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Sample records for recombinant corynebacterium glutamicum

  1. Expression of recombinant protein using Corynebacterium Glutamicum: progress, challenges and applications.

    PubMed

    Liu, Xiuxia; Yang, Yankun; Zhang, Wei; Sun, Yang; Peng, Feng; Jeffrey, Laura; Harvey, Linda; McNeil, Brian; Bai, Zhonghu

    2016-08-01

    Corynebacterium glutamicum (C. glutamicum) is a highly promising alternative prokaryotic host for recombinant protein expression, as it possesses several significant advantages over Escherichia coli (E. coli), the currently leading bacterial protein expression system. During the past decades, several experimental techniques and vector components for genetic manipulation of C. glutamicum have been developed and validated, including strong promoters for tightly regulating target gene expression, various types of plasmid vectors, protein secretion systems and methods of genetically modifying the host strain genome to improve protein production potential. This review critically discusses current progress in establishing C. glutamicum as a host for recombinant protein expression, and examines, in depth, some successful case studies of actual application of this expression system. The established "expression tool box" for developing novel constructs based on C. glutamicum as a host are also evaluated. Finally, the existing issues and solutions in process development with C. glutamicum as a host are specifically addressed. PMID:25714007

  2. Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli

    PubMed Central

    2014-01-01

    Background Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. Result In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. Conclusions The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt. PMID:24885047

  3. Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production.

    PubMed

    Seibold, Gerd; Auchter, Marc; Berens, Stephan; Kalinowski, Jörn; Eikmanns, Bernhard J

    2006-07-13

    Corynebacterium glutamicum, well known for the industrial production of amino acids, grows aerobically on a variety of mono- and disaccharides and on alcohols and organic acids as single or combined sources of carbon and energy. Members of the genera Corynebacterium and Brevibacterium were here tested for their ability to use the homopolysaccharide starch as a substrate for growth. None of the 24 type strains tested showed growth on or degradation of this substrate, indicating that none of the strains synthesized and secreted starch-degrading enzymes. Introducing the Streptomyces griseus amy gene on an expression vector into the lysine-producer C. glutamicum DM1730, we constructed a C. glutamicum strain synthesizing and secreting alpha-amylase into the culture broth. Although some high-molecular-weight degradation products remained in the culture broth, this recombinant strain effectively used soluble starch as carbon and energy substrate for growth and also for lysine production. Thus, employment of our construct allows avoidance of the cost-intensive enzymatic hydrolysis of the starch, which commercially is used as a substrate in industrial amino acid fermentations. PMID:16488498

  4. High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.

    PubMed

    Cheng, Fangyu; Gong, Qianying; Yu, Huimin; Stephanopoulos, Gregory

    2016-03-01

    Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw . The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA-hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects. PMID:26709615

  5. Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

    PubMed

    Yim, Sung Sun; Choi, Jae Woong; Lee, Roo Jin; Lee, Yong Jae; Lee, Se Hwa; Kim, So Young; Jeong, Ki Jun

    2016-01-01

    Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum. PMID:26134574

  6. Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation.

    PubMed

    Binder, Stephan; Siedler, Solvej; Marienhagen, Jan; Bott, Michael; Eggeling, Lothar

    2013-07-01

    Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause L-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology. PMID:23630315

  7. 3-Amino-4-hydroxybenzoic acid production from sweet sorghum juice by recombinant Corynebacterium glutamicum.

    PubMed

    Kawaguchi, Hideo; Sasaki, Kengo; Uematsu, Kouji; Tsuge, Yota; Teramura, Hiroshi; Okai, Naoko; Nakamura-Tsuruta, Sachiko; Katsuyama, Yohei; Sugai, Yoshinori; Ohnishi, Yasuo; Hirano, Ko; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko

    2015-12-01

    The production of the bioplastic precursor 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from sweet sorghum juice, which contains amino acids and the fermentable sugars sucrose, glucose and fructose, was assessed to address the limitations of producing bio-based chemicals from renewable feedstocks. Recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI derived from Streptomyces griseus produced 3,4-AHBA from the sweet sorghum juice of cultivar SIL-05 at a final concentration (1.0 g l(-1)) that was 5-fold higher than that from pure sucrose. Fractionation of sweet sorghum juice by nanofiltration (NF) membrane separation (molecular weight cut-off 150) revealed that the NF-concentrated fraction, which contained the highest concentrations of amino acids, increased 3,4-AHBA production, whereas the NF-filtrated fraction inhibited 3,4-AHBA biosynthesis. Amino acid supplementation experiments revealed that leucine specifically enhanced 3,4-AHBA production by strain KT01. Taken together, these results suggest that sweet sorghum juice is a potentially suitable feedstock for 3,4-AHBA production by recombinant C. glutamicum. PMID:26409852

  8. 4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp. lactofermentum under optimal corn steep liquor limitation.

    PubMed

    Shi, Feng; Niu, Tengfei; Fang, Huimin

    2015-05-01

    4-Hydroxyisoleucine (4-HIL) is a nonproteinogenic amino acid that exhibits insulinotropic biological activity. Here, L-isoleucine dioxygenase gene (ido) derived from Bacillus thuringiensis YBT-1520 was cloned and expressed in an L-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, in order to directly convert its endogenous L-isoleucine (Ile) into 4-HIL through single-step fermentation. The effects of corn steep liquor limitation as well as ido and truncated idoΔ6 overexpression on 4-HIL production were researched. 4-HIL production by ido-overexpressing strain was improved to 65.44 ± 2.27 mM after fermented for 144 h under corn steep liquor-subsufficient condition, obviously higher than that under corn steep liquor-rich and insufficient conditions. The conversion ratio of Ile to 4-HIL increased to 0.85 mol/mol. In addition, 4-HIL production by ido-overexpressing strain was higher than that by idoΔ6-overexpressing strain, in accord with the relatively higher affinity of Ido as compared to IdoΔ6. This research generated a novel system for 4-HIL de novo biosynthesis and demonstrated corn steep liquor limitation as a useful strategy for improving 4-HIL production in recombinant C. glutamicum ssp. lactofermentum. PMID:25725632

  9. Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum.

    PubMed

    Zhang, Yuan-Yuan; Bu, Yi-Fan; Liu, Jian-Zhong

    2015-09-01

    Sucrose and molasses are attractive raw materials for industrial fermentation. Although Corynebacterium glutamicum shows sucrose-utilizing activity, sucrose or molasses is only a fraction of carbon source used in the fermentation medium in most works. An engineered C. glutamicum strain was constructed for producing L-ornithine with sucrose or molasses as a sole carbon source by transferring Mannheimia succiniciproducens β-fructofuranosidase gene (sacC). The engineered strain, C. glutamicum ΔAPE6937R42 (pEC-sacC), produced 22.0 g/L of L-ornithine with sucrose as the sole carbon source, which is on par with that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. The resulting strain C. glutamicum ΔAPE6937R42 (pEC-sacC) produced 27.0 g/L of L-ornithine with molasses as the sole carbon source, which is higher than that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. This strategy can be applied for developing sucrose- or molasses-utilizing industrial strains. PMID:25527174

  10. Construction of Synthetic Promoter-Based Expression Cassettes for the Production of Cadaverine in Recombinant Corynebacterium glutamicum.

    PubMed

    Oh, Young Hoon; Choi, Jae Woo; Kim, Eun Young; Song, Bong Keun; Jeong, Ki Jun; Park, Kyungmoon; Kim, Il-Kwon; Woo, Han Min; Lee, Seung Hwan; Park, Si Jae

    2015-08-01

    Corynebacterium glutamicum is an important microorganism in the biochemical industry for the production of various platform chemicals. However, despite its importance, a limited number of studies have been conducted on how to constitute gene expression cassettes in engineered C. glutamicum to obtain desired amounts of the target products. Therefore, in this study, six expression cassettes for the expression of the second lysine decarboxylase of Escherichia coli, LdcC, were constructed using six synthetic promoters with different strengths and were examined in C. glutamicum for the production of cadaverine. Among six expression cassettes, the expression of the E. coli ldcC gene under the PH30 promoter supported the highest production of cadaverine in flask and fed-batch cultivations. A fed-batch fermentation of recombinant C. glutamicum expressing E. coli ldcC gene under the PH30 promoter resulted in the production of 40.91 g/L of cadaverine in 64 h. This report is expected to contribute toward developing engineered C. glutamicum strains to have desired features. PMID:26047931

  11. Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum.

    PubMed

    Gopinath, Vipin; Meiswinkel, Tobias M; Wendisch, Volker F; Nampoothiri, K Madhavan

    2011-12-01

    Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the L-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more L-glutamate and L-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM L-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM L-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production. PMID:21796382

  12. Production of the amino acids l-glutamate, l-lysine, l-ornithine and l-arginine from arabinose by recombinant Corynebacterium glutamicum.

    PubMed

    Schneider, Jens; Niermann, Karin; Wendisch, Volker F

    2011-07-10

    Amino acid production processes with Corynebacterium glutamicum are based on media containing glucose from starch hydrolysis or fructose and sucrose as present in molasses. Simultaneous utilization of various carbon sources, including glucose, fructose and sucrose, in blends is a typical characteristic of this bacterium. The renewable non-food carbon source arabinose, which is present in hemicellulosic hydrolysates, cannot be utilized by most C. glutamicum strains. Heterologous expression of the araBAD operon from Escherichia coli in the wild-type and in an l-lysine producing strain of C. glutamicum was shown to enable production of l-glutamate and l-lysine, respectively, from arabinose as sole carbon source. l-Ornithine and l-arginine producing strains were constructed and shown to produce l-ornithine and l-arginine from arabinose when araBAD from E. coli was expressed. Moreover, the recombinant strains produced l-glutamate, l-lysine, l-ornithine and l-arginine respectively, from arabinose also when glucose-arabinose blends were used as carbon sources. PMID:20638422

  13. The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse.

    PubMed

    Lee, Joo-Young; Na, Yoon-Ah; Kim, Eungsoo; Lee, Heung-Shick; Kim, Pil

    2016-05-28

    Starting as a glutamate producer, Corynebacterium glutamicum has played a variety of roles in the industrial production of amino acids, one of the most important areas of white biotechnology. From shortly after its genome information became available, C. glutamicum has been applied in various production processes for value-added chemicals, fuels, and polymers, as a key organism in industrial biotechnology alongside the surprising progress in systems biology and metabolic engineering. In addition, recent studies have suggested another potential for C. glutamicum as a synthetic biology platform chassis that could move the new era of industrial microbial biotechnology beyond the classical field. Here, we review the recent progress and perspectives in relation to C. glutamicum, which demonstrate it as one of the most promising and valuable workhorses in the field of industrial biotechnology. PMID:26838341

  14. D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii

    PubMed Central

    Park, Chul-Soon; Kim, Taeyong; Hong, Seung-Hye; Shin, Kyung-Chul; Kim, Kyoung-Rok; Oh, Deok-Kun

    2016-01-01

    A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively. PMID:27467527

  15. Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation.

    PubMed

    Mimitsuka, Takashi; Sawai, Hideki; Hatsu, Masahiro; Yamada, Katsushige

    2007-09-01

    Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum. PMID:17895539

  16. Glycerol-3-phosphatase of Corynebacterium glutamicum.

    PubMed

    Lindner, Steffen N; Meiswinkel, Tobias M; Panhorst, Maren; Youn, Jung-Won; Wiefel, Lars; Wendisch, Volker F

    2012-06-15

    Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg²⁺ or Mn²⁺ for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg⁻¹ with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and

  17. Construction of l-Isoleucine Overproducing Strains of Corynebacterium glutamicum

    NASA Astrophysics Data System (ADS)

    Sahm, H.; Eggeling, L.; Morbach, S.; Eikmanns, B.

    Nowadays the gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of the amino acids l-glutamate (1×106tons/year) and l-lysine (300×103tons/year). The classical approach to obtain amino acid overproducing strains of C. glutamicum was mutagenesis and then a selection of mutants. In the past 10 years the genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for constructing microbial strains with desired genotypes. To obtain l-isoleucine overproducing strains of C. glutamicum we therefore studied the l-isoleucine biosynthesis by overexpression of the various corresponding genes. To enable a flux increase in recombinant strains all genes specific for l-threonine and l-isoleucine biosynthesis were cloned from this bacterium. We demonstratet that amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enable the channeling of the carbon flow from the intermediate l-aspartate semialdehyde towards homoserine, resulting in an accumulation of l-threonine. To obtain effective l-isoleucine overproduction a deregulated threonine dehydratase was overexpressed in l-threonine producing strains of C. glutamicum. In this way the l-threonine was converted to l-isoleucine, which was secreted up to 30g/l into the culture medium.

  18. 3-Methyl-1-butanol Biosynthesis in an Engineered Corynebacterium glutamicum.

    PubMed

    Xiao, Shiyuan; Xu, Jingliang; Chen, Xiaoyan; Li, Xiekun; Zhang, Yu; Yuan, Zhenhong

    2016-05-01

    Biofuel offers a promising solution to the adverse environmental problems and depletion in reserves of fossil fuels. Higher alcohols including 3-methyl-1-butanol were paid much more attention as fuel substitute in recent years, due to its similar properties to gasoline. In the present work, 3-methyl-1-butanol production in engineered Corynebacterium glutamicum was studied. α-Ketoisovalerate decarboxylase gene (kivd) from Lactococcus lactis combined with alcohol dehydrogenase gene (adh2, adhA, and adh3) from three organisms were overexpressed in C. glutamicum. Enzymatic assay and alcohol production results showed that adh3 from Zymomonas mobilis was the optimum candidate for 3-methyl-1-butanol production in C. glutamicum. The recombinant with kivd and adh3 could produce 0.182 g/L of 3-methyl-1-butanol and 0.144 g/L of isobutanol after 12 h of incubation. Further inactivation of the E1 subunit of pyruvate dehydrogenase complex gene (aceE) and lactic dehydrogenase gene (ldh) in the above C. glutamicum strain would improve the 3-Methyl-1-butanol titer to 0.497 g/L after 12 h of incubation. PMID:26961908

  19. Synthetic promoter libraries for Corynebacterium glutamicum.

    PubMed

    Rytter, Jakob Vang; Helmark, Søren; Chen, Jun; Lezyk, Mateusz Jakub; Solem, Christian; Jensen, Peter Ruhdal

    2014-03-01

    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 with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms. PMID:24458563

  20. Corynebacterium glutamicum promoters: a practical approach

    PubMed Central

    Pátek, Miroslav; Holátko, Jiří; Busche, Tobias; Kalinowski, Jörn; Nešvera, Jan

    2013-01-01

    Summary Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate ‘green waves’ in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined. PMID:23305350

  1. Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

    NASA Astrophysics Data System (ADS)

    Wittmann, Christoph

    The Gram-positive soil bacterium Corynebacterium glutamicum was discovered as a natural overproducer of glutamate about 50 years ago. Linked to the steadily increasing economical importance of this microorganism for production of glutamate and other amino acids, the quest for efficient production strains has been an intense area of research during the past few decades. Efficient production strains were created by applying classical mutagenesis and selection and especially metabolic engineering strategies with the advent of recombinant DNA technology. Hereby experimental and computational approaches have provided fascinating insights into the metabolism of this microorganism and directed strain engineering. Today, C. glutamicum is applied to the industrial production of more than 2 million tons of amino acids per year. The huge achievements in recent years, including the sequencing of the complete genome and efficient post genomic approaches, now provide the basis for a new, fascinating era of research - analysis of metabolic and regulatory properties of C. glutamicum on a global scale towards novel and superior bioprocesses.

  2. Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)

    PubMed Central

    2016-01-01

    Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection. PMID:26829286

  3. Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi).

    PubMed

    Cleto, Sara; Jensen, Jaide Vk; Wendisch, Volker F; Lu, Timothy K

    2016-05-20

    Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection. PMID:26829286

  4. Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

    PubMed Central

    Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan

    2015-01-01

    Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate. PMID:25595770

  5. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    SciTech Connect

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.

  6. Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum.

    PubMed

    Hu, Jinyu; Tan, Yanzhen; Li, Yanyan; Hu, Xiaoqing; Xu, Daqing; Wang, Xiaoyuan

    2013-11-01

    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) for replicating in C. glutamicum only at low temperatures; it has high transformation efficiency in C. glutamicum and can be easily eliminated by growing at 37°C. pDTW201 and pDTW202 carry loxp-flanked or mutant lox-flanked kan, respectively. This deletion system combines homologous recombination and Cre/lox site-specific recombination, could efficiently delete the aceE gene from the chromosome of C. glutamicum ATCC13032, ATCC13869 or ATCC14067, respectively, and could also delete both genes of aceE and ilvA from the chromosome of C. glutamicum ATCC14067. The system is simple and efficient, and can be easily implemented for multiple-gene-deletion in C. glutamicum. PMID:23856168

  7. Crystal Structure of Amylomaltase from Corynebacterium glutamicum.

    PubMed

    Joo, Seongjoon; Kim, Sangwoo; Seo, Hogyun; Kim, Kyung-Jin

    2016-07-20

    Amylomaltase is an essential enzyme in maltose utilization and maltodextrin metabolism, and it has been industrially used for the production of cyclodextrin and modification of starch. We determined the crystal structure of amylomaltase from Corynebacterium glutamicum (CgAM) at a resolution of 1.7 Å. Although CgAM forms a dimer without NaCl, it exists as a monomer in physiological concentration of NaCl. CgAM is composed of N- and C-terminal domains, which can be further divided into two and four subdomains, respectively. It exhibits a unique structural feature at the functionally unknown N-domain and also shows two striking differences at the C-domain compared to other amylomaltases. These differences at extended edge of the substrate-binding site might affect substrate specificity for large cyclodextrin formation. The bis-tris methane and sulfate molecules bound at the substrate-binding site of our current structure mimic the binding of the hydroxyl groups of glucose bound at subsites -1 and -2, respectively. PMID:27366969

  8. Improving the secretion of cadaverine in Corynebacterium glutamicum by cadaverine-lysine antiporter.

    PubMed

    Li, Ming; Li, Dongxia; Huang, Yunyan; Liu, Meng; Wang, Hongxin; Tang, Qi; Lu, Fuping

    2014-04-01

    Cadaverine (1,5-pentanediamine, diaminopentane), the desired raw material of bio-polyamides, is an important industrial chemical with a wide range of applications. Biosynthesis of cadaverine in Corynebacterium glutamicum has been a competitive way in place of petroleum-based chemical synthesis method. To date, the cadaverine exporter has not been found in C. glutamicum. In order to improve cadaverine secretion, the cadaverine-lysine antiporter CadB from Escherichia coli was studied in C. glutamicum. Fusion expression of cadB and green fluorescent protein (GFP) gene confirmed that CadB could express in the cell membrane of C. glutamicum. Co-expression of cadB and ldc from Hafnia alvei in C. glutamicum showed that the cadaverine secretion rate increased by 22 % and the yield of total cadaverine and extracellular cadaverine increased by 30 and 73 %, respectively. Moreover, the recombinant strain cultured at acid and neutral pH separately hardly had any difference in cadaverine concentrations. These results suggested that CadB could be expressed in the cell membrane of C. glutamicum and that recombinant CadB could improve cadaverine secretion and the yield of cadaverine. Moreover, the pH value did not affect the function of recombinant CadB. These results may be a promising metabolic engineering strategy for improving the yield of the desired product by enhancing its export out of the cell. PMID:24510022

  9. Metabolic engineering Corynebacterium glutamicum to produce triacylglycerols.

    PubMed

    Plassmeier, Jens; Li, Youyuan; Rueckert, Christian; Sinskey, Anthony J

    2016-01-01

    In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38 ± 0.05 g/L intracellular fatty acids and 0.64 ± 0.09 g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8 ± 0.5% of the dry cell. PMID:26645801

  10. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

    PubMed Central

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki

    2015-01-01

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  11. Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum.

