (σ(a)), the primary-like σ(b) and five alternative sigma factors (σ(c), σ(d), σ(e), σ(h) and σ(m)). we have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. in vitro transcription assays and measurements of ...201728651382
crispr/cas9-coupled recombineering for metabolic engineering of corynebacterium glutamicum.genome engineering of corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in c. glutamicum in sequential and iterative manner. recombinase rect is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and crispr/cas9 to counter-select negative mutants ...201728649005
the linkage between nutrient supply, intracellular enzyme abundances and bacterial growth: new evidences from the central carbon metabolism of corynebacterium glutamicum.corynebacterium glutamicum serves as important production host for small molecular compounds that are derived from precursor molecules of the central carbon metabolism. it is therefore a well-studied model organism of industrial biotechnology. however, a deeper understanding of the regulatory principles underlying the synthesis of central metabolic enzymes under different environmental conditions as well as its impact on cell growth is still missing. we studied enzyme abundances in c. glutamicum ...201728647528
biotransformation of ferulic acid to protocatechuic acid by corynebacterium glutamicum atcc 21420 engineered to express vanillate o-demethylase.ferulic acid (4-hydroxy-3-methoxycinnamic acid, fa) is a lignin-derived phenolic compound abundant in plant biomass. the utilization of fa and its conversion to valuable compounds is desired. protocatechuic acid (3,4-dihydroxybenzoic acid, pca) is a precursor of polymers and plastics and a constituent of food. a microbial conversion system to produce pca from fa was developed in this study using a pca-producing strain of corynebacterium glutamicum f (atcc 21420). c. glutamicum strain f grown at ...201728641405
selective oxidation of aliphatic c-h bonds in alkylphenols by a chemomimetic biocatalytic system.selective oxidation of aliphatic c-h bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. here, we report the development of a unique chemomimetic bi ...201728607077
enhancement of anaerobic lysine production in corynebacterium glutamicum electrofermentations.it has been suggested that application of electric potential can affect lysine producing fermentations, although experimental evidence is lacking. to study this hypothesis we used the lysine producer corynebacterium glutamicum zw04, and we exposed it to 12 different conditions regarding anaerobic gas environment, applied electrode potential (cathodic, open circuit, anodic), redox mediator and nitrate presence. the gas environment was found to play a major role, with co2 leading to double the lys ...201728599233
novel chromosome organization pattern in actinomycetales-overlapping replication cycles combined with diploidy.bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of dna to daughter generations. the underlying mechanisms have been addressed in several model species. it became apparent that bacteria have evolved quite different strategies to regulate dna segregation and chromosomal organization. we have investigated here how the actinobacterium corynebacterium glutamicum organizes chromosome segregation and dna replication. unexpectedly, we fo ...201728588128
metabolic design of corynebacterium glutamicum for production of l-cysteine with consideration of sulfur-supplemented animal feed.l-cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. however, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. in this study, we constructed recombinant corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. the aims of this work were to investigate the effect of the combined overexpression of serine acetyl ...201728560868
miniaturized and automated adaptive laboratory evolution: evolving corynebacterium glutamicum towards an improved d-xylose utilization.adaptive laboratory evolution (ale) is increasingly being used as a technique for untargeted strain optimization. this work aimed at developing an automated and miniaturized ale approach based on repetitive batch cultivations in microtiter plates. the new method is applied to the recently published strain corynebacterium glutamicum pekex3-xylxabcdcc, which is capable of utilizing d-xylose via the weimberg (wmb) pathway. as a result, the significantly improved strain wmb2evo was obtained, showing ...201728552568
enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in corynebacterium glutamicum.to construct a strain of corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ala) via the c4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.201728536938
overexpression of ppc or deletion of mdh for improving production of γ-aminobutyric acid in recombinant corynebacterium glutamicum.l-glutamate decarboxylase (gad) transforms l-glutamate into γ-aminobutyric acid (gaba). corynebacterium glutamicum that expresses exogenous gad gene(s) can synthesize gaba from its own produced l-glutamate. to enhance gaba production in recombinant c. glutamicum strain sh, metabolic engineering strategies were used to improve the supply of the gaba precursor, l-glutamate. five new strains were constructed here. first, the ppc gene was coexpressed with two gad genes (gadb1 and gadb2). then, the m ...201728534111