    PubMed

    Henke, Nadja A; Heider, Sabine A E; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-01-01

    Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L(-1)·h(-1) which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L(-1), the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum. PMID:27376307

  12. Production and glucosylation of C50 and C 40 carotenoids by metabolically engineered Corynebacterium glutamicum.

    PubMed

    Heider, Sabine A E; Peters-Wendisch, Petra; Netzer, Roman; Stafnes, Marit; Brautaset, Trygve; Wendisch, Volker F

    2014-02-01

    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 from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C50 carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C50 carotenoids were glucosylated. We here also demonstrate production of the C40 carotenoids β-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C40 and C50 carotenoids. PMID:24270893

  13. Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum

    PubMed Central

    Henke, Nadja A.; Heider, Sabine A. E.; Peters-Wendisch, Petra; Wendisch, Volker F.

    2016-01-01

    Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L−1·h−1 which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L−1, the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum. PMID:27376307

  14. Identification of a suppressor gene for the arginine-auxotrophic argJ mutation in Corynebacterium glutamicum.

    PubMed

    Hwang, Gui-Hye; Cho, Jae-Yong

    2010-11-01

    We recently proposed a metabolic engineering strategy for L-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance L-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg(-1) protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted L-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during L-ornithine production in C. glutamicum. PMID:20544254

  15. Overexpression of ppc and lysC to improve the production of 4-hydroxyisoleucine and its precursor l-isoleucine in recombinant Corynebacterium glutamicum ssp. lactofermentum.

    PubMed

    Shi, Feng; Fang, Huimin; Niu, Tengfei; Lu, Zhengke

    2016-06-01

    4-hydroxyisoleucine (4-HIL) exhibits unique insulinotropic and insulin-sensitizing activities and is an attractive candidate for the treatment of type II and type I diabetes. In our previous study, l-isoleucine dioxygenase gene (ido) was cloned and overexpressed in an l-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, and 4-HIL was produced from the endogenous l-isoleucine (Ile). In this study, ppc and lysC were co-expressed with ido to increase the supply of Ile, the direct precursor of 4-HIL, and to further improve the 4-HIL yield. After 144h of fermentation, the ido-ppc-expressing strain produced 95.72±1.52mM 4-HIL, 29% higher than the ido-expressing strain. The co-expression of lysC and ppc with ido resulted in a further 35% increment of carbon flux to l-aspartate family amino acids biosynthesis pathway. However, the conversion ratio of Ile to 4-HIL and the 4-HIL yield decreased to 0.31mol/mol and 30.16±2.01mM, respectively, likely due to the decreased IDO activity caused by lower pH and higher intracellular Ile concentration. Therefore, co-expression of ido and ppc was benefit for 4-HIL de novo biosynthesis, while co-expression of lysC with ido and ppc decreased the conversion ratio of Ile to 4-HIL. PMID:27178798

  16. Rapid assessment of oxygen transfer impact for Corynebacterium glutamicum.

    PubMed

    Käß, Friedrich; Prasad, Arjun; Tillack, Jana; Moch, Matthias; Giese, Heiner; Büchs, Jochen; Wiechert, Wolfgang; Oldiges, Marco

    2014-12-01

    Oxygen supply is crucial in industrial application of microbial systems, such as Corynebacterium glutamicum, but oxygen transfer is often neglected in early strain characterizations, typically done under aerobic conditions. In this work, a new procedure for oxygen transfer screening is presented, assessing the impact of maximum oxygen transfer conditions (OTRmax) within microtiter plate-based cultivation for enhanced throughput. Oxygen-dependent growth and productivity were characterized for C. glutamicum ATCC13032 and C. glutamicum DM1933 (lysine producer). Biomass and lysine product yield are affected at OTRmax below 14 mmol L(-1) h(-1) in a standardized batch process, but not by further increase of OTRmax above this threshold value indicating a reasonable tradeoff between power input and oxygen transfer capacity OTRmax. The described oxygen transfer screening allows comparative determination of metabolic robustness against oxygen transfer limitation and serves identification of potential problems or opportunities later created during scale-up. PMID:24981020

  17. Ohr Protects Corynebacterium glutamicum against Organic Hydroperoxide Induced Oxidative Stress

    PubMed Central

    Xiao, Xiao; Guan, Jingyuan; Zhang, Yaoling; Ding, Wei; Chaudhry, Muhammad Tausif; Wang, Yao; Shen, Xihui

    2015-01-01

    Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum. PMID:26121694

  18. Improvement of L-citrulline production in Corynebacterium glutamicum by ornithine acetyltransferase.

    PubMed

    Hao, N; Mu, J; Hu, N; Xu, S; Yan, M; Li, Y; Guo, K; Xu, L

    2015-02-01

    In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce L-citrulline through a metabolic engineering strategy. To prevent the flux away from L-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene (argG) and the repressor gene (argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2) produced higher amounts of L-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of L-citrulline production, the effect of L-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19-argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during L-citrulline production in C. glutamicum. PMID:25492493

  19. Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum.

    PubMed

    Tzvetkov, Mladen V; Liebl, Wolfgang

    2008-04-30

    Heterologous expression of a phytase gene (phyC) from Bacillus amyloliquefaciens DSM 7 enabled the growth of Corynebacterium glutamicum with phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) as a new, sole source of phosphorus. Phytate was not used as a carbon source. During growth of the phyC-expressing amino acid (l-lysine)-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) with phytate as the source of phosphorus, merely a small, transient accumulation of inorganic phosphate was observed in the fermentation broth. At the later stages of fermentation, free inorganic phosphate from phytate degradation was no longer detectable. Growth and l-lysine production by the phytase-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) in phytate medium did not differ significantly from control experiments with strain C. glutamicum ATCC 21253 (pWLQ2) conducted with an excess of inorganic phosphate, demonstrating that there was no phosphate limitation when phytate was used as the phosphorus source. Under the expression conditions employed, only part of PhyC was secreted to the culture broth by C. glutamicum, but this did not significantly affect growth or lysine production. PMID:18374441

  20. Construction of a novel expression system for use in Corynebacterium glutamicum.

    PubMed

    Hu, Jinyu; Li, Yanyan; Zhang, Hailing; Tan, Yanzhen; Wang, Xiaoyuan

    2014-09-01

    Corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. Suitable vectors are needed for metabolic engineering in C. glutamicum. Most available vectors used in C. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. These vectors, though excellent for laboratory use, are not preferable choices for industry-scale fermentation. In this work, we developed a novel expression system for use in C. glutamicum, which do not require antibiotics when used for industrial fermentation. This system includes two vectors: the shuttle vector pJYW-4 for expression of genes and the vector pJYW-6 for deletion of the essential gene alr in C. glutamicum. The vector pJYW-4 contains a large multiple cloning site for cloning multiple genes and two selective markers: one is the kanamycin-resistant gene kan and the other is an essential gene alr. The selective marker kan facilitates molecular manipulation or fermentations in the laboratory, and the selection marker alr is good for use in industry-scale fermentation, allowing in vivo maintenance of the expression vector through auxotrophic complementation; therefore, the two selection markers in pJYW-4 make it useful for both laboratory research and industrial fermentation, and convenient to transfer valuable laboratory-developed strains into industrial production. This newly-constructed expression system was successfully used to increase L-valine production in C. glutamicum ATCC 14067, indicating its potential on developing amino acid-producing C. glutamicum strains. PMID:25108235

  1. Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

    PubMed

    Peters-Wendisch, P; Götker, S; Heider, S A E; Komati Reddy, G; Nguyen, A Q; Stansen, K C; Wendisch, V F

    2014-12-20

    The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-negative Escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of bioF together with bioC and bioH from E. coli did not entail biotin prototrophy. Heterologous expression of bioWAFDBI from Bacillus subtilis encoding another biotin synthesis pathway in C. glutamicum allowed for growth in biotin-depleted media. Stable growth of the recombinant was observed without biotin addition for eight transfers to biotin-depleted medium while the empty vector control stopped growth after the first transfer. Expression of bioWAFDBI from B. subtilis in C. glutamicum strains overproducing the amino acids l-lysine and l-arginine, the diamine putrescine, and the carotenoid lycopene, respectively, enabled formation of these products under biotin-depleted conditions. Thus, biotin-prototrophic growth and production by recombinant C. glutamicum were achieved. PMID:24486440

  2. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    PubMed Central

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-01

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum. PMID:25622822

  3. Effect of Corynebacterium glutamicum on Livestock Material Burial Treatment.

    PubMed

    Kim, Bit-Na; Cho, Ho-Seong; Cha, Yougin; Park, Joon-Kyu; Kim, Geonha; Kim, Yang-Hoon; Min, Jiho

    2016-08-28

    In recent years, foot-and-mouth disease has occurred in all parts of the world. The animals with the disease are buried in the ground; therefore, their concentration could affect ground or groundwater. Moreover, the complete degradation of carcasses is not a certainty, and their disposal is important to prevent humans, livestock, and the environment from being affected with the disease. The treatment of Corynebacterium glutamicum is a feasible method to reduce the risk of carcass decomposition affecting humans or the environment. Therefore, this study aimed to investigate the effect of C. glutamicum on the soil environment with a carcass. The composition of amino acids in the soil treated with C. glutamicum was generally higher than those in the untreated soil. Moreover, the plant root in the soil samples treated with C. glutamicum had 84.0% amino acids relative to the standard value and was similar to that of the control. The results of this study suggest the possibility to reduce the toxicity of a grave land containing animals with this disease. PMID:27160580

  4. Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum

    PubMed Central

    Kulis-Horn, Robert K; Persicke, Marcus; Kalinowski, Jörn

    2014-01-01

    l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production. PMID:23617600

  5. Engineering of Corynebacterium glutamicum for growth and succinate production from levoglucosan, a pyrolytic sugar substrate.

    PubMed

    Kim, Eun-Mi; Um, Youngsoon; Bott, Michael; Woo, Han Min

    2015-10-01

    Thermochemical processing provides continuous production of bio-oils from lignocellulosic biomass. Levoglucosan, a pyrolytic sugar substrate C6H10O5 in a bio-oil, has been used for ethanol production using engineered Escherichia coli. Here we provide the first example for succinate production from levoglucosan with Corynebacterium glutamicum, a well-known industrial amino acid producer. Heterologous expression of a gene encoding a sugar kinase from Lipomyces starkeyi, Gibberella zeae or Pseudomonas aeruginosa was employed for levoglucosan conversion in C. glutamicum because the wild type was unable to utilize levoglucosan as sole carbon source. As result, expression of a levoglucosan kinase (LGK) of L. starkeyi only enabled growth with levoglucosan as sole carbon source in CgXII minimal medium by catalyzing conversion of levoglucosan to glucose-6-phosphate. Subsequently, the lgk gene was expressed in an aerobic succinate producer of C. glutamicum, strain BL-1. The recombinant strain showed a higher succinate yield (0.25 g g(-1)) from 2% (w/v) levoglucosan than the reference strain BL-1 from 2% (w/v) glucose (0.19 g g(-1)), confirming that levoglucosan is an attractive carbon substrate for C. glutamicum producer strains. In summary, we demonstrated that a pyrolytic sugar could be a potential carbon source for microbial cell factories. PMID:26363018

  6. Formation of volutin granules in Corynebacterium glutamicum.

    PubMed

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted. PMID:15668011

  7. A method for gene amplification and simultaneous deletion in Corynebacterium glutamicum genome without any genetic markers.

    PubMed

    Xu, Jianzhong; Xia, Xiuhua; Zhang, Junlan; Guo, Yanfeng; Qian, He; Zhang, Weiguo

    2014-03-01

    A method for the simultaneous replacement of a given gene by a target gene, leaving no genetic markers, has been developed. The method is based on insertional inactivation and double-crossover homologous recombination. With this method, the lysC(T311I), fbp and ddh genes were inserted into Corynebacterium glutamicum genome, and the pck, alaT and avtA genes were deleted. Mobilizable plasmids with lysC(T311I), fbp and ddh cassettes and two homologous arms on the ends of pck, alaT and avtA were constructed, and then transformed into C. glutamicum. The target-expression cassettes were inserted in the genome via the first homologous recombination, and the genetic markers were removed via the second recombination. The target-transformants were sequentially screened from kanamycin-resistance and sucrose-resistance plates. The enzyme activities of transformants were stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated cassettes in host were successfully expressed and maintained as stable chromosomal insertions in C. glutamicum. The target-transformants were used to optimize the l-lysine production, showing that the productions were strongly increased because the selected genes were closely linked to l-lysine production. In short, this method can be used to construct amino acid high-producing strains with unmarked gene amplification and simultaneous deletion in genome. PMID:24613758

  8. Characteristics of methionine production by an engineered Corynebacterium glutamicum strain.

    PubMed

    Park, Soo-Dong; Lee, Joo-Young; Sim, Soo-Yeon; Kim, Younhee; Lee, Heung-Shick

    2007-07-01

    A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene (hom(FBR)) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (delta thrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/hom(FBR)/delta thrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity. PMID:17604670

  9. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain.

    PubMed

    Gui, Yongli; Ma, Yuechao; Xu, Qingyang; Zhang, Chenglin; Xie, Xixian; Chen, Ning

    2016-02-20

    Here, we report the complete genome sequence of Corynebacterium glutamicum CP, an industrial l-leucine producing strain in China. The whole genome consists of a circular chromosome and a plasmid. The comparative genomics analysis shows that there are many mutations in the key enzyme coding genes relevant to l-leucine biosynthesis compared to C. glutamicum ATCC 13032. PMID:26784991

  10. Corynebacterium glutamicum Tailored for Efficient Isobutanol Production ▿ †

    PubMed Central

    Blombach, Bastian; Riester, Tanja; Wieschalka, Stefan; Ziert, Christian; Youn, Jung-Won; Wendisch, Volker F.; Eikmanns, Bernhard J.

    2011-01-01

    We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (YP/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the YP/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the YP/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H+ to NADPH+H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h−1, and showed an overall YP/S of about 0.48 mol per mol of glucose in the production phase. PMID:21441331

  11. The Zur regulon of Corynebacterium glutamicum ATCC 13032

    PubMed Central

    2010-01-01

    Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc

  12. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.

    PubMed

    Marx, Achim; Hans, Stephan; Möckel, Bettina; Bathe, Brigitte; de Graaf, Albert A; McCormack, Ashling C; Stapleton, Cliona; Burke, Kevin; O'Donohue, Michael; Dunican, L K

    2003-09-01

    A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential

  13. Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production

    PubMed Central

    2014-01-01

    Background Corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation. Results C. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGKHis was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg2+ for its activity. PGKHis is thermo labile and it has shown maximal activity at 50–65°C. The maximal activity of PGKHis was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively. Conclusion Unlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains. PMID:24593686

  14. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms. PMID:26300047

  15. Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine.

    PubMed

    Meiswinkel, Tobias M; Gopinath, Vipin; Lindner, Steffen N; Nampoothiri, K Madhavan; Wendisch, Volker F

    2013-03-01

    Because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h(-1), followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h(-1) and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased. PMID:23164409

  16. Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

    PubMed Central

    2013-01-01

    Background 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 release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems. Results Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media. Conclusions The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the

  17. Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

    PubMed

    Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

    2013-06-01

    Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity. PMID:23404100

  18. High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

    PubMed

    Ravasi, Pablo; Braia, Mauricio; Eberhardt, Florencia; Elena, Claudia; Cerminati, Sebastián; Peirú, Salvador; Castelli, Maria Eugenia; Menzella, Hugo G

    2015-12-20

    Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme. PMID:26519562

  19. The Corynebacterium glutamicum aconitase repressor: scratching around for crystals

    PubMed Central

    García-Nafría, Javier; Baumgart, Meike; Bott, Michael; Wilkinson, Anthony J.; Wilson, Keith S.

    2010-01-01

    Imperfections on the surfaces of crystallization containers are known to influence crystal formation and are thought to do so by helping to overcome the nucleation barrier. The intentional creation of imperfections has been widely applied to induce crystallization of small molecules, but has not been reported for protein crystallization. Here, the crystallization and preliminary X-ray analysis of the TetR-type aconitase repressor are reported. This regulator was the first transcription factor to be identified in the regulation of the tricarboxylic acid cycle in Corynebacterium glutamicum, an organism that is of special industrial interest and is an emerging model organism for Corynebacterineae. Successful crystallization involved introducing manual scratches on the surface of standard commercial plates, which led to a substantial improvement in crystal nucleation and quality. PMID:20823530

  20. Anaerobic growth of Corynebacterium glutamicum via mixed-acid fermentation.

    PubMed

    Michel, Andrea; Koch-Koerfges, Abigail; Krumbach, Karin; Brocker, Melanie; Bott, Michael

    2015-11-01

    Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. PMID:26276118

  1. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum.

    PubMed

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    "Lonely guy" (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a "PGGXGTXXE" motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms. PMID:27507425

  2. Metabolic engineering of Corynebacterium glutamicum for the production of itaconate.

    PubMed

    Otten, Andreas; Brocker, Melanie; Bott, Michael

    2015-07-01

    The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg(-1) and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l(-1)), a molar yield of 0.4 mol mol(-1), and a volumetric productivity of 2.1 mmol l(-1) h(-1). PMID:26100077

  3. Bio-based production of organic acids with Corynebacterium glutamicum.

    PubMed

    Wieschalka, Stefan; Blombach, Bastian; Bott, Michael; Eikmanns, Bernhard J

    2013-03-01

    The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, L- and D-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. PMID:23199277

  4. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum

    PubMed Central

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    “Lonely guy” (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a “PGGXGTXXE” motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms. PMID:27507425

  5. Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

    PubMed Central

    Michel, Andrea; Koch-Koerfges, Abigail; Krumbach, Karin; Brocker, Melanie

    2015-01-01

    Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. PMID:26276118

  6. Regulons of global transcription factors in Corynebacterium glutamicum.

    PubMed

    Toyoda, Koichi; Inui, Masayuki

    2016-01-01

    Corynebacterium glutamicum, a high GC content gram-positive soil bacterium in Actinobacteria, has been used for the industrial production of amino acids and engineered to produce various compounds, including polymer building blocks and biofuels. Since its genome sequence was first published, its versatile metabolic pathways and their genetic components and regulatory mechanisms have been extensively studied. Previous studies on transcriptional factors, including two-component systems and σ factors, in the bacterium have revealed transcriptional regulatory links among the metabolic pathways and those among the stress response systems, forming a complex transcriptional regulatory network. The regulatory links are based on knowledge of the transcription factors, such as their target genes (regulons), DNA sequence motifs for recognition, and effector molecules controlling their activities, all of which are fundamental for understanding their physiological functions. Recent advances in chromatin immunoprecipitation (ChIP)-based genome-wide analyses provide an opportunity to comprehensively identify the transcription factor regulon, composed of its direct target genes, and its precise consensus binding motif. A common feature among the regulon constituents may provide clues to identify an effector molecule targeting the factor. In this mini-review, we summarize the current knowledge of the regulons of the C. glutamicum transcription factors that have been analyzed via ChIP-based technologies. The regulons consisting of direct target genes revealed new physiological roles of the transcription factors and new regulatory interactions, contributing to refinement and expansion of the transcriptional regulatory network and the development of guidelines and genetic tools for metabolic engineering of C. glutamicum. PMID:26496920

  7. Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background 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 overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production. Results Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium. Conclusions The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach. PMID:23806148

  8. Characterization of the mannitol catabolic operon of Corynebacterium glutamicum.

    PubMed

    Peng, Xue; Okai, Naoko; Vertès, Alain A; Inatomi, Ken-Ichi; Inui, Masayuki; Yukawa, Hideaki

    2011-09-01

    Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5'-TCTAACA-3' being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene. PMID:21655984

  9. Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum.