y418 in 410s loop is required for high transglucosylation activity and large-ring cyclodextrin production of amylomaltase from corynebacterium glutamicum.amylomaltase catalyzes α-1,4 glucosyl transfer reaction to yield linear or cyclic oligosaccharide products. the aim of this work is to investigate functional roles of 410s loop unique to amylomaltase from corynebacterium glutamicum (cgam). site-directed mutagenesis of y418, the residue at the loop tip, was performed. y418a/s/d/r/w/f - cgams were characterized and compared to the wild-type (wt). a significant decrease in starch transglucosylation, disproportionation and cyclization activities was ...201728522291
crystal structure of the 2-iminoglutarate-bound complex of glutamate dehydrogenase from corynebacterium glutamicum.the nadp(+) -dependent glutamate dehydrogenase from corynebacterium glutamicum (cggdh) is considered to be one of the key enzymes in the industrial fermentation of glutamate due to its high glutamate-producing activity. we determined the crystal structure of cggdh complexed with nadp(+) and 2-iminoglutarate. among six subunits of hexameric cggdh-binding nadp(+) , only four subunits bind 2-iminoglutarate in a closed form, while the other two are in an open form. in the closed form, 2-iminoglutara ...201728486765
isoprenoid pyrophosphate-dependent transcriptional regulation of carotenogenesis in corynebacterium glutamicum.corynebacterium glutamicum is a natural producer of the c50 carotenoid decaprenoxanthin. the crtecg0722crtbiyeb operon comprises most of its genes for terpenoid biosynthesis. the marr-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. this regulator, named crtr in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. crtr was shown to repress the crt operon of c. gluta ...201728484430
improved fermentative production of the compatible solute ectoine by corynebacterium glutamicum from glucose and alternative carbon sources.the cyclic amino acid ectoine is a compatible solute serving as a protective substance against osmotic stress. ectoine finds various applications due to its moisturizing effect. to avoid the disadvantages of the prevailing so-called "bacterial milking ectoine production process" caused by the high salt concentration, low salt fermentation strategies are sought after. as l-lysine and ectoine biosynthesis share l-aspartate-semialdehyde as common precursor, l-lysine producing strains can be convert ...201728478080
complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.the complete genome of ϕ16, a temperate corynephage from corynebacterium glutamicum atcc 21792, was sequenced and annotated (genbank: ky250482). the electron microscopy study of ϕ16 virion confirmed that it belongs to the family siphoviridae. the ϕ16 genome consists of a linear double-stranded dna molecule of 58,200 bp (g+c = 52.2%) with protruding cohesive 3'-ends of 14 nt. four major structural proteins were separated by sds-page and identified by peptide mass fingerprinting technique. using b ...201728455670
the arsenic detoxification system in corynebacteria: basis and application for bioremediation and redox control.arsenic (as) is widespread in the environment and highly toxic. it has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. to survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (as(v)) to arsenite (as(iii)) by arsenate reductases, which is then extruded by arsenite permeases. one of t ...201728438267
genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete corynebacterium glutamicum.the genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cdna library enriched for native 5' transcript ends, is ideally suited for the analysis of promoters. here, the transcriptome of corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5'-ends of native transcripts. total rna samples were harvested from ...201728412515
dynamic flux balance analysis with nonlinear objective function.dynamic flux balance analysis (dfba) extends flux balance analysis and enables the combined simulation of both intracellular and extracellular environments of microbial cultivation processes. a dfba model contains two coupled parts, a dynamic part at the upper level (extracellular environment) and an optimization part at the lower level (intracellular environment). both parts are coupled through substrate uptake and product secretion rates. this work proposes a karush-kuhn-tucker condition based ...201728401266
analysis of strain-specific genes in glutamic acid-producing corynebacterium glutamicum ssp. lactofermentum aj 1511.strains of the bacterium, corynebacterium glutamicum, are widely used for the industrial production of l-glutamic acid and various other substances. c. glutamicum ssp. lactofermentum aj 1511, formerly classified as brevibacterium lactofermentum, and the closely related c. glutamicum atcc 13032 have been used as industrial strains for more than 50 years. we determined the whole genome sequence of c. glutamicum aj 1511 and performed genome-wide comparative analysis with c. glutamicum atcc 13032 to ...201728392541
identification of specific posttranslational o-mycoloylations mediating protein targeting to the mycomembrane.the outer membranes (oms) of members of the corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (omps), including those with α-helical structure. the signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. we report here the molecular features responsible for omp targeting to the mycomembrane of corynebacterium glutamicum, a nonpathogenic member of the corynebacteriales order. to ...201728373551
transcriptome analysis of corynebacterium glutamicum in the process of recombinant protein expression in bioreactors.corynebacterium glutamicum (c. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of c. glutamicum. two c. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (egfp), were cultured in ...201728369109
a novel genetic tool for metabolic optimization of corynebacterium glutamicum: efficient and repetitive chromosomal integration of synthetic promoter-driven expression libraries.fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. we have developed a tool for this purpose for the important industrial workhorse corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. we first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pjs31 serving as the carrier. the pjs31 vector ...201728361238
a newly determined member of the meso-diaminopimelate dehydrogenase family with a broad substrate spectrum.meso-diaminopimelate dehydrogenase (meso-dapdh) from symbiobacterium thermophilum (stdapdh) is the first member of the meso-dapdh family known to catalyze the asymmetric reductive amination of 2-keto acids to produce d-amino acids. it is important to understand the catalytic mechanisms of stdapdh and other enzymes in this family. in this study, based on an evolutionary analysis and examination of catalytic activity, the meso-dapdh enzymes can be divided into two types. type i showed highly prefe ...201728341677
proteomics of facs-sorted heterogeneous corynebacterium glutamicum populations.the metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. however, it is hardly understood in molecular detail due to limitations of current analytical tools. here, we demonstrate that facs in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. a previously established gfp reporter system was used to detect and sort single corynebacterium glutamicum ...201728323243
similarities in the structure of the transcriptional repressor amtr in two different space groups suggest a model for the interaction with glnk.amtr belongs to the tetr family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions in corynebacterium glutamicum. amtr regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. the recognition of operator dna by homodimeric amtr is not regulated by small-molecule effectors as in other tetr-family members but by a trimeric adenylylated p ...201728291750
characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in corynebacterium glutamicum.protein nε-acylation is emerging as a ubiquitous post-translational modification. in corynebacterium glutamicum, which is utilized for industrial production of l-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. here, the acylation of phosphoenolpyruvate carboxylase (pepc), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction was characterized. it was shown that acetylation of pepc ...201728256782
complete genome sequencing of newly isolated thermotolerant corynebacterium glutamicum n24 provides a new insights into its thermotolerant phenotype.to understand the genetic background of thermotolerance, we determined the complete genome sequence of a thermotolerant corynebacterium glutamicum n24 strain isolated from soil. the whole genome based phylogenetic analysis between n24 and other related species revealed that n24 diverged from other c. glutamicum strains at earlier stages. comparisons of thermotolerance between n24 and its related species showed that n24 and corynebacterium efficiens ys-314 have a higher thermotolerance than coryn ...201728249784
beyond amino acids: use of the corynebacterium glutamicum cell factory for the secretion of heterologous proteins.the gram-positive soil bacterium corynebacterium glutamicum has a long tradition in industry as a potent cell factory for the production of various amino acids. besides this, in the last few years it became increasingly clear that this microorganism can also efficiently be used as a host organism for the expression and secretion of biotechnologically or pharmaceutically relevant heterologous proteins. in this review, first a short overview is given on the two main protein export pathways (sec an ...201728238807
improvement of succinate production by release of end-product inhibition in corynebacterium glutamicum.succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. however, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. in this study, we report the development of metabolically engineered corynebacterium glutamicum for high production of succinate by release of ...201728232033