    PubMed

    Meiswinkel, Tobias M; Rittmann, Doris; Lindner, Steffen N; Wendisch, Volker F

    2013-10-01

    Corynebacterium glutamicum possesses genes for glycerol kinase and glycerol-3-phosphate dehydrogenase that were shown to support slow growth with glycerol only when overexpressed from a plasmid. Pure glycerol and crude glycerol from biodiesel factories were tested for growth of recombinant strains expressing glpF, glpK and glpD from Escherichia coli. Some, but not all crude glycerol lots served as good carbon sources. Although the inhibitory compound(s) present in these crude glycerol lots remained unknown, the addition of substoichiometric glucose concentrations (below 10% by weight) enabled the utilization of some of the inhibitory crude glycerol lots. Besides growth, production of the amino acids L-glutamate, L-lysine, L-ornithine and L-arginine as well as of the diamine putrescine based on crude glycerol qualities from biodiesel factories was demonstrated. PMID:23562176

  10. Implication of ornithine acetyltransferase activity on l-ornithine production in Corynebacterium glutamicum.

    PubMed

    Hao, Ning; Mu, Jingrui; Hu, Nan; Xu, Sheng; Shen, Peng; Yan, Ming; Li, Yan; Xu, Lin

    2016-01-01

    l-Ornithine is an intermediate of the l-arginine biosynthetic pathway in Corynebacterium glutamicum. The effect of ornithine acetyltransferase (OATase; ArgJ) on l-ornithine production was investigated, and C. glutamicum 1006 was engineered to overproduce l-ornithine as a major product by inactivating regulatory repressor argR gene and overexpressing argJ gene. A genome sequence analysis indicated that the argF gene encoding ornithine carbamoyltransferase in C. glutamicum 1006 was mutated, resulting in the accumulation of a certain amount of l-ornithine (20.5 g/L). The assays using a crude extract of C. glutamicum 1006 indicated that the l-ornithine concentration for 50% inhibition of OAT was 5 mM. To enhance l-ornithine production, the argJ gene from C. glutamicum ATCC 13032 was overexpressed. In flask cultures, the resulting strain, C. glutamicum 1006∆argR-argJ, produced 31.6 g/L l-ornithine, which is 54.15% more than that produced by C. glutamicum 1006. The OAT activity of C. glutamicum 1006∆argR-argJ was significantly greater than that of C. glutamicum 1006, and this study achieved the highest conversion ratio of sugar to acid (0.396 g/g) compared with those of previous reports. ArgJ strongly influences the production of l-ornithine in C. glutamicum. PMID:25630515

  11. Bio-based production of organic acids with Corynebacterium glutamicum

    PubMed Central

    Wieschalka, Stefan; Blombach, Bastian; Bott, Michael; Eikmanns, Bernhard J

    2013-01-01

    The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG. PMID

  12. Complete genome sequence of Corynebacterium glutamicum B253, a Chinese lysine-producing strain.

    PubMed

    Wu, Yong; Li, Pengpeng; Zheng, Ping; Zhou, Wenjuan; Chen, Ning; Sun, Jibin

    2015-08-10

    We disclosed the complete genome sequence of Corynebacterium glutamicum B253, an important lysine-producing strain in China. The genome consists a circular chromosome (3,159,203bp) and a plasmid (24,775bp), encoding 2767 protein coding genes in total. The genome contains all genes for lysine biosynthesis, and some mutations potentially relevant to lysine production were detected in comparison with sequence of other C. glutamicum. PMID:25953304

  13. Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids.

    PubMed

    Wendisch, Volker F; Bott, Michael; Eikmanns, Bernhard J

    2006-06-01

    Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine. PMID:16617034

  14. Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

    PubMed

    Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

    2011-07-01

    Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

  15. Updates on industrial production of amino acids using Corynebacterium glutamicum.

    PubMed

    Wendisch, Volker F; Jorge, João M P; Pérez-García, Fernando; Sgobba, Elvira

    2016-06-01

    L-Amino acids find various applications in biotechnology. L-Glutamic acid and its salts are used as flavor enhancers. Other L-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. L-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources. PMID:27116971

  16. Properties of Cassava Starch Modified by Amylomaltase from Corynebacterium glutamicum.

    PubMed

    Suriyakul Na Ayudhaya, Pitcha; Pongsawasdi, Piamsook; Laohasongkram, Kalaya; Chaiwanichsiri, Saiwarun

    2016-06-01

    Amylomaltase (α-1,4-glucanotransferase, AM; EC 2.4.1.25) from Corynebacterium glutamicum expressed in Escherichia coli was used to prepare the enzyme-modified cassava starch for food application. About 5% to 15% (w/v) of cassava starch slurries were incubated with 1, 3, or 5 units of amylomaltase/g starch. Apparent amylose, amylopectin chain length distribution, thermal properties, freeze-thaw stability, thermo-reversibility, and gel strength of the obtained modified starches were measured. The apparent amylose content and retrogradation enthalpy were lower, whereas the retrogradation temperatures, freeze-thaw stability, and thermo-reversibility were higher than those of the native cassava starch. However, when amylomaltase content was increased to 20 units of amylomaltase/g starch and for 24 h, the modified starch showed an improvement in the thermo-reversibility property. When used in panna cotta, the gel strength of the sample using the 20 units/24 h modified cassava starch was similar to that of using gelatin. PMID:27105125

  17. Recent progress in development of synthetic biology platforms and metabolic engineering of Corynebacterium glutamicum.

    PubMed

    Woo, Han Min; Park, Jin-Byung

    2014-06-20

    The paradigm of synthetic biology has been evolving, along with relevant engineering, to achieve designed bio-systems. Synthetic biology has reached the point where it is possible to develop microbial strains to produce desired chemicals. Recent advances in this field have promoted metabolic engineering of Corynebacterium glutamicum as an amino-acid producer for use in intelligent microbial-cell factories. Here, we review recent advances that address C. glutamicum as a potential model organism for synthetic biology, and evaluate their industrial applications. Finally, we highlight the perspective of developing C. glutamicum as a step toward advanced microbial-cell factories that could produce valuable chemicals from renewable resources. PMID:24632177

  18. Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum.

    PubMed

    Lindner, Steffen N; Seibold, Gerd M; Krämer, Reinhard; Wendisch, Volker F

    2011-01-01

    Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes. PMID:22008639

  19. Development of novel cell surface display in Corynebacterium glutamicum using porin.

    PubMed

    Tateno, Toshihiro; Hatada, Kazuki; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-09-01

    We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum. PMID:19430772

  20. Co-expression of endoglucanase and β-glucosidase in Corynebacterium glutamicum DM1729 towards direct lysine fermentation from cellulose.

    PubMed

    Anusree, Murali; Wendisch, Volker F; Nampoothiri, K Madhavan

    2016-08-01

    The aim of the present study is the development of a consolidated bioprocess for the production of lysine with recombinant Corynebacterium glutamicum DM1729 strains expressing endoglucanase and β-glucosidase genes. Here, the endoglucanase genes from Xanthomonas campestris XCC3521 and XCC2387 and betaglucosidase gene from Saccharophagus degradans Sde1394 were cloned in C. glutamicum DM1729 and expressed either extracellularly or on cell surface. The highest β-glucosidase activity of 9±0.5U/OD600 of 1 and endoglucanase activity of 5.5±0.8U was obtained in C. glutamicum DM 1729 (pVWEx1-TATXCC2387) (pEKEx3-PorC-Sde1394) when cellobiose (20g/L) alone or in combination with carboxymethyl cellulose (20g/L) was used as the carbon sources respectively. The overall efforts resulted in a lysine titre of 5.9±0.5mM. The ability of the constructs to utilize carboxymethyl cellulose and cellobiose for growth and amino acid production proves the concept of utilization of C. glutamicum as a biocatalyst in the lignocellulosic biorefinery. PMID:27020126

  1. Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress.

    PubMed

    Si, Meiru; Zhao, Chao; Zhang, Bing; Wei, Dawei; Chen, Keqi; Yang, Xu; Xiao, He; Shen, Xihui

    2016-01-01

    Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress. PMID:27383057

  2. Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress

    PubMed Central

    Si, Meiru; Zhao, Chao; Zhang, Bing; Wei, Dawei; Chen, Keqi; Yang, Xu; Xiao, He; Shen, Xihui

    2016-01-01

    Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress. PMID:27383057

  3. Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

    PubMed Central

    Heider, Sabine A. E.; Wolf, Natalie; Hofemeier, Arne; Peters-Wendisch, Petra; Wendisch, Volker F.

    2014-01-01

    The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca. PMID:25191655

  4. Complete Sucrose Metabolism Requires Fructose Phosphotransferase Activity in Corynebacterium glutamicum To Ensure Phosphorylation of Liberated Fructose

    PubMed Central

    Dominguez, H.; Lindley, N. D.

    1996-01-01

    Sucrose uptake by Corynebacterium glutamicum involves a phosphoenolpyruvate-dependent sucrose phosphotransferase (PTS), but in the absence of fructokinase, further metabolism of the liberated fructose requires efflux of the fructose and reassimilation via the fructose PTS. Mutant strains lacking detectable fructose-transporting PTS activity accumulated fructose extracellularly but consumed sucrose at rates comparable to those of the wild-type strain. PMID:16535429

  5. Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum.

    PubMed

    Rittmann, Doris; Lindner, Steffen N; Wendisch, Volker F

    2008-10-01

    The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures. PMID:18757581

  6. Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

    PubMed Central

    Takeno, Seiki; Takasaki, Manami; Urabayashi, Akinobu; Mimura, Akinori; Muramatsu, Tetsuhiro; Mitsuhashi, Satoshi

    2013-01-01

    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 surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63up, and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the β-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63up mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63up fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic

  7. Phosphotransferase system-mediated glucose uptake is repressed in phosphoglucoisomerase-deficient Corynebacterium glutamicum strains.

    PubMed

    Lindner, Steffen N; Petrov, Dimitar P; Hagmann, Christian T; Henrich, Alexander; Krämer, Reinhard; Eikmanns, Bernhard J; Wendisch, Volker F; Seibold, Gerd M

    2013-04-01

    Corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum Δpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum Δpgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized l-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmid-encoded ptsG. l-Lysine yields and productivity with C. glutamicum Δpgi(pBB1-ptsG) were significantly higher than those with C. glutamicum Δpgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTS-mediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast l-lysine production. PMID:23396334

  8. Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum.

    PubMed

    Nimpiboon, Pitchanan; Krusong, Kuakarun; Kaulpiboon, Jarunee; Kidokoro, Shun-Ichi; Pongsawasdi, Piamsook

    2016-09-16

    Amylomaltase catalyzes intermolecular and intramolecular transglucosylation reactions to form linear and cyclic oligosaccharides, respectively. The aim of this work is to investigate the structure-function relationship of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM). Site-directed mutagenesis was performed to substitute Tyr for Asn287 (N287Y) to determine its role in controlling amylomaltase activity and product formation. Expression of the wild-type (WT) and N287Y was achieved by cultivating recombinant cells in the medium containing lactose at 16 °C for 14 h. The purified mutated enzyme showed a significant decrease in all transglucosylation activities while hydrolysis activity was not changed. Optimum temperature and pH for disproportionation reaction were slightly changed upon mutation while those for cyclization reaction were not changed. Interestingly, N287Y showed a change in large-ring cyclodextrin (LR-CD) product profile in which the larger size was observed together with an increase in thermostability and substrate preference for G5 in addition to G3. The secondary structure of the mutated enzyme was slightly changed in related to the WT as evidenced from circular dichroism analysis. This work thus demonstrates that N287 is required for transglucosylation activities of CgAM. Having an aromatic residue in this position increased thermostability, changed product profile and substrate preference but demolished most enzyme activities. PMID:27507216

  9. Comparisons of potentials for L-lysine production among different Corynebacterium glutamicum strains.

    PubMed

    Ohnishi, Junko; Ikeda, Masato

    2006-04-01

    Corynebacterium glutamicum is an industrially important organism that is most widely used for the production of various amino acids. A defined L-lysine-producing mutant was generated by introduction of the lysC mutation (T311I) into each of six representative C. glutamicum strains. The resulting six isogenic mutants were compared for L-lysine production under traditional 30 degrees C conditions and industrially more advantageous 40 degrees C conditions. It was found that there were significant differences in yield and productivity, especially at 40 degrees C. These results indicate the diversity among C. glutamicum strains in fermentative characters, as well as the importance of selecting a strain with industrially best performance. PMID:16636474

  10. Physiological roles of mycothiol in detoxification and tolerance to multiple poisonous chemicals in Corynebacterium glutamicum.

    PubMed

    Liu, Ying-Bao; Long, Ming-Xiu; Yin, Ya-Jie; Si, Mei-Ru; Zhang, Lei; Lu, Zhi-Qiang; Wang, Yao; Shen, Xi-Hui

    2013-06-01

    Mycothiol (MSH) plays important roles in maintaining cytosolic redox homeostasis and in adapting to reactive oxygen species in the high-(G + C)-content Gram-positive Actinobacteria. However, its physiological roles are ill defined compared to glutathione, the functional analog of MSH in Gram-negative bacteria and most eukaryotes. In this research, we explored the impact of intracellular MSH on cellular physiology by using MSH-deficient mutants in the model organism Corynebacterium glutamicum. We found that intracellular MSH contributes significantly to resistance to alkylating agents, glyphosate, ethanol, antibiotics, heavy metals and aromatic compounds. In addition, intracellular MSH is beneficial for withstanding oxidative stress induced by various oxidants in C. glutamicum. This study greatly expanded our current knowledge on the physiological functions of mycothiol in C. glutamicum and could be applied to improve the robustness of this scientifically and commercially important species in the future. PMID:23615850

  11. Production of protocatechuic acid by Corynebacterium glutamicum expressing chorismate-pyruvate lyase from Escherichia coli.

    PubMed

    Okai, Naoko; Miyoshi, Takanori; Takeshima, Yasunobu; Kuwahara, Hiroaki; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation. PMID:26392137

  12. Production of L-Lysine from starch by Corynebacterium glutamicum displaying alpha-amylase on its cell surface.

    PubMed

    Tateno, Toshihiro; Fukuda, Hideki; Kondo, Akihiko

    2007-04-01

    We engineered a Corynebacterium glutamicum strain displaying alpha-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of alpha-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed L-lysine fermentation at various temperatures (30-40 degrees C) and pHs (6.0-7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest L-lysine yield was recorded at 30 degrees C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l L-lysine was produced in 24 h. The L-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying alpha-amylase has a potential to directly convert soluble starch to amino acids. PMID:17216452

  13. [Construction and structural analysis of integrated cellular network of Corynebacterium glutamicum].

    PubMed

    Jiang, Jinguo; Song, Lifu; Zheng, Ping; Jia, Shiru; Sun, Jibin

    2012-05-01

    Corynebacterium glutamicum is one of the most important traditional industrial microorganisms and receiving more and more attention towards a novel cellular factory due to the recently rapid development in genomics and genetic operation toolboxes for Corynebacterium. However, compared to other model organisms such as Escherichia coli, there were few studies on its metabolic regulation, especially a genome-scale integrated cellular network model currently missing for Corynebacterium, which hindered the systematic study of Corynebacterium glutamicum and large-scale rational design and optimization for strains. Here, by gathering relevant information from a number of public databases, we successfully constructed an integrated cellular network, which was composed of 1384 reactions, 1276 metabolites, 88 transcriptional factors and 999 pairs of transcriptional regulatory relationships. The transcriptional regulatory sub-network could be arranged into five layers and the metabolic sub-network presented a clear bow-tie structure. We proposed a new method to extract complex metabolic and regulatory sub-network for product-orientated study taking lysine biosynthesis as an example. The metabolic and regulatory sub-network extracted by our method was more close to the real functional network than the simplex biochemical pathways. The results would be greatly helpful for understanding the high-yielding biomechanism for amino acids and the re-design of the industrial strains. PMID:22916496

  14. Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence.

    PubMed

    Tateno, Toshihiro; Fukuda, Hideki; Kondo, Akihiko

    2007-12-01

    Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes alpha-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40 degrees C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37 degrees C, respectively. The alpha-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence. PMID:17891388

  15. Reconstruction and analysis of a genome-scale metabolic network of Corynebacterium glutamicum S9114.

    PubMed

    Mei, Jie; Xu, Nan; Ye, Chao; Liu, Liming; Wu, Jianrong

    2016-01-10

    Corynebacterium glutamicum S9114 is commonly used for industrial glutamate production. Therefore, a comprehensive understanding of the physiological and metabolic characteristics of C. glutamicum is important for developing its potential for industrial production. A genome-scale metabolic model, iJM658, was reconstructed based on genome annotation and literature mining. The model consists of 658 genes, 984 metabolites and 1065 reactions. The model quantitatively predicted C. glutamicum growth on different carbon and nitrogen sources and determined 129 genes to be essential for cell growth. The iJM658 model predicted that C. glutamicum had two glutamate biosynthesis pathways and lacked eight key genes in biotin synthesis. Robustness analysis indicated a relative low oxygen level (1.21mmol/gDW/h) would improve glutamate production rate. Potential metabolic engineering targets for improving γ-aminobutyrate and isoleucine production rate were predicted by in silico deletion or overexpression of some genes. The iJM658 model is a useful tool for understanding and optimizing the metabolism of C. glutamicum and a valuable resource for future metabolic and physiological research. PMID:26392034

  16. Monitoring of population dynamics of Corynebacterium glutamicum by multiparameter flow cytometry

    PubMed Central

    Neumeyer, Andrea; Hübschmann, Thomas; Müller, Susann; Frunzke, Julia

    2013-01-01

    Summary Phenotypic variation of microbial populations is a well-known phenomenon and may have significant impact on the success of industrial bioprocesses. Flow cytometry (FC) and the large repertoire of fluorescent dyes bring the high-throughput analysis of multiple parameters in single bacterial cells into reach. In this study, we evaluated a set of different fluorescent dyes for suitability in FC single cell analysis of the biotechnological platform organism Corynebacterium glutamicum. Already simple scattering properties of C. glutamicum cells in the flow cytometer were shown to provide valuable information on the growth activity of analysed cells. Furthermore, we used DAPI staining for a FC-based determination of the DNA content of C. glutamicum cells grown on standard minimal or complex media. Characteristic DNA patterns were observed mirroring the typical uncoupled DNA synthesis in the logarithmic (log) growth phase and are in agreement with a symmetric type of cell division of C. glutamicum. Application of the fluorescent dyes Syto 9, propidium iodide, and DiOC2(3) allowed the identification of subpopulations with reduced viability and membrane potential within early log and stationary phase populations. The presented data highlight the potential of FC-based analyses for online monitoring of C. glutamicum bioprocesses and provide a first reference for future applications and protocols. PMID:23279937

  17. Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid.

    PubMed

    Feng, Lili; Zhang, Ya; Fu, Jing; Mao, Yufeng; Chen, Tao; Zhao, Xueming; Wang, Zhiwen

    2016-06-01

    5-Aminolevulinic acid (5-ALA) has recently attracted attention for its potential applications in the fields of medicine and agriculture. In this study, Corynebacterium glutamicum was firstly engineered for 5-ALA production via the C4 pathway. HemA encoding 5-aminolevulinic acid synthase from Rhodobacter sphaeroides was codon optimized and expressed in C. glutamicum ATCC13032, resulting in accumulation of 5-ALA. Deletion of all known genes responsible for the formation of acetate and lactate further enhanced production of 5-ALA. Overexpression of ppc gene encoding phoenolpyruvate carboxylase resulted in an accumulation of 5-ALA up to 2.06 ± 0.05 g/L. Furthermore, deletion of high-molecular-weight penicillin-binding proteins (HMW-PBPs) genes pbp1a, pbp1b, and pbp2b led to an increase in 5-ALA production of 13.53%, 29.47%, and 22.22%, respectively. Finally, 5-ALA production was enhanced to 3.14 ± 0.02 g/L in shake flask by heterologously expressing rhtA encoding threonine/homoserine exporter, and 86.77% of supplemented glycine was channeled toward 5-ALA production in shake flask. The engineered C. glutamicum ALA7 strain produced 7.53 g/L 5-ALA in a 5 L bioreactor. This study demonstrated the potential utility of C. glutamicum as a platform for metabolic production of 5-ALA. Change of cell permeability by metabolic engineering HMW-PBPs may provide a new strategy for biochemicals production in Corynebacterium glutamicum. Biotechnol. Bioeng. 2016;113: 1284-1293. © 2015 Wiley Periodicals, Inc. PMID:26616115

  18. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    PubMed

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  19. Current knowledge on mycolic acids in Corynebacterium glutamicum and their relevance for biotechnological processes.

    PubMed

    Lanéelle, Marie-Antoinette; Tropis, Maryelle; Daffé, Mamadou

    2013-12-01

    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-negative microorganisms. As the amino acids have to cross both membranes, their integrity, composition and fluidity influence the export process. While the precise mechanism of the export of the amino acids by C. glutamicum is not fully understood, the excretion of amino acids through the inner membrane involved at least a major export system mechanosensitive channel MscS family (MscCG) encoded by NCgl1221. As the various industrial treatments have been shown to affect the lipid content of the bacterial cell, it is strongly believed that defects in the hallmark of the outer membrane, 2-alkyl, 3-hydroxylated long-chain fatty acids (mycolic acids), could be key factors in the glutamate overproduction. This review aims at giving an overview of the current knowledge on mycolic acids structure, biosynthesis and transfer in C. glutamicum and their relevance for amino acid biotechnological production. PMID:24113823

  20. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products.

    PubMed

    Zahoor, Ahmed; Lindner, Steffen N; Wendisch, Volker F

    2012-01-01

    Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols. PMID:24688664

  1. In vitro functional characterization of the Na+/H+ antiporters in Corynebacterium glutamicum.

    PubMed

    Xu, Ning; Wang, Lei; Cheng, Haijiao; Liu, Qingdai; Liu, Jun; Ma, Yanhe

    2016-02-01

    Corynebacterium glutamicum, typically used as industrial workhorse for amino acid production, is a moderately salt-alkali-tolerant microorganism with optimal growth at pH 7-9. However, little is known about the mechanisms of salt-alkali tolerance in C. glutamicum. Here, the catalytic capacity of three putative Na(+)/H(+) antiporters from C. glutamicum (designated as Cg-Mrp1, Cg-Mrp2 and Cg-NhaP) were characterized in an antiporter-deficient Escherichia coli KNabc strain. Only Cg-Mrp1 was able to effectively complement the Na(+)-sensitive of E. coli KNabc. Cg-Mrp1 exhibited obvious Na(+)(Li(+))/H(+) antiport activities with low apparent Km values of 1.08 mM and 1.41 mM for Na(+) and Li(+), respectively. The Na(+)/H(+) antiport activity of Cg-Mrp1 was optimal in the alkaline pH range. All three antiporters showed detectable K(+)/H(+) antiport activitiy. Cg-NhaP also exhibited Na(+)(Li(+),Rb(+))/H(+) antiport activities but at lower levels of activity. Interestingly, overexpression of Cg-Mrp2 exhibited clear Na(+)(K(+))/H(+) antiport activities. These results suggest that C. glutamicum Na(+)(K(+))/H(+) antiporters may have overlapping roles in coping with salt-alkali and perhaps high-osmolarity stress. PMID:26667218

  2. Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production.

    PubMed

    Kabus, Armin; Niebisch, Axel; Bott, Michael

    2007-02-01

    Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1 -aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by approximately 45% and of the maximal biomass by approximately 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by approximately 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum. PMID:17142369

  3. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products

    PubMed Central

    Zahoor, Ahmed; Lindner, Steffen N.; Wendisch, Volker F.

    2012-01-01

    Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols PMID:24688664

  4. Next-generation sequencing-based transcriptome analysis of L-lysine-producing Corynebacterium glutamicum ATCC 21300 strain.

    PubMed

    Kim, Hong-Il; Nam, Jae-Young; Cho, Jae-Yong; Lee, Chang-Soo; Park, Young-Jin

    2013-12-01

    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 identified to have mutations including SNP (9 genes) and InDel (1 gene). This information will be useful for genome breeding of C. glutamicum to develop an industrial amino acid-producing strain with minimal mutation. PMID:24385368

  5. Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

    SciTech Connect

    Schoepe, Jan Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar

    2006-07-01

    The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.

  6. A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum.

    PubMed

    Eggeling, Lothar; Bott, Michael

    2015-04-01

    L-lysine is made in an exceptional large quantity of currently 2,200,000 tons/year and belongs therefore to one of the leading biotechnological products. Production is done almost exclusively with mutants of Corynebacterium glutamicum. The increasing L-lysine market forces companies to improve the production process fostering also a deeper understanding of the microbial physiology of C. glutamicum. Current major challenges are the identification of ancillary mutations not intuitively related with product increase. This review gives insights on how cellular characteristics enable to push the carbon flux in metabolism towards its theoretical maximum, and this example may also serve as a guide to achieve and increase the formation of other products of interest in microbial biotechnology. PMID:25761623

  7. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering.

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

    The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. This review summarizes the current knowledge and achievements on metabolic engineering approaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine, 2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production. PMID:24486441

  8. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

    PubMed

    Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

    2012-03-01

    Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain. PMID:22159614

  9. Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Schreiner, Mark E; Moch, Matthias; Oldiges, Marco; Eikmanns, Bernhard J

    2007-09-01

    Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway. PMID:17333167

  10. Crystallization and initial crystallographic characterization of the Corynebacterium glutamicum nitrilotriacetate monooxygenase component A

    SciTech Connect

    Kim, Kyung-Jin; Kim, Sujin; Lee, Sujin; Kang, Beom Sik; Lee, Heung-Soo; Oh, Tae-Kwang; Kim, Myung Hee

    2006-11-01

    The Corynebacterium glutamicum NTA monooxygenase component A protein, which plays the central role in NTA biodegradation, was crystallized. The initial X-ray crystallographic characterization is reported. Safety and environmental concerns have recently dictated the proper disposal of nitrilotriacetate (NTA). Biodegradation of NTA is initiated by NTA monooxygenase, which is composed of two proteins: component A and component B. The NTA monooxygenase component A protein from Corynebacterium glutamicum was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate as the precipitant. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 111.04, b = 98.51, c = 171.61 Å, β = 101.94°. The asymmetric unit consists of four molecules, corresponding to a packing density of 2.3 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  11. Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

    SciTech Connect

    Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa; Kim, Kyung-Jin

    2007-11-01

    PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  12. Phenotypic characterization of Corynebacterium glutamicum under osmotic stress conditions using elementary mode analysis.

    PubMed

    Rajvanshi, Meghna; Venkatesh, K V

    2011-09-01

    Corynebacterium glutamicum, a soil bacterium, is used to produce amino acids such as lysine and glutamate. C. glutamicum is often exposed to osmolality changes in its medium, and the bacterium has therefore evolved several adaptive response mechanisms to overcome them. In this study we quantify the metabolic response of C. glutamicum under osmotic stress using elementary mode analysis (EMA). Further, we obtain the optimal phenotypic space for the synthesis of lysine and formation of biomass. The analysis demonstrated that with increasing osmotic stress, the flux towards trehalose formation and energy-generating pathways increased, while the flux of anabolic reactions diminished. Nodal analysis indicated that glucose-6-phosphate, phosphoenol pyruvate, and pyruvate nodes were capable of adapting to osmotic stress, whereas the oxaloacetic acid node was relatively unresponsive. Fewer elementary modes were active under stress indicating the rigid behavior of the metabolism in response to high osmolality. Optimal phenotypic space analysis revealed that under normal conditions the organism optimized growth during the initial log phase and lysine and trehalose formation during the stationary phase. However, under osmotic stress, the analysis demonstrated that the organism operates under suboptimal conditions for growth, and lysine and trehalose formation. PMID:21132515

  13. Metabolic engineering of Corynebacterium glutamicum ATCC13032 to produce S-adenosyl-L-methionine.

    PubMed

    Han, Guoqiang; Hu, Xiaoqing; Qin, Tianyu; Li, Ye; Wang, Xiaoyuan

    2016-02-01

    As an important biological methyl group donor, S-adenosyl-L-methionine is used as nutritional supplement or drug for various diseases, but bacterial strains that can efficiently produce S-adenosyl-L-methionine are not available. In this study, Corynebacterium glutamicum strain HW104 which can accumulate S-adenosyl-L-methionine was constructed from C. glutamicum ATCC13032 by deleting four genes thrB, metB, mcbR and Ncgl2640, and six genes metK, vgb, lysC(m), hom(m), metX and metY were overexpressed in HW104 in different combinations, forming strains HW104/pJYW-4-metK-vgb, HW104/pJYW-4-SAM2C-vgb, HW104/pJYW-4-metK-vgb-metYX, and HW104/pJYW-4-metK-vgb-metYX-hom(m)-lysC(m). Fermentation experiments showed that HW104/pJYW-4-metK-vgb produced more S-adenosyl-L-methionine than other strains, and the yield achieved 196.7 mg/L (12.15 mg/g DCW) after 48h. The results demonstrate the potential application of C. glutamicum for production of S-adenosyl-L-methionine without addition of L-methionine. PMID:26777246

  14. Glycerol as a substrate for aerobic succinate production in minimal medium with Corynebacterium glutamicum

    PubMed Central

    Litsanov, Boris; Brocker, Melanie; Bott, Michael

    2013-01-01

    Corynebacterium glutamicum, an established microbial cell factory for the biotechnological production of amino acids, was recently genetically engineered for aerobic succinate production from glucose in minimal medium. In this work, the corresponding strains were transformed with plasmid pVWEx1-glpFKD coding for glycerol utilization genes from Escherichia coli. This plasmid had previously been shown to allow growth of C. glutamicum with glycerol as sole carbon source. The resulting strains were tested in minimal medium for aerobic succinate production from glycerol, which is a by-product in biodiesel synthesis. The best strain BL-1/pVWEx1-glpFKD formed 79 mM (9.3 g l−1) succinate from 375 mM glycerol, representing 42% of the maximal theoretical yield under aerobic conditions. A specific succinate production rate of 1.55 mmol g−1 (cdw) h−1 and a volumetric productivity of 3.59 mM h−1 were obtained, the latter value representing the highest one currently described in literature. The results demonstrate that metabolically engineered strains of C. glutamicum are well suited for aerobic succinate production from glycerol. PMID:22513227

  15. Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering.

    PubMed

    Neuner, Andreas; Heinzle, Elmar

    2011-03-01

    The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates. PMID:21370474

  16. Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis.

    PubMed

    Küberl, Andreas; Fränzel, Benjamin; Eggeling, Lothar; Polen, Tino; Wolters, Dirk Andreas; Bott, Michael

    2014-06-01

    In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins. PMID:24737727

  17. Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis.

    PubMed

    Polen, Tino; Schluesener, Daniela; Poetsch, Ansgar; Bott, Michael; Wendisch, Volker F

    2007-08-01

    Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate. PMID:17559405

  18. Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level.

    PubMed

    Li, Ying; Cong, Hua; Liu, Bingnan; Song, Jinzhu; Sun, Xueying; Zhang, Junzheng; Yang, Qian

    2016-09-01

    Relieving the feedback inhibition of key enzymes in a metabolic pathway is frequently the first step of producer-strain construction by genetic engineering. However, the strict feedback regulation exercised by microorganisms in methionine biosynthesis often makes it difficult to produce methionine at a high level. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for methionine production. First, the metD gene encoding the methionine uptake system was deleted to achieve extracellular accumulation of methionine. Then, random mutagenesis was performed to remove feedback inhibition by metabolic end-products. The resulting strain C. glutamicum ENM-16 was further engineered to block or decrease competitive branch pathways by deleting the thrB gene and changing the start codon of the dapA gene, followed by point mutations of lysC (C932T) and pyc (G1A, C1372T) to increase methionine precursor supply. To enrich the NADPH pool, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway were mutated to reduce their sensitivity to inhibition by intracellular metabolites. The resultant strain C. glutamicum LY-5 produced 6.85 ± 0.23 g methionine l(-1) with substrate-specific yield (Y P/S) of 0.08 mol per mol of glucose after 72 h fed-batch fermentation. The strategies described here will be useful for construction of methionine engineering strains. PMID:27255137

  19. FudC, a protein primarily responsible for furfural detoxification in Corynebacterium glutamicum.

    PubMed

    Tsuge, Yota; Kudou, Motonori; Kawaguchi, Hideo; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-03-01

    Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli. PMID:26541332

  20. Cathodes enhance Corynebacterium glutamicum growth with nitrate and promote acetate and formate production.

    PubMed

    Xafenias, Nikolaos; Kmezik, Cathleen; Mapelli, Valeria

    2016-09-01

    The industrially important Corynebacterium glutamicum can only incompletely reduce nitrate into nitrite which then accumulates and inhibits growth. Herein we report that cathodes can resolve this problem and enhance glucose fermentation and growth by promoting nitrite reduction. Cell growth was inhibited at relatively high potentials but was significant when potentials were more reductive (-1.20V with anthraquinone-2-sulfonate as redox mediator or -1.25V vs. Ag/AgCl). Under these conditions, glucose was consumed up to 6 times faster and acetate was produced at up to 11 times higher yields (up to 1.1mol/mol-glucose). Acetate concentrations are the highest reported so far for C. glutamicum under anaerobic conditions, reaching values up to 5.3±0.3g/L. Herein we also demonstrate for the first time formate production (up to 3.4±0.3g/L) by C. glutamicum under strongly reducing conditions, and we attribute this to a possible mechanism of CO2 bioreduction that was electrochemically triggered. PMID:27235972

  1. RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane

    PubMed Central

    Yang, Liang; Lu, Shuo; Belardinelli, Juan; Huc-Claustre, Emilie; Jones, Victoria; Jackson, Mary; Zgurskaya, Helen I

    2014-01-01

    Corynebacterium–Mycobacterium–Nocardia (CMN) group are the causative agents of a broad spectrum of diseases in humans. A distinctive feature of these Gram-positive bacteria is the presence of an outer membrane of unique structure and composition. Recently, resistance–nodulation–division (RND) transporters (nicknamed MmpLs, Mycobacterial membrane protein Large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. In this study, we investigated the role of RND transporters in the physiology of Corynebacterium glutamicum and analyzed properties of these proteins. Our results show that in contrast to Gram-negative species, in which RND transporters actively extrude antibiotics from cells, in C. glutamicum and relatives these transporters protect cells from antibiotics by playing essential roles in the biogenesis of the low-permeability barrier of the outer membrane. Conditional C. glutamicum mutants lacking RND proteins and with the controlled expression of either NCgl2769 (CmpL1) or NCgl0228 (CmpL4) are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellularly trehalose monocorynomycolates, free corynomycolates, and the previously uncharacterized corynomycolate-containing lipid. Our results also suggest that similar to other RND transporters, Corynebacterial membrane proteins Large (CmpLs) functions are dependent on a proton-motive force. PMID:24942069

  2. Codon Usage Patterns in Corynebacterium glutamicum: Mutational Bias, Natural Selection and Amino Acid Conservation.

    PubMed

    Liu, Guiming; Wu, Jinyu; Yang, Huanming; Bao, Qiyu

    2010-01-01

    The alternative synonymous codons in Corynebacterium glutamicum, a well-known bacterium used in industry for the production of amino acid, have been investigated by multivariate analysis. As C. glutamicum is a GC-rich organism, G and C are expected to predominate at the third position of codons. Indeed, overall codon usage analyses have indicated that C and/or G ending codons are predominant in this organism. Through multivariate statistical analysis, apart from mutational selection, we identified three other trends of codon usage variation among the genes. Firstly, the majority of highly expressed genes are scattered towards the positive end of the first axis, whereas the majority of lowly expressed genes are clustered towards the other end of the first axis. Furthermore, the distinct difference in the two sets of genes was that the C ending codons are predominate in putatively highly expressed genes, suggesting that the C ending codons are translationally optimal in this organism. Secondly, the majority of the putatively highly expressed genes have a tendency to locate on the leading strand, which indicates that replicational and transciptional selection might be invoked. Thirdly, highly expressed genes are more conserved than lowly expressed genes by synonymous and nonsynonymous substitutions among orthologous genes fromthe genomes of C. glutamicum and C. diphtheriae. We also analyzed other factors such as the length of genes and hydrophobicity that might influence codon usage and found their contributions to be weak. PMID:20445740

  3. Engineering of Corynebacterium glutamicum to utilize methyl acetate, a potential feedstock derived by carbonylation of methanol with CO.