click-chemistry approach to study mycoloylated proteins: evidence for porb and porc porins mycoloylation in corynebacterium glutamicum.protein mycoloylation is a recently identified, new form of protein acylation. this post-translational modification consists in the covalent attachment of mycolic acids residues to serine. mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of corynebacteriales, a bacterial order that includes important genera such as mycobacterium, nocardia or corynebacterium. so far, only 3 mycoloylated proteins have been identified: pora, porh a ...201728199365
comparative analysis of corynebacterium glutamicum genomes: a new perspective for the industrial production of amino acids.corynebacterium glutamicum is a non-pathogenic bacterium widely used in industrial amino acid production and metabolic engineering research. although the genome sequences of some c. glutamicum strains are available, comprehensive comparative genome analyses of these species have not been done. six wild type c. glutamicum strains were sequenced using next-generation sequencing technology in our study. together with 20 previously reported strains, we present a comprehensive comparative analysis of ...201728198668
cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered corynebacterium glutamicum.production of 1,3-propanediol (1,3-pdo) from glycerol is a promising route toward glycerol biorefinery. however, the yield of 1,3-pdo is limited due to the requirement of nadh regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. glutamate fermentation by corynebacterium glutamicum is an important oxidation process generating excess nadh. in this study, we proposed a novel strategy to couple the process of 1,3-pdo synthesis with glutamate production ...201728176878
structural basis for redox sensitivity in corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis.diaminopimelate epimerase (dapf) is one of the crucial enzymes involved in l-lysine biosynthesis, where it converts l,l-diaminopimelate (l,l-dap) into d,l-dap. dapf is also considered as an attractive target for the development of antibacterial drugs. here, we report the crystal structure of dapf from corynebacterium glutamicum (cgdapf). structures of cgdapf obtained under both oxidized and reduced conditions reveal that the function of cgdapf is regulated by redox-switch modulation via reversib ...201728176858
fermentative production of l-pipecolic acid from glucose and alternative carbon sources.corynebacterium glutamicum is used for the million-ton scale production of amino acids and has recently been engineered for production of the cyclic non-proteinogenic amino acid l-pipecolic acid (l-pa). in this synthetic pathway l-lysine was converted to l-pa by oxidative deamination, dehydration and reduction by l-lysine 6-dehydrogenase (deaminating) from silicibacter pomeroyi and pyrroline 5-carboxylate reductase from c. glutamicum. however, production of l-pa occurred as by-product of l-lysin ...201728169491
l-lysine production by bacillus methanolicus: genome-based mutational analysis and l-lysine secretion engineering.bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. this bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. the genomes of two different l-lysine secreting b. methanolicus classical mutant strains, noa2#13a52-8a66 and m168-20, were sequenced. we focused on mutational mapping in g ...201728163092
modular pathway engineering of corynebacterium glutamicum to improve xylose utilization and succinate production.xylose-negative corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (xi) pathway or the weimberg pathway. heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. in this study, we show that two functionally-redundant transcriptional regulators (gntr1 and gntr2) present on xylose repress the pentose phosphate pathway genes. for efficient xylose ...201728153765
comprehensive and accurate tracking of carbon origin of lc-tandem mass spectrometry collisional fragments for (13)c-mfa.in recent years the benefit of measuring positionally resolved (13)c-labeling enrichment from tandem mass spectrometry (ms/ms) collisional fragments for improved precision of (13)c-metabolic flux analysis ((13)c-mfa) has become evident. however, the usage of positional labeling information for (13)c-mfa faces two challenges: (1) the mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) the positional identity of carb ...201728116490
global transcriptomic analysis of the response of corynebacterium glutamicum to ferulic acid.corynebacterium glutamicum can survive by using ferulic acid as the sole carbon source. in this study, we assessed the response of c. glutamicum to ferulic acid stress by means of a global transcriptional response analysis. the transcriptional data showed that several genes involved in degradation of ferulic acid were affected. moreover, several genes related to the stress response; protein protection or degradation and dna repair; replication, transcription and translation; and the cell envelop ...201727766354
comparing galactan biosynthesis in mycobacterium tuberculosis and corynebacterium diphtheriae.the suborder corynebacterineae encompasses species like corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as corynebacterium diphtheriae and mycobacterium tuberculosis, which cause devastating human diseases. a distinctive component of the corynebacterineae cell envelope is the mycolyl-arabinogalactan (mag) complex. the mag is composed of lipid mycolic acids, and arabinofuranose (araf) and galactofuranose (galf) carbohydrate residues. elucidat ...201728039359