    PubMed

    Choo, Seungjung; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

    2016-04-20

    The possibilities to utilize one-carbon substrates (C1) like CO, methane and methanol have been explored as a cheap alternative feedstock in the biotechnology. For the first time, methyl acetate (MeOAc), which can be formed from carbonylation of methanol with CO, was demonstrated to be an alternative carbon source for the cell growth of Corynebacterium glutamicum as a model microbial cell factory. To do so, a carboxyl esterase activity was necessary to hydrolyze MeOAc to methanol and acetate. Although the wild-type has an unknown esterase activity to MeOAc, the activity was not high enough to grow from 270mM MeOAc as sole carbon source, reaching OD600 of 5.28±0.2 in 32h. Based on the literatures studied for the esterase, we chose three esterases (MekB of Pseudomonas veronii MEK700, AcmB of Gordonia sp. Strain TY-5, and Est of Pyrobaculum calidifontis VA1) and cloned into the wild-type. As a result, the recombinant C. glutamicum expressing the highly active MekB esterase (28.6±0.77U/mg protein) showed complete degradation of MeOAc and utilization of acetate, resulting in OD600 of 16.5±0.02at 24h. In addition, the recombinant strain exhibited the rapid degradation of MeOAc to methanol and acetate in 2h under anaerobic condition. Therefore, MeOAc can be used as another C1-derived carbon source in the biotechnology. PMID:26970052

  4. Succinate production from CO2-grown microalgal biomass as carbon source using engineered Corynebacterium glutamicum through consolidated bioprocessing

    PubMed Central

    Lee, Jungseok; Sim, Sang Jun; Bott, Michael; Um, Youngsoon; Oh, Min-Kyu; Woo, Han Min

    2014-01-01

    The potential for production of chemicals from microalgal biomass has been considered as an alternative route for CO2 mitigation and establishment of biorefineries. This study presents the development of consolidated bioprocessing for succinate production from microalgal biomass using engineered Corynebacterium glutamicum. Starch-degrading and succinate-producing C. glutamicum strains produced succinate (0.16 g succinate/g total carbon source) from a mixture of starch and glucose as a model microalgal biomass. Subsequently, the engineered C. glutamicum strains were able to produce succinate (0.28 g succinate/g of total sugars including starch) from pretreated microalgal biomass of CO2-grown Chlamydomonas reinhardtii. For the first time, this work shows succinate production from CO2 via sequential fermentations of CO2-grown microalgae and engineered C. glutamicum. Therefore, consolidated bioprocessing based on microalgal biomass could be useful to promote variety of biorefineries. PMID:25056811

  5. The small ribosomal protein S12P gene rpsL as an efficient positive selection marker in allelic exchange mutation systems for Corynebacterium glutamicum.

    PubMed

    Kim, Il Kwon; Jeong, Weol Kyu; Lim, Seong Han; Hwang, In Kwan; Kim, Young Ho

    2011-01-01

    We report that the mutant rpsL K43R in streptomycin-resistant and lysine-producing Corynebacterium glutamicum is responsible for streptomycin resistance. In addition, we describe its effective application in gene modification in C. glutamicum. PMID:20951172

  6. Corynebacterium glutamicum as a host for synthesis and export of D-Amino Acids.

    PubMed

    Stäbler, Norma; Oikawa, Tadao; Bott, Michael; Eggeling, Lothar

    2011-04-01

    A number of d-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established l-amino-acid-producing bacterium Corynebacterium glutamicum to study whether d-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates d-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an l-arginine, l-ornithine, or l-lysine producer, equimolar mixtures of the d- and l-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an l-serine producer resulted in extracellular accumulation of a surplus of d-serine (81 mM d-serine and 37 mM l-serine) at intracellular concentrations of 125 mM d-serine plus 125 mM l-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for d-serine. Export of d-lysine relies fully on the presence of lysE, encoding the exporter for l-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that d-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible. PMID:21257776

  7. Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum.

    PubMed

    Kallscheuer, Nicolai; Vogt, Michael; Kappelmann, Jannick; Krumbach, Karin; Noack, Stephan; Bott, Michael; Marienhagen, Jan

    2016-02-01

    Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum. PMID:26610800

  8. Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum.

    PubMed

    Uhde, Andreas; Youn, Jung-Won; Maeda, Tomoya; Clermont, Lina; Matano, Christian; Krämer, Reinhard; Wendisch, Volker F; Seibold, Gerd M; Marin, Kay

    2013-02-01

    Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS(Glc) transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid L-lysine and the diamine putrescine from glucosamine was demonstrated. PMID:22854894

  9. Development and experimental verification of a genome-scale metabolic model for Corynebacterium glutamicum

    PubMed Central

    Shinfuku, Yohei; Sorpitiporn, Natee; Sono, Masahiro; Furusawa, Chikara; Hirasawa, Takashi; Shimizu, Hiroshi

    2009-01-01

    Background In silico genome-scale metabolic models enable the analysis of the characteristics of metabolic systems of organisms. In this study, we reconstructed a genome-scale metabolic model of Corynebacterium glutamicum on the basis of genome sequence annotation and physiological data. The metabolic characteristics were analyzed using flux balance analysis (FBA), and the results of FBA were validated using data from culture experiments performed at different oxygen uptake rates. Results The reconstructed genome-scale metabolic model of C. glutamicum contains 502 reactions and 423 metabolites. We collected the reactions and biomass components from the database and literatures, and made the model available for the flux balance analysis by filling gaps in the reaction networks and removing inadequate loop reactions. Using the framework of FBA and our genome-scale metabolic model, we first simulated the changes in the metabolic flux profiles that occur on changing the oxygen uptake rate. The predicted production yields of carbon dioxide and organic acids agreed well with the experimental data. The metabolic profiles of amino acid production phases were also investigated. A comprehensive gene deletion study was performed in which the effects of gene deletions on metabolic fluxes were simulated; this helped in the identification of several genes whose deletion resulted in an improvement in organic acid production. Conclusion The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities and prediction of the metabolic characteristics of C. glutamicum. This can form a basis for the in silico design of C. glutamicum metabolic networks for improved bioproduction of desirable metabolites. PMID:19646286

  10. Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine.

    PubMed

    Matano, Christian; Uhde, Andreas; Youn, Jung-Won; Maeda, Tomoya; Clermont, Lina; Marin, Kay; Krämer, Reinhard; Wendisch, Volker F; Seibold, Gerd M

    2014-06-01

    Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid L-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed. PMID:24668244

  11. Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production.

    PubMed

    Lindner, Steffen N; Niederholtmeyer, Henrike; Schmitz, Katja; Schoberth, Siegfried M; Wendisch, Volker F

    2010-06-01

    Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 +/- 0.006 mol lysine (mol glucose)(-1), which was 12% higher than that of the empty vector control strain. PMID:20180116

  12. Process inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum

    PubMed Central

    2014-01-01

    Background 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 uncertain impact on metabolism. Scale-down bioreactors can be applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale. Results In this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth and intracellular metabolite characteristics. Conclusions Carbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a futile cycle over extracellular side products and back into

  13. Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences.

    PubMed

    Watanabe, Keiro; Tsuchida, Yoshiki; Okibe, Naoko; Teramoto, Haruhiko; Suzuki, Nobuaki; Inui, Masayuki; Yukawa, Hideaki

    2009-03-01

    Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form alpha-amylase derived from Geobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted alpha-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency. PMID:19246745

  14. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  15. Transcriptome analysis reveals global expression changes in an industrial L-lysine producer of Corynebacterium glutamicum.

    PubMed

    Hayashi, Mikiro; Ohnishi, Junko; Mitsuhashi, Satoshi; Yonetani, Yoshiyuki; Hashimoto, Shin-Ichi; Ikeda, Masato

    2006-02-01

    Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production. PMID:16495679

  16. [Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

    PubMed

    Zhang, Xiaomei; Dou, Wenfang; Xu, Hongyu; Xu, Zhenghong

    2010-10-01

    Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity. PMID:21218623

  17. Purification and characterization of the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase from Corynebacterium glutamicum.

    PubMed

    Yang, Yi-Fan; Zhang, Jun-Jie; Wang, Song-He; Zhou, Ning-Yi

    2010-12-01

    Corynebacterium glutamicum ATCC 13032 metabolizes 3-hydroxybenzoate via gentisate. We have now characterized the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase involved in the initial step of 3-hydroxybenzoate catabolism by this strain, a first 3-hydroxybenzoate 6-hydroxylase molecularly and biochemically characterized from a Gram-positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3-hydroxybenzoate 6-hydroxylase, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N-terminal six-His tag and purified to apparent homogeneity by Ni²(+)-nitrilotriacetic acid affinity chromatography. The purified H₆-Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg⁻¹ against 3-hydroxybenzoate and with a K(m) value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3-hydroxybenzoate hydroxylation catalyzed by H₆-Ncgl2923 was identified by high-performance liquid chromatography as gentisate, a ring-cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three-fold. Although this enzyme shares no more than 33% identity with any of reported 3-hydroxybenzoate 6-hydroxylases from Gram-negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them. PMID:20806251

  18. Direct L-lysine production from cellobiose by Corynebacterium glutamicum displaying beta-glucosidase on its cell surface.

    PubMed

    Adachi, Noriko; Takahashi, Chihiro; Ono-Murota, Naoko; Yamaguchi, Rie; Tanaka, Tsutomu; Kondo, Akihiko

    2013-08-01

    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 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of L-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced L-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum. PMID:23749228

  19. Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing alpha-amylase and lysine decarboxylase.

    PubMed

    Tateno, Toshihiro; Okada, Yusuke; Tsuchidate, Takeyuki; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-02-01

    Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 alpha-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli-C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5'-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert L-lysine to cadaverine, as there was no accumulation of L-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources. PMID:18989633

  20. Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

    PubMed

    Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

    2016-08-10

    Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. PMID:27297548

  1. tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

    PubMed

    Smith, Angela M; Harrison, Jesse S; Grube, Christopher D; Sheppe, Austin E F; Sahara, Nahoko; Ishii, Ryohei; Nureki, Osamu; Roy, Hervé

    2015-11-01

    Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells. PMID:26235234

  2. Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose.

    PubMed

    Chen, Zhen; Huang, Jinhai; Wu, Yao; Liu, Dehua

    2016-01-01

    Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production. PMID:26556130

  3. Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

    PubMed

    Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

    2014-02-01

    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 (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

  4. Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale.

    PubMed

    Wittmann, Christoph; Kim, Hyung Min; Heinzle, Elmar

    2004-07-01

    We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale. This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543). Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments. These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C. glutamicum ATCC 21543 compared with ATCC 13287. The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates. PMID:15211482

  5. Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes.

    PubMed

    Sindelar, Georg; Wendisch, Volker F

    2007-09-01

    For the biotechnological production of L: -lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis. PMID:17364200

  6. Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

    PubMed

    Neuner, Andreas; Wagner, Ines; Sieker, Tim; Ulber, Roland; Schneider, Konstantin; Peifer, Susanne; Heinzle, Elmar

    2013-01-20

    Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine. PMID:22898177

  7. Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states.

    PubMed

    Rajvanshi, Meghna; Gayen, Kalyan; Venkatesh, K V

    2013-06-01

    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 of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality. PMID:24432142

  8. Disruption of malate:quinone oxidoreductase increases L-lysine production by Corynebacterium glutamicum.

    PubMed

    Mitsuhashi, Satoshi; Hayashi, Mikiro; Ohnishi, Junko; Ikeda, Masato

    2006-11-01

    Genomic analysis of a classically derived L-lysine-producing mutant, Corynebacterium glutamicum B-6, identified a nonsense mutation in the mqo gene, which encodes malate:quinone oxidoreductase (MQO). The effect of mqo disruption on L-lysine production was investigated in a defined L-lysine producer, C. glutamicum AHP-3, showing approximately 18% increased production. To explore the underlying mechanisms of the increase, the mqo-disrupted strain was analyzed from the viewpoints of redox balance, activities of membrane-bound dehydrogenases, and transcriptome. The intracellular [NADH]/[NAD] ratio in the strain remained unchanged. Also, there were no significant differences in the activities of the membrane-bound dehydrogenases examined. However, transcriptome analysis showed that some TCA cycle genes, such as acn, sucC, and sucD, were down-regulated in the strain. These results suggest that the loss of MQO activity down-regulates the flux of the TCA cycle to maintain the redox balance and results in redirection of oxaloacetate into L-lysine biosynthesis. PMID:17090916

  9. Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum.

    PubMed

    Dietrich, Christiane; Nato, Aimé; Bost, Bruno; Le Maréchal, Pierre; Guyonvarch, Armel

    2009-04-01

    Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding. PMID:19332837

  10. Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum, Revealed by Metabolomics▿ †

    PubMed Central

    Woo, Han Min; Noack, Stephan; Seibold, Gerd M.; Willbold, Sabine; Eikmanns, Bernhard J.; Bott, Michael

    2010-01-01

    In this study, we analyzed the influence of phosphate (Pi) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under Pi limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under Pi excess, the glycogen level in Pi-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under Pi excess, did so under Pi limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under Pi limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited Pi concentrations. PMID:20802079

  11. Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis.

    PubMed

    Pátek, Miroslav; Muth, Günther; Wohlleben, Wolfgang

    2003-09-01

    The function of seven promoters from Corynebacterium glutamicum, P-hom, P-leuA, P-per, P-aes1, P-aes2, P-45, and P-104, was analyzed in a heterologous background. DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C. glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394). With the exception of P-hom, P-leuA and P-104 in B. subtilis, all promoters were found to be active in all species. Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs). All TSPs were determined by primer extension and in two promoters (P-45 and P-hom) the main TSPs were confirmed by S1-mapping. While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S. lividans. Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background. PMID:12948649

  12. DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR

    PubMed Central

    Muhl, Daniela; Jeßberger, Nadja; Hasselt, Kristin; Jardin, Christophe; Sticht, Heinrich; Burkovski, Andreas

    2009-01-01

    Background The TetR family member AmtR is the central regulator of nitrogen starvation response in Corynebacterium glutamicum. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif. Results Site-directed mutagenesis, the characterization of corresponding mutant proteins by gel retardation assays and surface plasmon resonance and molecular modelling revealed several amino acids, which are directly involved in DNA binding, while others have more structural function. Furthermore, we could show that the spacing of the binding motif half sites is crucial for repression of transcription by AmtR. Conclusion Although the DNA binding domain of TetR-type repressors is highly conserved and a core binding motif was identified for AmtR and TetR(D), the AmtR binding domain shows individual properties compared to other TetR proteins. Besides by distinct amino acids of AmtR, DNA binding is influenced by nucleotides not only of the conserved binding motif but also by spacing nucleotides in C. glutamicum. PMID:19627583

  13. Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

    PubMed

    Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

    2015-11-01

    Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains. PMID:26453945

  14. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    PubMed

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division. PMID:27362471

  15. Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

    PubMed

    Tsuge, Yota; Hori, Yoshimi; Kudou, Motonori; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2014-10-01

    The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032. PMID:25112225

  16. Glucose-6-Phosphate Dehydrogenase and Its Deficiency in Mutants of Corynebacterium glutamicum

    PubMed Central

    Ihnen, Ernel D.; Demain, Arnold L.

    1969-01-01

    Corynebacterium glutamicum is a member of a group of taxonomically related glutamate-excreting bacteria which utilize glucose both by the Embden-Meyerhof and the pentose phosphate pathways, the latter sequence accounting for 10 to 38% of the glucose metabolized. Some of the properties of glucose-6-phosphate dehydrogenase in crude extracts of C. glutamicum were studied. The enzyme was rapidly inactivated by dilution in tris (hydroxymethyl)aminomethane-hydrochloride buffer. This inactivation was prevented by the presence of 0.45 m NaCl. Mg++ was required for enzyme activity, but Mn++, Ca++, Sr++, and Ba++ were equally effective. Growth of the organism under differing conditions did not markedly affect the specific activity of the enzyme. A generally applicable method for detecting colonies deficient in glucose-6-phosphate dehydrogenase was developed. Mutants so obtained were found to be auxotrophic for tryptophan. Upon reversion of the tryptophan requirement, the revertants still retained the property of glucose-6-phosphate dehydrogenase deficiency. Neither the mutants nor the revertants could grow as rapidly as the parent culture in glucose, in gluconate, or in a complex medium. PMID:5788701

  17. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

    PubMed

    Ramzi, Ahmad Bazli; Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-12-01

    ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C5 pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering. PMID:26453466

  18. Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source.

    PubMed

    Wittmann, Christoph; Kiefer, Patrick; Zelder, Oskar

    2004-12-01

    Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-(13C)Fru]sucrose, [1-(13C)Glc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated. PMID:15574927

  19. Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for L-lysine production.

    PubMed

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-11-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP(+) to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  20. Systems metabolic engineering of xylose-utilizing Corynebacterium glutamicum for production of 1,5-diaminopentane.

    PubMed

    Buschke, Nele; Becker, Judith; Schäfer, Rudolf; Kiefer, Patrick; Biedendieck, Rebekka; Wittmann, Christoph

    2013-05-01

    The sustainable production of industrial platform chemicals is one of the great challenges facing the biotechnology field. Ideally, fermentation feedstocks would rather rely on industrial waste streams than on food-based raw materials. Corynebacterium glutamicum was metabolically engineered to produce the bio-nylon precursor 1,5-diaminopentane from the hemicellulose sugar xylose. Comparison of a basic diaminopentane producer strain on xylose and glucose feedstocks revealed a 30% reduction in diaminopentane yield and productivity on the pentose sugar. The integration of in vivo and in silico metabolic flux analysis by (13) C and elementary modes identified bottlenecks in the pentose phosphate pathway and the tricarboxylic acid cycle that limited performance on xylose. By the integration of global transcriptome profiling, this could be specifically targeted to the tkt operon, genes that encode for fructose bisphosphatase (fbp) and isocitrate dehydrogenase (icd), and to genes involved in formation of lysine (lysE) and N-acetyl diaminopentane (act). This was used to create the C. glutamicum strain DAP-Xyl1 icd(GTG) Peftu fbp Psod tkt Δact ΔlysE. The novel producer, designated DAP-Xyl2, exhibited a 54% increase in product yield to 233 mmol mol(-1) and a 100% increase in productivity to 1 mmol g(-1) h(-1) on the xylose substrate. In a fed-batch process, the strain achieved 103 g L(-1) of diaminopentane from xylose with a product yield of 32%. Xylose utilization is currently one of the most relevant metabolic engineering subjects. In this regard, the current work is a milestone in industrial strain engineering of C. glutamicum. See accompanying commentary by Hiroshi Shimizu DOI: 10.1002/biot.201300097. PMID:23447448

  1. A proteomic study of Corynebacterium glutamicum AAA+ protease FtsH

    PubMed Central

    Lüdke, Alja; Krämer, Reinhard; Burkovski, Andreas; Schluesener, Daniela; Poetsch, Ansgar

    2007-01-01

    Background The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis. Results In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization. Conclusion The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria. PMID:17254330

  2. Fermentative Production of the Diamine Putrescine: System Metabolic Engineering of Corynebacterium Glutamicum

    PubMed Central

    Nguyen, Anh Q. D.; Schneider, Jens; Reddy, Gajendar Komati; Wendisch, Volker F.

    2015-01-01

    Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1. PMID:25919117

  3. Fermentative production of the diamine putrescine: system metabolic engineering of corynebacterium glutamicum.

    PubMed

    Nguyen, Anh Q D; Schneider, Jens; Reddy, Gajendar Komati; Wendisch, Volker F

    2015-01-01

    Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g-1·h-1 and a yield on glucose of 0.26 g·g-1. PMID:25919117

  4. Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle extraction

    PubMed Central

    Bansal-Mutalik, Ritu; Nikaido, Hiroshi

    2011-01-01

    Cells of the Corynebacterium-Nocardia-Mycobacterium group of bacteria are surrounded by an outer membrane (OM) containing mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. This OM presumably acts as a permeability barrier that imparts high levels of intrinsic drug resistance to some members of this group, such as Mycobacterium tuberculosis, and its component lipids have been studied intensively in a qualitative manner over the years. However, the quantitative lipid composition of this membrane has remained obscure, mainly because of difficulties in isolating it without contamination from the inner cytoplasmic membrane. Here we use the extraction, with reverse surfactant micelles, of intact cells of Corynebacterium glutamicum and show that this method extracts the free OM lipids quantitatively with no contamination from lipids of the cytoplasmic membrane, such as phosphatidylglycerol. Although only small amounts of corynomycolate were esterified to arabinogalactan, a large amount of cardiolipin was present in a nonextractable form, tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex. Furthermore, we show that the OM contains just enough lipid hydrocarbons to produce a bilayer covering the cell surface, with its inner leaflet composed mainly of the aforementioned nonextractable cardiolipin and its outer leaflet composed of trehalose dimycolates, phosphatidylinositol mannosides, and highly apolar lipids, similar to the Minnikin model of 1982. The reverse micelle extraction method is also useful for extracting proteins associated with the OM, such as porins. PMID:21876124

  5. Engineering Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid.

    PubMed

    Pérez-García, Fernando; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-09-01

    The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose. PMID:27345060

  6. Size exclusion chromatography: an improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites.

    PubMed

    Persicke, Marcus; Plassmeier, Jens; Neuweger, Heiko; Rückert, Christian; Pühler, Alfred; Kalinowski, Jörn

    2011-07-10

    The efficient separation of Corynebacterium glutamicum cells from culture medium by size exclusion chromatography (SEC) is presented. Residue analysis demonstrated that this method effectively depletes extracellular compounds. For evaluation, SEC was compared with the common methods cold methanol treatment, fast centrifugation and fast filtration. For this purpose, samples of C. glutamicum cells from fermenter cultures were harvested and subjected to a metabolome analysis. In particular, the wild type strain C. glutamicum ATCC13032 and the lysine production strain C. glutamicum DM1730 were grown in a minimal or in a complex medium. Comparison of metabolite pool sizes after harvesting C. glutamicum cells by the methods mentioned above by gas chromatography coupled to mass spectrometry (GC-MS) revealed that SEC is the most suitable method when intracellular metabolite pools are to be measured during growth in complex media or in the presence of significant amounts of secreted metabolites. In contrast to the other methods tested, the SEC method turned out to be fast and able to remove extracellular compounds almost completely. PMID:20817050

  7. Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate.

    PubMed

    Witthoff, Sabrina; Eggeling, Lothar; Bott, Michael; Polen, Tino

    2012-09-01

    Here, we show that Corynebacterium glutamicum ATCC 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. CO(2) measurements during bioreactor cultivation and use of (13)C-labelled formate demonstrated that formate is almost completely oxidized to CO(2). The deletion of fdhF (cg0618), annotated as formate dehydrogenase (FDH) and located in a cluster of genes conserved in the family Corynebacteriaceae, prevented formate utilization. Similarly, deletion of fdhD (cg0616) resulted in the inability to metabolize formate and deletion of cg0617 markedly reduced formate utilization. These results illustrated that all three gene products are required for FDH activity. Growth studies with molybdate and tungstate indicated that the FDH from C. glutamicum ATCC 13032 is a molybdenum-dependent enzyme. The presence of 100 mM formate caused a 25 % lowered growth rate during cultivation of C. glutamicum ATCC 13032 wild-type in glucose minimal medium. This inhibitory effect was increased in the strains lacking FDH activity. Our data demonstrate that C. glutamicum ATCC 13032 possesses an FDH with a currently unknown electron acceptor. The presence of the FDH might help the soil bacterium C. glutamicum ATCC 13032 to alleviate growth retardation caused by formate, which is ubiquitously present in the environment. PMID:22767548

  8. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. PMID:26168906

  9. Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

    PubMed

    Kind, Stefanie; Jeong, Weol Kyu; Schröder, Hartwig; Wittmann, Christoph

    2010-07-01

    In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an

  10. Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum

    PubMed Central

    Litsanov, Boris; Kabus, Armin; Brocker, Melanie; Bott, Michael

    2012-01-01

    Summary Corynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l−1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by‐product. By deleting genes for all known acetate‐producing pathways (pta‐ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l−1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l−1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)−1 h−1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta‐ackAΔsdhCABΔcat/pAN6‐pycP458Sppc) led to an additional increase of the product yield to 0.45 mol succinate mol−1 glucose and a titre of 10.6 g l−1 (90 mM) succinate. PMID:22018023

  11. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.

    PubMed

    Vogt, Michael; Haas, Sabine; Klaffl, Simon; Polen, Tino; Eggeling, Lothar; van Ooyen, Jan; Bott, Michael

    2014-03-01

    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, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application. PMID:24333966

  12. Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Uematsu, Kimio; Yamamoto, Shogo; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-07-01

    Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation. PMID:25808520

  13. Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

    PubMed

    Šilar, Radoslav; Holátko, Jiří; Rucká, Lenka; Rapoport, Andrey; Dostálová, Hana; Kadeřábková, Pavla; Nešvera, Jan; Pátek, Miroslav

    2016-09-01

    Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum. PMID:27270733

  14. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    PubMed

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene. PMID:27119954

  15. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum

    PubMed Central

    Shah, Adnan; Eikmanns, Bernhard J.

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene. PMID:27119954

  16. Evaluation of the food grade expression systems NICE and pSIP for the production of 2,5-diketo-D-gluconic acid reductase from Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production. PMID:23356419

  17. Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity.

    PubMed

    Walter, Frederik; Grenz, Sebastian; Ortseifen, Vera; Persicke, Marcus; Kalinowski, Jörn

    2016-08-20

    In this work the role of γ-glutamyl transpeptidase in the metabolism of γ-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate γ-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45°C. The KM value was 123μM. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of γ-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five γ-glutamyl dipeptides (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Val, γ-Glu-Leu, γ-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo. PMID:26528625

  18. Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

    SciTech Connect

    Vetting,M.; Frantom, P.; Blanchard, J.

    2008-01-01

    The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97 rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the {beta}-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.

  19. Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

    SciTech Connect

    Xu Ying; Yan Dazhong; Zhou Ningyi . E-mail: n.zhou@pentium.whiov.ac.cn

    2006-07-28

    Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.

  20. Structure of a GTP-dependent Bacterial PEP-carboxykinase from Corynebacterium glutamicum

    SciTech Connect

    Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

    2008-06-23

    GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.

  1. MscCG from Corynebacterium glutamicum: functional significance of the C-terminal domain.

    PubMed

    Becker, Michael; Krämer, Reinhard

    2015-10-01

    Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, e.g., glutamate and lysine. Excretion of glutamate into the surrounding medium under production conditions is mediated by MscCG, an MscS-type mechanosensitive channel. In difference to most other MscS-type channel proteins, MscCG carries, in addition to the N-terminal pore domain, a long C-terminal domain that amounts to about half of the size of the protein and harbors an additional transmembrane segment. Here we study the impact of the C-terminal domain on both functions of MscCG as mechanosensitive channel and as glutamate exporter. Sequential truncations of the C-terminal domain were applied, as well as deletion of particular subdomains, replacement of these segments by other amino acid sequences, and sequence randomization. Several parameters of cell physiology and bioenergetics of the obtained mutants related to both glutamate excretion and response to osmotic stress were quantified. All three subdomains of the C-terminal domain, i.e., the periplasmic loop, the fourth transmembrane segment, and the cytoplasmic loop, proved to be of core significance for MscCG function, in particular for glutamate excretion. PMID:26033538

  2. Crystal Structure and Biochemical Characterization of Tetrahydrodipicolinate N-Succinyltransferase from Corynebacterium glutamicum.

    PubMed

    Sagong, Hye-Young; Kim, Kyung-Jin

    2015-12-16

    Tetrahydrodipicolinate N-succinyltransferase (DapD) is an enzyme involved in the biosynthesis of l-lysine by converting tetrahydrodipicolinate into N-succinyl-l-2-amino-6-oxopimelate, using succinyl-CoA as a cofactor. We determined the crystal structure of DapD from Corynebacterium glutamicum (CgDapD). CgDapD functions as a trimer, and each monomer consists of three domains: an N-terminal helical domain (NTD), a left-handed β-helix (LβH) domain, and a β C-terminal domain (CTD). The mode of cofactor binding to CgDapD, elucidated by determining the structure in complex with succinyl-CoA, reveals that the position of the CTD changes slightly as the cofactor binds to the enzyme. The superposition of this structure with that of Mycobacterium tuberculosis shows differences in residues that make up cofactor-binding sites. Moreover, we determined the structure of CgDapD in complex with the substrate analogue 2-aminopimelate and revealed that the analogue was stabilized by conserved residues. The catalytic and substrate binding sites of CgDapD were confirmed by site-directed mutagenesis experiments. PMID:26602189

  3. Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase.

    PubMed

    Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen

    2016-05-01

    Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83g/l (0.06g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84g/l (0.41g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain. PMID:26992791

  4. Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

    PubMed

    Nimpiboon, Pitchanan; Kaulpiboon, Jarunee; Krusong, Kuakarun; Nakamura, Shigeyoshi; Kidokoro, Shun-Ichi; Pongsawasdi, Piamsook

    2016-05-01

    This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50°C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40°C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme. PMID:26875536

  5. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum.

    PubMed

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  6. The three tricarboxylate synthase activities of Corynebacterium glutamicum and increase of L-lysine synthesis.

    PubMed

    Radmacher, Eva; Eggeling, Lothar

    2007-09-01

    Corynebacterium glutamicum owns a citrate synthase and two methylcitrate synthases. Characterization of the isolated enzymes showed that the two methylcitrate synthases have comparable catalytic efficiency, k (cat)/K (m), as the citrate synthase with acetyl-CoA as substrate, although these enzymes are only synthesized during growth on propionate-containing media. Thus, the methylcitrate synthases have a relaxed substrate specifity, as also demonstrated by their activity with butyryl-CoA, whereas the citrate synthase does not accept acyl donors other than acetyl-CoA. A double mutant deleted of the citrate synthase gene gltA and one of the methylcitrate synthase genes, prpC1, was made unable to grow on glucose. From this mutant, a collection of suppressor mutants could be isolated which were demonstrated to have regained citrate synthase activity due to the relaxed specificity of the methylcitrate synthase PrpC2. Molecular characterization of these mutants showed that the regulator PrpR (Cg0800) located downstream of prpC1 is mutated with mutations likely to effect the secondary structure of the regulator, thus, resulting in expression of prpC2. This expression results in a citrate synthase activity, which is lower than that due to gltA in the original strain and results in increased L-lysine accumulation. PMID:17653710

  7. Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of L-threonine.

    PubMed

    Dong, Xunyan; Quinn, Peter J; Wang, Xiaoyuan

    2011-01-01

    L-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize L-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of L-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including L-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined L-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered. PMID:20688145

  8. Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock.

    PubMed

    Wellerdiek, Max; Winterhoff, Dajana; Reule, Waldemar; Brandner, Jürgen; Oldiges, Marco

    2009-08-01

    Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps. PMID:19050933

  9. Enhancing Corynebacterium glutamicum robustness by over-expressing a gene, mshA, for mycothiol glycosyltransferase.

    PubMed

    Liu, Ying-Bao; Chen, Can; Chaudhry, Muhammad Tausif; Si, Mei-Ru; Zhang, Lei; Wang, Yao; Shen, Xi-Hui

    2014-07-01

    Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), Cd(2+) (0.01 mM), Mn(2+) (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals. PMID:24737070

  10. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum.

    PubMed

    Du, Lei; Ma, Li; Qi, Feifei; Zheng, Xianliang; Jiang, Chengying; Li, Ailei; Wan, Xiaobo; Liu, Shuang-Jiang; Li, Shengying

    2016-03-18

    4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (Km and kcat) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route. PMID:26817843

  11. Proteome turnover in bacteria: current status for Corynebacterium glutamicum and related bacteria

    PubMed Central

    Trötschel, Christian; Albaum, Stefan P; Poetsch, Ansgar

    2013-01-01

    With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established ‘steady state’ proteomics approaches. In this article, we summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques. Although the number of available case studies for Corynebacterium glutamicum and related actinobacteria is still very limited, our review illustrates the potential of protein turnover studies for an improved understanding of questions in the area of biotechnology and biomedicine. Here, new insights from investigations of growth phase transition and different stress dynamics including iron, acid and heat stress for pathogenic but also for industrial actinobacteria are presented. Finally, we will comment on the advantages of integrated software solutions for biologists and briefly discuss the remaining technical challenges and upcoming possibilities for protein turnover analysis. PMID:23425033

  12. Interaction sites of DivIVA and RodA from Corynebacterium glutamicum

    PubMed Central

    Sieger, Boris; Bramkamp, Marc

    2015-01-01

    Elongation growth in actinobacteria is localized at the cell poles. This is in contrast to many classical model organisms where insertion of new cell wall material is localized around the lateral site. We previously described a role of RodA from Corynebacterium glutamicum in apical cell growth and morphogenesis. Deletion of rodA had drastic effects on morphology and growth, likely a result from misregulation of penicillin-binding proteins and cell wall precursor delivery. We identified the interaction of RodA with the polar scaffold protein DivIVA, thus explaining subcellular localization of RodA to the cell poles. In this study, we describe this interaction in detail and map the interaction sites of DivIVA and RodA. A single amino acid residue in the N-terminal domain of DivIVA was found to be crucial for the interaction with RodA. The interaction site of RodA was mapped to its cytoplasmic, C-terminal domain, in a region encompassing the last 10 amino acids (AAs). Deletion of these 10 AAs significantly decreased the interaction efficiency with DivIVA. Our results corroborate the interaction of DivIVA and RodA, underscoring the important role of DivIVA as a spatial organizer of the elongation machinery in Corynebacterineae. PMID:25709601

  13. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  14. The transcriptional regulatory repertoire of Corynebacterium glutamicum: reconstruction of the network controlling pathways involved in lysine and glutamate production.

    PubMed

    Brinkrolf, Karina; Schröder, Jasmin; Pühler, Alfred; Tauch, Andreas

    2010-09-01

    Corynebacterium glutamicum is one of the best studied organisms of the high G+C branch of Gram-positive bacteria and an emerging model system for the suborder Corynebacterineae. To gain insights into the regulatory gene composition and architecture of the transcriptional regulatory network of C. glutamicum, components of the transcriptional regulatory repertoire were intensively studied by many scientific groups in recent years. In this mini-review, we summarize the present knowledge about the deduced transcriptional regulatory repertoire of C. glutamicum and the current status of transcriptional regulatory network reconstruction with regard to the genome-wide detection of transcriptional regulations, coregulatory interactions and hierarchical cross-regulations. Moreover, we provide an overview of those regulators and their transcriptional regulations controlling genes involved in the conversion of the carbon sources glucose, fructose and sucrose into the industrially relevant products l-lysine and l-glutamate. This data will contribute to our understanding of l-lysine and l-glutamate production by C. glutamicum from the perspective of systems biology and may provide the basis for computational modeling of the respective biotechnologically important metabolic pathways. PMID:19963020

  15. Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum.

    PubMed

    Huang, Yuanyuan; Zhang, Hao; Tian, Hongming; Li, Cheng; Han, Shuangyan; Lin, Ying; Zheng, Suiping

    2015-09-01

    N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine that is inhibited by its end product L-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized L-arginine producing strain. PMID:25750030

  16. Requirement of de novo synthesis of the OdhI protein in penicillin-induced glutamate production by Corynebacterium glutamicum.

    PubMed

    Kim, Jongpill; Fukuda, Hirohisa; Hirasawa, Takashi; Nagahisa, Keisuke; Nagai, Kazuo; Wachi, Masaaki; Shimizu, Hiroshi

    2010-04-01

    We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. (3)H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production. PMID:19956942

  17. Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.

    PubMed

    Lindner, Steffen N; Seibold, Gerd M; Henrich, Alexander; Krämer, Reinhard; Wendisch, Volker F

    2011-06-01

    Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield. PMID:21478323

  18. Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer.

    PubMed

    Ikeda, Masato; Mitsuhashi, Satoshi; Tanaka, Kenji; Hayashi, Mikiro

    2009-03-01

    Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92(up), and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (Delta argR), the production was further increased. The best mutation set, Delta argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance

  19. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway.

    PubMed

    Xu, Jianzhong; Han, Mei; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-09-01

    The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and L-lysine production drastically improved. Moreover, increasing the flux through L-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and L-methionine biosynthesis, further improved L-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the L-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45% by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., L-threonine, L-methionine and L-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce L-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The L-lysine productivity was 2.73 g l(-1) h(-1) and the α was 47.06% after 48 h. However, the attenuation of MurE was not beneficial to increase the L-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through L-lysine biosynthetic pathway and DCW are beneficial to improve L-lysine production in C. glutamicum. PMID:24879631

  20. The transcriptional regulator SsuR activates expression of the Corynebacterium glutamicum sulphonate utilization genes in the absence of sulphate.

    PubMed

    Koch, Daniel J; Rückert, Christian; Albersmeier, Andreas; Hüser, Andrea T; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2005-10-01

    In a recent study, the putative regulatory gene cg0012 was shown to belong to the regulon of McbR, a global transcriptional regulator of sulphur metabolism in Corynebacterium glutamicum ATCC 13032. A deletion of cg0012, now designated ssuR (sulphonate sulphur utilization regulator), led to the mutant strain C. glutamicum DK100, which was shown to be blocked in the utilization of sulphonates as sulphur sources. According to DNA microarray hybridizations, transcription of the ssu and seu genes, encoding the sulphonate utilization system of C. glutamicum, was considerably decreased in C. glutamicum DK100 when compared with the wild-type strain. Electrophoretic mobility shift assays with purified SsuR protein demonstrated that the upstream regions of ssuI, seuABC, ssuD2 and ssuD1CBA contain SsuR binding sites. A nucleotide sequence alignment of the four DNA fragments containing the SsuR binding sites revealed a common 21 bp motif consisting of T-, GC- and A-rich domains. Mapping of the transcriptional start sites in front of ssuI, seuABC, ssuD2 and ssuD1CBA indicated that the SsuR binding sites are located directly upstream of identified promoter sequences and that the ssu genes are expressed by leaderless transcripts. Binding of the SsuR protein to its operator was shown to be diminished in vitro by the effector substance sulphate and its direct assimilation products adenosine 5'-phosphosulphate, sulphite and sulphide. Real-time reverse transcription polymerase chain reaction experiments verified that the expression of the ssu and seu genes was also repressed in vivo by the presence of sulphate or sulphite. Therefore, the regulatory protein SsuR activates the expression of the ssu and seu genes in C. glutamicum in the absence of the preferred sulphur source sulphate. PMID:16194234

  1. The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

    PubMed

    Vogt, Michael; Krumbach, Karin; Bang, Won-Gi; van Ooyen, Jan; Noack, Stephan; Klein, Bianca; Bott, Michael; Eggeling, Lothar

    2015-01-01

    L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids. PMID:25301583

  2. A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum.

    PubMed

    Ohnishi, Junko; Katahira, Ritsuko; Mitsuhashi, Satoshi; Kakita, Shingo; Ikeda, Masato

    2005-01-15

    Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation. PMID:15621447

  3. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    PubMed

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  4. Engineering of Corynebacterium glutamicum for xylitol production from lignocellulosic pentose sugars.

    PubMed

    Dhar, Kiran S; Wendisch, Volker F; Nampoothiri, Kesavan Madhavan

    2016-07-20

    Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L). PMID:27184428

  5. Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and class II aldolases.