inactivation of genes involved in base excision repair of corynebacterium glutamicum and survival of the mutants in presence of various mutagens.base excision repair (ber) is considered as the most active dna repair pathway in vivo, which is initiated by recognition of the nucleotide lesions and excision of the damaged dna base. the genome of corynebacterium glutamicum atcc 13032 contains various dna glycosylases encoding genes (ung, fpg/mutm, tagi, alka, muty), two ap-endonuclease encoding genes (nei and nth) and an exonuclease encoding gene xth. to investigate the role of these genes during dna repair in c. glutamicum, mutants with del ...201728391506
heterologous expression of the halothiobacillus neapolitanus carboxysomal gene cluster in corynebacterium glutamicum.compartmentalization represents a ubiquitous principle used by living organisms to optimize metabolic flux and to avoid detrimental interactions within the cytoplasm. proteinaceous bacterial microcompartments (bmcs) have therefore created strong interest for the encapsulation of heterologous pathways in microbial model organisms. however, attempts were so far mostly restricted to escherichia coli. here, we introduced the carboxysomal gene cluster of halothiobacillus neapolitanus into the biotech ...201728359868
redirecting carbon flux through pgi-deficient and heterologous transhydrogenase toward efficient succinate production in corynebacterium glutamicum.corynebacterium glutamicum is particularly known for its potentiality in succinate production. we engineered c. glutamicum for the production of succinate. to enhance c3-c4 carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain nc-4. to increase intracellular nadh pools, the pntab gene from escherichia coli, encoding for transhydrogenase, was chromosomally integrated into nc-4, leading to strain nc-5. furthermore, we deleted pgi gene in strain n ...201728303352
functional expression of plant-derived o-methyltransferase, flavanone 3-hydroxylase, and flavonol synthase in corynebacterium glutamicum for production of pterostilbene, kaempferol, and quercetin.plant polyphenols receive significant attention due to their anti-oxidative and health-promoting properties, and several microorganisms are currently engineered towards producing these valuable compounds. previously, corynebacterium glutamicum has been engineered for synthesizing polyphenol core structures such as the stilbene resveratrol and the (2s)-flavanone naringenin. decoration of these compounds by o-methylation or hydroxylation would provide access to polyphenols of even higher commercia ...201728143765
heat-killed cell preparation of corynebacterium glutamicum stimulates the immune activity and improves survival of mice against enterohemorrhagic escherichia coli.fermentation by corynebacterium glutamicum is used by various industries to produce l-glutamate, and the heat-killed cell preparation of this bacterium (hccg) is a by-product of the fermentation process. in present study, we evaluated the immunostimulating and survival effects against enterohemorrhagic escherichia coli (stec) infection of hccg. hccg significantly stimulated in vitro iga and interleukin-12 p70 production in murine peyer's patch cells and peritoneal macrophages, respectively. oral ...201728137189
biotin-independent strains of escherichia coli for enhanced streptavidin production.biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. however, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. here we describe the creation of biotin-independent strains of escherichia coli and corynebacterium glutamicum through installation of an optimized malonyl-coa b ...201728062280
polynucleotide phosphorylase, rnase e/g, and ybey are involved in the maturation of 4.5s rna in corynebacterium glutamicum.corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. to understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (srp) assembly. srp is found in all three domains of life and plays an important role in the membrane insertion of proteins. srp rna is initially transcribed as precursor molecules; however, relatively little is known about its maturation. in c. gl ...201728031281
enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant escherichia coli.poly(3-hydroxypropionate) (p3hp) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest p3hp production. however, exogenous supply of vitamin b12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. to avoid the addition of vb12, we have previously constructed a p3hp biosynthetic route with β-alanine as intermediate, and the presen ...201728253372
bacterial genome editing via a designed toxin-antitoxin cassette.manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. here, we reprogrammed the bacterial ta systems as the toxin counter-selectable cassette regulated by an antitoxin switch (tccras) for genetic modifications in the extensively studied and utilized gram-positive bacteria, b. subtilis and corynebacterium glutamicum. in the five characterized type ii ta systems, the relbe complex can specifically and efficiently regulate cell growth an ...201728094982
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