    PubMed

    von der Osten, C H; Barbas, C F; Wong, C H; Sinskey, A J

    1989-11-01

    The Corynebacterium glutamicum fda gene encoding fructose-1,6-biphosphate (FBP) aldolase has been isolated by complementation of an Escherichia coli mutant. The nucleotide sequence of a 3371 bp chromosomal fragment containing the C. glutamicum fda gene was determined. The N-terminal amino acid sequence of C. glutamicum FBP aldolase identified the correct initiation site for the fda gene, and a molecular weight of 37,092 was predicted for the fda polypeptide. S1 nuclease mapping identified the transcriptional start site, and Northern hybridization analysis indicated that the fda gene encodes a single 1.3 kb transcript. The primary structure of C. glutamicum FBP aldolase shows strong homology to class II FBP aldolases. Conservation of primary structure was observed between class I and class II aldolases, but several residues essential for catalytic activity in class I aldolases were absent from class II aldolases. PMID:2615658

  6. Cutting the Gordian Knot: Identifiability of anaplerotic reactions in Corynebacterium glutamicum by means of (13) C-metabolic flux analysis.

    PubMed

    Kappelmann, Jannick; Wiechert, Wolfgang; Noack, Stephan

    2016-03-01

    Corynebacterium glutamicum is the major workhorse for the microbial production of several amino and organic acids. As long as these derive from tricarboxylic acid cycle intermediates, the activity of anaplerotic reactions is pivotal for a high biosynthetic yield. To determine single anaplerotic activities (13) C-Metabolic Flux Analysis ((13) C-MFA) has been extensively used for C. glutamicum, however with different network topologies, inconsistent or poorly determined anaplerotic reaction rates. Therefore, in this study we set out to investigate whether a focused isotopomer model of the anaplerotic node can at all admit a unique solution for all fluxes. By analyzing different scenarios of active anaplerotic reactions, we show in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from (13) C-isotopomer data. We stress that the result of this analysis for different assumptions on active enzymes is directly transferable to other compartment-free organisms. Our results demonstrate that there exist biologically relevant metabolic network topologies for which the flux distribution cannot be inferred by classical (13) C-MFA. PMID:26375179

  7. Enhancing (L)-isoleucine production by thrABC overexpression combined with alaT deletion in Corynebacterium glutamicum.

    PubMed

    Wang, Jing; Wen, Bing; Wang, Jian; Xu, Qingyang; Zhang, Chenglin; Chen, Ning; Xie, Xixian

    2013-09-01

    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, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher L-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of L-lysine, L-alanine, and L-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to L-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly. PMID:23813403

  8. Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase

    PubMed Central

    2014-01-01

    Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L−1 glucose and 0.1 mM pyridoxal 5′-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L−1 (0.259 g L−1 h−1) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol−1. PMID:24949255

  9. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions

    PubMed Central

    2013-01-01

    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 compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  10. The physiological role of riboflavin transporter and involvement of FMN-riboswitch in its gene expression in Corynebacterium glutamicum.

    PubMed

    Takemoto, Norihiko; Tanaka, Yuya; Inui, Masayuki; Yukawa, Hideaki

    2014-05-01

    Riboflavin is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which work as cofactors of numerous enzymes. Understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. In this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expression of riboflavin transporter in Corynebacterium glutamicum. Disruption of the ribA gene that encodes bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase in C. glutamicum suppressed growth in the absence of supplemental riboflavin. The growth was fully recovered upon supplementation with 1 μM riboflavin, albeit at reduced intracellular concentrations of FMN and FAD during the log phase. Concomitant disruption of the ribA and ribM gene that encodes a riboflavin transporter exacerbated supplemental riboflavin requirement from 1 μM to 50 μM. RibM expression in FMN-rich cells was about 100-fold lower than that in FMN-limited cells. Mutations in putative FMN-riboswitch present immediately upstream of the ribM gene abolished the FMN response. This 5'UTR sequence of ribM constitutes a functional FMN-riboswitch in C. glutamicum. PMID:24531272

  11. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions.

    PubMed

    Tsuge, Yota; Tateno, Toshihiro; Sasaki, Kengo; Hasunuma, Tomohisa; Tanaka, Tsutomu; Kondo, Akihiko

    2013-01-01

    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 compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  12. Characterization of a Corynebacterium glutamicum dnaB mutant that shows temperature-sensitive growth and mini-cell formation.

    PubMed

    Uchida, Makoto; Hirasawa, Takashi; Wachi, Masaaki

    2014-12-01

    Corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. In order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from C. glutamicum wild-type strain ATCC 31831, and found that one of them, M45, produced high frequencies of mini-cells with no nucleoids. Cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally observed. The temperature sensitivity and mini-cell formation of M45 was complemented by a 2-kb DraI-EcoRI fragment derived from the ATCC 31831 chromosomal DNA, which carried a dnaB homolog encoding a replicative DNA helicase. DNA sequence analysis revealed that M45 carried a missense mutation in the dnaB gene, which caused a substitution of Thr364 to Ile. Microscopic observation after 4',6-diamidino-2-phenylindole staining revealed that the DNA content of single cells was decreased by culturing at the restrictive temperature, suggesting that the mutation affects chromosomal replication. These results suggest that the C. glutamicum dnaB mutant performs an asymmetric cell division even after DNA replication is inhibited, which results in the production of mini-cells. PMID:25141796

  13. A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background 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. Results Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. Conclusions The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising

  14. A gene homologous to beta-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions.

    PubMed

    Mitsuhashi, S; Ohnishi, J; Hayashi, M; Ikeda, M

    2004-02-01

    Carbonic anhydrase catalyzes the interconversion of CO(2) and bicarbonate. We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for L-lysine overproduction. A whole-genome sequence revealed two genes encoding putative beta-type and gamma-type carbonic anhydrases in C. glutamicum. These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively. Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO(2)) on both complete and minimal media. The growth defect of the Delta bca strain was restored under elevated CO(2) conditions (5% CO(2)). Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene ( pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO(2). In contrast, the Delta gca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO(2) conditions. Attempts to increase the dosage of bca, gca, and pca in the defined L-lysine-producing strain C. glutamicum AHD-2 led to no discernable effects on growth and production. Northern blot analysis indicated that the bca transcript in strain AHD-2 and another L-lysine producer, C. glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases. These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product's ability to maintain favorable intracellular bicarbonate/CO(2) levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of L-lysine overproduction, both of which are

  15. Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

    PubMed Central

    Molenaar, Douwe; van der Rest, Michel E.; Drysch, André; Yücel, Raif

    2000-01-01

    Like many other bacteria, Corynebacterium glutamicum possesses two types of l-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure. PMID:11092846

  16. Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in Corynebacterium glutamicum.

    PubMed

    Lee, Jung-Hoon; Kim, Yong-Jae; Shin, Hee-Sung; Lee, Heung-Shick; Jin, Shouguang; Ha, Un-Hwan

    2016-06-01

    Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum. PMID:27225460

  17. A TatABC-Type Tat Translocase Is Required for Unimpaired Aerobic Growth of Corynebacterium glutamicum ATCC13032

    PubMed Central

    Oertel, Dan; Schmitz, Sabrina; Freudl, Roland

    2015-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria. PMID:25837592

  18. Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

    PubMed Central

    Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Ma, Yanhe

    2015-01-01

    The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars. PMID:25888171

  19. Gene expression analysis of Corynebacterium glutamicum subjected to long-term lactic acid adaptation.

    PubMed

    Jakob, Kinga; Satorhelyi, Peter; Lange, Christian; Wendisch, Volker F; Silakowski, Barbara; Scherer, Siegfried; Neuhaus, Klaus

    2007-08-01

    Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced >or=2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low p

  20. Gene Expression Analysis of Corynebacterium glutamicum Subjected to Long-Term Lactic Acid Adaptation▿ ¶

    PubMed Central

    Jakob, Kinga; Satorhelyi, Peter; Lange, Christian; Wendisch, Volker F.; Silakowski, Barbara; Scherer, Siegfried; Neuhaus, Klaus

    2007-01-01

    Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced ≥2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low p

  1. Osmo-sensing by N- and C-terminal extensions of the glycine betaine uptake system BetP of Corynebacterium glutamicum.

    PubMed

    Peter, H; Burkovski, A; Krämer, R

    1998-01-30

    The major uptake carrier for the compatible solute glycine betaine in Corynebacterium glutamicum is the secondary transport system BetP. It is effectively regulated by the external osmolality both on the level of expression and of activity. BetP carries highly charged domains both at the N and at the C terminus. We investigated the role of these extensions in the regulatory response to hyperosmotic stress. Mutants of the betP gene coding for proteins with truncated N- and C-terminal extensions were expressed in the C. glutamicum betP deletion strain DHP1 and were functionally characterized with respect to regulation of activity. The optimum of activation at 1.3 osmol/kg in wild type was shifted in the recombinant strains to about 2.6 osmol/kg in mutants with deletions in the N-terminal part. Deletions in the C-terminal domain resulted in a complete loss of regulation. The altered response to changes in osmolality led to severe consequences in the cellular adaption to hyperosmotic stress. Whereas in the wild type, the steady state level of glycine betaine accumulation is maintained by activity regulation of the BetP system itself, in the mutant with BetP proteins carrying truncations in the C-terminal domain, the observed steady state betaine accumulation was found to be due to a kinetic balance of unregulated glycine betaine uptake by the modifed BetP and efflux via the mechanosensitive efflux channel for compatible solutes at the same time. PMID:9446558

  2. Transcriptional response of Corynebacterium glutamicum ATCC 13032 to hydrogen peroxide stress and characterization of the OxyR regulon.

    PubMed

    Milse, Johanna; Petri, Kathrin; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The aerobic soil bacterium Corynebacterium glutamicum ATCC 13032 has a remarkable natural resistance to hydrogen peroxide. A major player in hydrogen peroxide defense is the LysR type transcriptional regulator OxyR, homologs of which are present in a wide range of bacteria. In this study, the global transcriptional response of C. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome DNA microarrays, demonstrating the dynamic reaction of the regulatory networks. Deletion of oxyR resulted in an increased resistance of the C. glutamicum mutant to hydrogen peroxide. By performing DNA microarray hybridizations and RT-qPCR, differentially expressed genes were detected in the mutant. The direct control by OxyR was verified by electrophoretic mobility shift assays for 12 target regions. The results demonstrated that OxyR in C. glutamicum acts as a transcriptional repressor under non-stress conditions for a total of 23 genes. The regulated genes encode proteins related to oxidative stress response (e.g. katA), iron homeostasis (e.g. dps) and sulfur metabolism (e.g. suf cluster). Besides the regulator of the suf cluster, SufR, OxyR regulated the gene cg1695 encoding a putative transcriptional regulator, indicating the role of OxyR as a master regulator in defense against oxidative stress. Using a modified DNase footprint approach, the OxyR-binding sites in five target promoter regions, katA, cydA, hemH, dps and cg1292, were localized and in each upstream region at least two overlapping binding sites were found. The DNA regions protected by the OxyR protein are about 56bp in length and do not have evident sequence similarities. Still, by giving an insight in the H2O2 stimulon and extending the OxyR regulon this study considerably contributes to the understanding of the response of C. glutamicum to hydrogen peroxide-mediated oxidative stress. PMID:25107507

  3. Engineering of nitrogen metabolism and its regulation in Corynebacterium glutamicum: influence on amino acid pools and production.

    PubMed

    Rehm, Nadine; Burkovski, Andreas

    2011-01-01

    Nitrogen is one of the macronutrients necessary for living cells, and consequently, assimilation of nitrogen is a crucial step for metabolism. To satisfy their nitrogen demand and to ensure a sufficient nitrogen supply even in situations of nitrogen limitation, microorganisms have evolved sophisticated uptake and assimilation mechanisms for different nitrogen sources. This mini-review focuses on nitrogen metabolism and its control in the biotechnology workhorse Corynebacterium glutamicum, which is used for the industrial production of more than 2 million tons of L: -amino acids annually. Ammonium assimilation and connected control mechanisms on activity and transcription level are summarized, and the influence of mutations on amino acid pools and production is described with emphasis on L: -glutamate, L: -glutamine, and L: -lysine. PMID:20922371

  4. Characterization of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain.

    PubMed

    Ohnishi, Junko; Mizoguchi, Hiroshi; Takeno, Seiki; Ikeda, Masato

    2008-01-01

    Mutations induced by classical whole-cell mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were determined for all genes of pathways from glucose to L-lysine in an industrial L-lysine producer of Corynebacterium glutamicum. A total of 50 mutations with a genome-wide distribution were identified and characterized for mutational types and mutagenic specificities. Those mutations were all point mutations with single-base substitutions and no deletions, frame shifts, and insertions were found. Among six possible types of base substitutions, the mutations consisted of only two types: 47 G.C-->A.T transitions and three A.T-->G.C transitions with no transversion. The findings indicate a limited repertoire of amino acid substitutions by classical NTG mutagenesis and thus raise a new possibility of further improving industrial strains by optimizing key mutations through PCR-mediated site-directed mutagenesis. PMID:18037338

  5. Improved L-lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity.

    PubMed

    van Ooyen, Jan; Noack, Stephan; Bott, Michael; Reth, Alexander; Eggeling, Lothar

    2012-08-01

    We here developed a series of Corynebacterium glutamicum strains with gradual decreased specific citrate synthase (CS) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on L-lysine formation and growth. We achieved an intended gradual L-lysine yield increase and recognized and overcame further new limitations in the L-lysine biosynthesis pathway to result in a strain with the highest yield reported so far when assayed under comparable conditions. As a non-intended outcome, a detailed flux analysis revealed an almost constant flux through CS at 10% remaining CS activity, whereas the metabolome data revealed an increase in the oxaloacetate and acetyl-CoA concentrations. Hence reduced CS activity is apparently efficiently buffered by increased concentrations of CS substrates, implying a certain robustness of the central metabolism in response of the imposed gene expressions. PMID:22392073

  6. Involvement of the osrR gene in the hydrogen peroxide-mediated stress response of Corynebacterium glutamicum.

    PubMed

    Hong, Eun-Ji; Kim, Pil; Kim, Eung-Soo; Kim, Younhee; Lee, Heung-Shick

    2016-01-01

    A transcriptional profile of the H2O2-adapted Corynebacterium glutamicum HA strain reveals a list of upregulated regulatory genes. Among them, we selected ORF NCgl2298, designated osrR and analyzed its role in H2O2 adaptation. The osrR-deleted (ΔosrR) mutant had defective growth in minimal medium, which was even more pronounced in an osrR deletion mutant of an HA strain. The ΔosrR strain displayed increased sensitivity to H2O2. In addition to H2O2 sensitivity, the ΔosrR strain was found to be temperature-sensitive at 37 °C. 2D-PAGE analysis of the ΔosrR mutant found that MetE and several other proteins involved in redox metabolism were affected by the mutation. Accordingly, the NADPH/NADP(+) ratio of the ΔosrR strain (0.85) was much lower than that of the wild-type strain (2.01). In contrast, the NADH/NAD(+) ratio of the mutant (0.54) was considerably higher than that of the wild-type (0.21). Based on these findings, we propose that H2O2-detoxifying metabolic systems, excluding those involving catalase, are present in C. glutamicum and are regulated, in part, by osrR. PMID:26433092

  7. Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate.

    PubMed

    Leßmeier, Lennart; Pfeifenschneider, Johannes; Carnicer, Marc; Heux, Stephanie; Portais, Jean-Charles; Wendisch, Volker F

    2015-12-01

    Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine. PMID:26276544

  8. Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.

    PubMed

    Jensen, Jaide V K; Eberhardt, Dorit; Wendisch, Volker F

    2015-11-20

    The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes. PMID:26393954

  9. Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

    PubMed Central

    Vrljic, M; Kronemeyer, W; Sahm, H; Eggeling, L

    1995-01-01

    We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine. PMID:7608075

  10. From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production.

    PubMed

    Becker, Judith; Zelder, Oskar; Häfner, Stefan; Schröder, Hartwig; Wittmann, Christoph

    2011-03-01

    Here, we describe the development of a genetically defined strain of l-lysine hyperproducing Corynebacterium glutamicum by systems metabolic engineering of the wild type. Implementation of only 12 defined genome-based changes in genes encoding central metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L(-1) lysine and a productivity of 4.0 g L(-1) h(-1) in fed-batch culture. The specific glucose uptake rate of the wild type could be completely maintained during the engineering process, providing a highly viable producer. For these key criteria, the genetically defined strain created in this study lies at the maximum limit of classically derived producers developed over the last fifty years. This is the first report of a rationally derived lysine production strain that may be competitive with industrial applications. The design-based strategy for metabolic engineering reported here could serve as general concept for the rational development of microorganisms as efficient cellular factories for bio-production. PMID:21241816

  11. Modular Optimization of a Hemicellulose-Utilizing Pathway in Corynebacterium glutamicum for Consolidated Bioprocessing of Hemicellulosic Biomass.

    PubMed

    Yim, Sung Sun; Choi, Jae Woong; Lee, Se Hwa; Jeong, Ki Jun

    2016-04-15

    Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass. PMID:26808593

  12. Osmotic stress response: quantification of cell maintenance and metabolic fluxes in a lysine-overproducing strain of Corynebacterium glutamicum.

    PubMed

    Varela, Cristian A; Baez, Mauricio E; Agosin, Eduardo

    2004-07-01

    Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability. PMID:15240305

  13. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    PubMed

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes. PMID:26494188

  14. RosR (Cg1324), a Hydrogen Peroxide-sensitive MarR-type Transcriptional Regulator of Corynebacterium glutamicum*

    PubMed Central

    Bussmann, Michael; Baumgart, Meike; Bott, Michael

    2010-01-01

    The cg1324 gene (rosR) of Corynebacterium glutamicum encodes a MarR-type transcriptional regulator. By a comparative transcriptome analysis with DNA microarrays of a ΔrosR mutant and the wild type and subsequent EMSAs with purified RosR protein, direct target genes of RosR were identified. The narKGHJI operon, which encodes a nitrate/nitrite transporter and the dissimilatory nitrate reductase complex, was activated by RosR. All other target genes were repressed by RosR. They encode four putative monooxygenases, two putative FMN reductases, a protein of the glutathione S-transferase family, a putative polyisoprenoid-binding protein, and RosR itself. The DNA binding site of RosR was characterized as an 18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA. The in vitro DNA binding activity of RosR was reversibly inhibited by the oxidant H2O2. Mutational analysis of the three cysteine residues present in RosR (Cys-64, Cys-92, and Cys-151) showed that these are responsible for the inhibition of DNA binding by H2O2. A deletion mutant (Δcg1322) lacking the putative polyisoprenoid-binding protein showed an increased sensitivity to H2O2, supporting the role of RosR in the oxidative stress response of C. glutamicum. PMID:20643656

  15. Corynebacterium glutamicum as a potent biocatalyst for the bioconversion of pentose sugars to value-added products.

    PubMed

    Gopinath, Vipin; Murali, Anusree; Dhar, Kiran S; Nampoothiri, K Madhavan

    2012-01-01

    Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals. PMID:22094976

  16. Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Kreye, Steffen; Wittmann, Christoph

    2011-09-01

    The present work describes the development of a superior strain of Corynebacterium glutamicum for diaminopentane (cadaverine) production via metabolic engineering of cellular transport processes. In C. glutamicum DAP-3c, a tailor-made producer, the diaminopentane forming enzyme, lysine decarboxylase, was inhibited in vivo by its end-product, suggesting a potential bottleneck at the level of the export. The previously proposed lysine exporter lysE was shown not to be involved in diaminopentane export. Its deletion did not reduce diaminopentane secretion and could therefore be exploited to completely eliminate the export of lysine, an undesired by-product. Genome-wide transcription profiling revealed the up-regulation of 35 candidate genes as response to diaminopentane overproduction, including several transporters. The highest expression increase (2.6-fold) was observed for a permease, encoded by cg2893. Targeted gene deletion in the producer resulted in a 90% reduced diaminopentane secretion. Genome-based overexpression of the exporter, however, revealed a 20% increased yield, a 75% reduced formation of the undesired by-product N-acetyl-diaminopentane and a substantially higher viability, reflected by increased specific rates for growth, glucose uptake and product formation. Similarly, deletion of cg2894, TetR type repressor neighboring the permease gene, resulted in improved production properties. The discovery and amplification of the permease, as presented here, displays a key contribution towards superior C. glutamicum strains for production of the platform chemical diaminopentane. The exact function of the permease remained unclear. Its genetic modification had pronounced effects on various intracellular pools of the biosynthetic pathway, which did not allow a final conclusion on its physiological role, although a direct contribution to diaminopentane export appears possible. PMID:21821142

  17. Regulation of the malic enzyme gene malE by the transcriptional regulator MalR in Corynebacterium glutamicum.

    PubMed

    Krause, Jens P; Polen, Tino; Youn, Jung-Won; Emer, Denise; Eikmanns, Bernhard J; Wendisch, Volker F

    2012-06-15

    Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR. PMID:22261175

  18. Maltose Uptake by the Novel ABC Transport System MusEFGK2I Causes Increased Expression of ptsG in Corynebacterium glutamicum

    PubMed Central

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W.; Krämer, Reinhard

    2013-01-01

    The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [14C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum. PMID:23543710

  19. Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system.

    PubMed

    Schneider, Jens; Eberhardt, Dorit; Wendisch, Volker F

    2012-07-01

    Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). Previously, we constructed the putrescine-producing strain PUT1 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the L-arginine repressor, combined with heterologous expression of the Escherichia coli gene for L-ornithine decarboxylase SpeC. As a consequence of argF deletion, this strain requires supplementation of L-arginine and shows growth-decoupled putrescine production. To avoid costly supplementation with L-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by L-arginine, a plasmid addiction system for low-level argF expression was developed. By fine-tuning argF expression through modifications of the promoter, the translational start codon and/or the ribosome binding site, high productivity and titer could be obtained. OTC activity varied almost thousandfold between 960 and 1 mU mg⁻¹ resulting in putrescine yields on glucose from less than 0.001 up to 0.26 g g⁻¹, the highest yield in bacteria reported to date. The most promising strain, designated PUT21, was characterized comprehensively. PUT21 strain grew with a rate of 0.19 h⁻¹ in mineral salt medium without the need for L-arginine supplementation and produced putrescine with a yield of 0.16 g g⁻¹ glucose at a volumetric productivity of 0.57 g L⁻¹ h⁻¹ and a specific productivity of 0.042 g g⁻¹ h⁻¹. The carbon balance suggested that no major unidentified by-product was produced. Compared to the first-generation strain PUT1, the putrescine yield observed with PUT21 was increased by 60%. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g L⁻¹ at a volumetric productivity of 0.55 g L⁻¹ h⁻¹ and a yield of 0.16 g g⁻¹ glucose could be achieved. Moreover, while plasmid segregation of the initial strain required antibiotic selection

  20. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation.

    PubMed

    Kabus, Armin; Georgi, Tobias; Wendisch, Volker F; Bott, Michael

    2007-05-01

    A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type. PMID:17216441

  1. Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey.

    PubMed

    Barrett, Eoin; Stanton, Catherine; Zelder, Oskar; Fitzgerald, Gerald; Ross, R Paul

    2004-05-01

    The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and beta-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability. PMID:15128544

  2. Production of L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168) and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and Reusability

    PubMed Central

    Shyamkumar, Rajaram; Moorthy, Innasi Muthu Ganesh; Ponmurugan, Karuppiah; Baskar, Rajoo

    2014-01-01

    Background L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food additive and flavor enhancer in the form of sodium salt. Corynebacterium glutamicum (C. glutamicum) is one of the major organisms widely used for glutamic acid production. Methods The study was dealing with immobilization of C. glutamicum and mixed culture of C. glutamicum and Pseudomonas reptilivora (P. reptilivora) for L-glutamic acid production using submerged fermentation. 2, 3 and 5% sodium alginate concentrations were used for production and reusability of immobilized cells for 5 more trials. Results The results revealed that 2% sodium alginate concentration produced the highest yield (13.026±0.247 g/l by C. glutamicum and 16.026±0.475 g/l by mixed immobilized culture). Moreover, reusability of immobilized cells was evaluated in 2% concentration with 5 more trials. However, when the number of cycles increased, the production of L-glutamic acid decreased. Conclusion Production of glutamic acid using optimized medium minimizes the time needed for designing the medium composition. It also minimizes external contamination. Glutamic acid production gradually decreased due to multiple uses of beads and consequently it reduces the shelf life. PMID:25215180

  3. Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum (D)-lactate productivity under oxygen deprivation.

    PubMed

    Tsuge, Yota; Yamamoto, Shougo; Suda, Masako; Inui, Masayuki; Yukawa, Hideaki

    2013-08-01

    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 glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on D-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect D-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The D-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of D-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the D-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on D-lactate production is discussed. PMID:23712891

  4. From zero to hero - production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Neubauer, Steffi; Becker, Judith; Yamamoto, Motonori; Völkert, Martin; Abendroth, Gregory von; Zelder, Oskar; Wittmann, Christoph

    2014-09-01

    Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2gL(-1)h(-1), and a final titer of 88gL(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production. PMID:24831706

  5. Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σH in Corynebacterium glutamicum

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Yukawa, Hideaki

    2014-01-01

    The extracytoplasmic function sigma factor σH is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of σH. Here, we determined the direct genome-wide targets of σH using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an anti-σ factor of σH. Seventy-five σH-dependent promoters, including 39 new ones, were identified. σH-dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5′ ends were mapped to the σH-dependent promoters identified. Interestingly, functional internal σH-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of σH. Furthermore, sigA encoding the primary σ factor was identified as a new target of σH. Reporter assays demonstrated that the σH-dependent promoter upstream of sigA was highly heat inducible but much weaker than the known σA-dependent one. Our ChIP-chip analysis also detected the σH-dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 σ factor, supporting the previous findings of their σH-dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum. PMID:25404703

  6. SpiE interacts with Corynebacterium glutamicum WhcE and is involved in heat and oxidative stress responses.

    PubMed

    Park, Jung Chul; Park, Joon-Song; Kim, Younhee; Kim, Pil; Kim, Eung Soo; Lee, Heung-Shick

    2016-05-01

    The gene whcE in Corynebacterium glutamicum positively responds to oxidative and heat stress. To search for proteins that interact with WhcE, we employed a two-hybrid system with WhcE as the bait. Sequencing analysis of the isolated clones revealed peptide sequences, one of which showed high sequence identity to a hydrophobe/amphiphile efflux-1 family transporter encoded by NCgl1497. The interaction of the NCgl1497-encoded protein with WhcE in vivo was verified using reporter gene expression by real-time quantitative PCR (RT-qPCR). The WhcE protein strongly interacted with the NCgl1497-encoded protein in the presence of oxidative and heat stress. Furthermore, purified WhcE and NCgl1497-encoded proteins interacted in vitro, especially in the presence of the oxidant diamide, and the protein-protein interaction was disrupted in the presence of the reductant dithiothreitol. In addition, the transcription of NCgl1497 was activated approximately twofold in diamide- or heat-treated cells. To elucidate the function of the NCgl497 gene, an NCgl1497-deleted mutant strain was constructed. The mutant showed decreased viability in the presence of diamide and heat stress. The mutant strain also exhibited reduced transcription of the thioredoxin reductase gene, which is known to be regulated by whcE. Based on the results, NCgl1497 was named spiE (stress protein interacting with WhcE). Collectively, our data suggest that spiE is involved in the whcE-mediated oxidative stress response pathway of C. glutamicum. PMID:26996627

  7. Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level.

    PubMed

    Nanda, Arun M; Heyer, Antonia; Krämer, Christina; Grünberger, Alexander; Kohlheyer, Dietrich; Frunzke, Julia

    2014-01-01

    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 level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages. PMID:24163339

  8. Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

    PubMed Central

    Chi, Bui Khanh; Busche, Tobias; Van Laer, Koen; Bäsell, Katrin; Becher, Dörte; Clermont, Lina; Seibold, Gerd M.; Persicke, Marcus; Kalinowski, Jörn; Messens, Joris

    2014-01-01

    Abstract Aims: 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. Results: Transcriptomics revealed the strong upregulation of the disulfide stress σH regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography–tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives. Antioxid. Redox Signal. 20, 589–605

  9. Negative transcriptional control of biotin metabolism genes by the TetR-type regulator BioQ in biotin-auxotrophic Corynebacterium glutamicum ATCC 13032.

    PubMed

    Brune, Iris; Götker, Susanne; Schneider, Jessica; Rodionov, Dmitry A; Tauch, Andreas

    2012-06-15

    Genomic context analysis in actinobacteria revealed that biotin biosynthesis and transport (bio) genes are co-localized in several genomes with a gene encoding a transcription regulator of the TetR protein family, now named BioQ. Comparative analysis of the upstream regions of bio genes identified the common 13-bp palindromic motif TGAAC-N3-GTTAC as candidate BioQ-binding site. To verify the role of BioQ in controlling the transcription of bio genes, a deletion in the bioQ coding region (cg2309) was constructed in Corynebacterium glutamicum ATCC 13032, resulting in the mutant strain C. glutamicum IB2309. Comparative whole-genome DNA microarray hybridizations and subsequent expression analyses by real-time reverse transcriptase PCR revealed enhanced transcript levels of all bio genes in C. glutamicum IB2309, when compared with the wild-type strain ATCC 13032. Accordingly, the BioQ protein of C. glutamicum acts as a repressor of ten genes that are organized in four transcription units: bioA-bioD, cg2884-cg2883, bioB-cg0096-cg0097, and bioY-bioM-bioN. DNA band shift assays with an intein-tagged BioQ protein demonstrated the specific binding of the purified protein to DNA fragments containing the candidate BioQ-binding sites, which were located within the mapped promoter regions of bioA, cg2884, bioB, and bioY. These data confirmed the direct regulatory role of BioQ in the control of biotin biosynthesis and transport genes in C. glutamicum. Differential expression of bio genes in C. glutamicum IB2309 was moreover complemented by bioQ genes cloned from other corynebacterial genomes. PMID:22178235

  10. Mechanism of concerted inhibition of alpha2beta2-type hetero-oligomeric aspartate kinase from Corynebacterium glutamicum.

    PubMed

    Yoshida, Ayako; Tomita, Takeo; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2010-08-27

    Aspartate kinase (AK) is the first and committed enzyme of the biosynthetic pathway producing aspartate family amino acids, lysine, threonine, and methionine. AK from Corynebacterium glutamicum (CgAK), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. To elucidate the mechanism of this unique regulation in CgAK, we determined the crystal structures in several forms: an inhibitory form complexed with both lysine and threonine, an active form complexed with only threonine, and a feedback inhibition-resistant mutant (S301F) complexed with both lysine and threonine. CgAK has a characteristic alpha(2)beta(2)-type heterotetrameric structure made up of two alpha subunits and two beta subunits. Comparison of the crystal structures between inhibitory and active forms revealed that binding inhibitors causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in the alpha subunit and beta subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of alpha(2)beta(2)-type AK but also the mechanism of concerted inhibition in CgAK. PMID:20573952

  11. The extracytoplasmic function σ factor σ(C) regulates expression of a branched quinol oxidation pathway in Corynebacterium glutamicum.

    PubMed

    Toyoda, Koichi; Inui, Masayuki

    2016-05-01

    Bacteria modify their expression of different terminal oxidases in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium of the phylum Actinobacteria, possesses aa3 -type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced by oxygen limitation. We report that an extracytoplasmic function σ factor, σ(C) , is responsible for the regulation of this process. Chromatin immunoprecipitation with microarray analysis detected eight σ(C) -binding regions in the genome, facilitating the identification of a consensus promoter sequence for σ(C) recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. However, one consensus promoter sequence found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a σ(C) -dependent negative effect on expression of the operon. The σ(C) regulon was induced by cytochrome aa3 deficiency without modifying sigC expression, but not by bc1 complex deficiency. These findings suggest that σ(C) is activated in response to impaired electron transfer via cytochrome aa3 and not directly to a shift in oxygen levels. Our results reveal a new paradigm for transcriptional regulation of the aerobic respiratory system in bacteria. PMID:26789738

  12. The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species-scavenging enzyme that shows promiscuity in thiol redox control.

    PubMed

    Pedre, Brandán; Van Molle, Inge; Villadangos, Almudena F; Wahni, Khadija; Vertommen, Didier; Turell, Lucía; Erdogan, Huriye; Mateos, Luis M; Messens, Joris

    2015-06-01

    Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (-SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H2 O2 reduction, a sulfenic acid (-SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S-S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx-MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general. PMID:25766783

  13. Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase▿†

    PubMed Central

    Baumgart, Meike; Mustafi, Nurije; Krug, Andreas; Bott, Michael

    2011-01-01

    The aconitase gene acn of Corynebacterium glutamicum is regulated by four transcriptional regulators, indicating that the synthesis of this enzyme is carefully controlled. To understand the causes for this elaborate regulation, the properties of the Δacn-1 deletion mutant were analyzed in detail. The mutant was glutamate auxotrophic in glucose minimal medium, showed a strong growth defect, and secreted large amounts of acetate. None of these phenotypes could be complemented by plasmid-encoded aconitase, suggesting the presence of a secondary mutation. In fact, a point mutation within the gltA gene encoding citrate synthase was identified that caused the instability of the protein and an almost complete lack of its enzymatic activity. Subsequently, 27 further, independent Δacn clones were isolated, and 15 of them were found to contain distinct mutations in gltA, causing the loss of citrate synthase activity. A similar result was observed for mutants lacking the isocitrate dehydrogenase gene icd. In this case, 8 of 24 Δicd clones contained additional mutations in gltA. Indirect evidence was obtained that elevated intracellular citrate concentrations could be the cause of this selection pressure. Accordingly, the careful control of aconitase synthesis might have evolved due to the necessity to avoid inhibitory cytoplasmic citrate levels on the one hand and to prevent the excessive synthesis of an oxygen-sensitive protein requiring both iron and sulfur on the other hand. PMID:21984793

  14. Chassis organism from Corynebacterium glutamicum--a top-down approach to identify and delete irrelevant gene clusters.

    PubMed

    Unthan, Simon; Baumgart, Meike; Radek, Andreas; Herbst, Marius; Siebert, Daniel; Brühl, Natalie; Bartsch, Anna; Bott, Michael; Wiechert, Wolfgang; Marin, Kay; Hans, Stephan; Krämer, Reinhard; Seibold, Gerd; Frunzke, Julia; Kalinowski, Jörn; Rückert, Christian; Wendisch, Volker F; Noack, Stephan

    2015-02-01

    For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application. PMID:25139579

  15. Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-06-19

    Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of Eschericia coli (ECRS) and Bacillus subtilis (BSRS) to control the expression of citrate synthase (gltA) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in E. coli. ECRS and BSRS were then successfully used to control the gltA gene and TCA cycle activity in a lysine producing strain Corynebacterium glutamicum LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-gltA mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-gltA and 38% higher in the mutant BSRS-gltA, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an "on-switch" to enhance the metabolic fluxes of desired metabolic pathways. PMID:25575181

  16. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  17. Chassis organism from Corynebacterium glutamicum – a top-down approach to identify and delete irrelevant gene clusters

    PubMed Central

    Unthan, Simon; Baumgart, Meike; Radek, Andreas; Herbst, Marius; Siebert, Daniel; Brühl, Natalie; Bartsch, Anna; Bott, Michael; Wiechert, Wolfgang; Marin, Kay; Hans, Stephan; Krämer, Reinhard; Seibold, Gerd; Frunzke, Julia; Kalinowski, Jörn; Rückert, Christian; Wendisch, Volker F; Noack, Stephan

    2015-01-01

    For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application. PMID:25139579

  18. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    PubMed

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg. PMID:23467831

  19. Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

    PubMed Central

    Rittmann, Doris; Sorger-Herrmann, Ulrike; Wendisch, Volker F.

    2005-01-01

    Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli, or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli, C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus. PMID:16085822

  20. Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE.

    PubMed

    Liu, Yingbao; Yang, Xiaobing; Yin, Yajie; Lin, Jinshui; Chen, Can; Pan, Junfeng; Si, Meiru; Shen, Xihui

    2016-07-14

    Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass. PMID:27250661

  1. The TetR-Type Transcriptional Repressor RolR from Corynebacterium glutamicum Regulates Resorcinol Catabolism by Binding to a Unique Operator, rolO

    PubMed Central

    Li, Tang; Zhao, Kexin; Huang, Yan; Li, Defeng; Jiang, Cheng-Ying; Zhou, Nan; Fan, Zheng

    2012-01-01

    The rol (designated for resorcinol) gene cluster rolRHMD is involved in resorcinol catabolism in Corynebacterium glutamicum, and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of the rol genes in C. glutamicum. The transcription start sites and promoters of rolR and rolHMD were identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription of rolHMD and of its own gene. Further, a 29-bp operator rolO was located at the intergenic region of rolR and rolHMD and was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR to rolO was affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR-rolO complex, thus releasing RolR from the complex and derepressing the transcription of rol genes in C. glutamicum. It is proposed that the binding of RolR to its operator rolO blocks the transcription of rolHMD and of its own gene, thus negatively regulated resorcinol degradation in C. glutamicum. PMID:22706057

  2. Genetic and functional analysis of the soluble oxaloacetate decarboxylase from Corynebacterium glutamicum.

    PubMed

    Klaffl, Simon; Eikmanns, Bernhard J

    2010-05-01

    Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO(2). Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 +/- 1 kDa and that the enzyme is composed of two approximately 29-kDa homodimeric subunits and has a K(m) for oxaloacetate of 1.4 mM and a V(max) of 201 micromol of oxaloacetate converted per min per mg of protein, resulting in a k(cat) of 104 s(-1). Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an L-lysine-producing strain of C. glutamicum led to accumulation of less L-lysine. However, inactivation of the odx gene did not improve L-lysine production under the conditions tested. PMID:20233922

  3. Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol-xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum.

    PubMed

    Scheele, Sandra; Oertel, Dan; Bongaerts, Johannes; Evers, Stefan; Hellmuth, Hendrik; Maurer, Karl-Heinz; Bott, Michael; Freudl, Roland

    2013-03-01

    Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol-xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies. PMID:23163932

  4. Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

    PubMed Central

    Scheele, Sandra; Oertel, Dan; Bongaerts, Johannes; Evers, Stefan; Hellmuth, Hendrik; Maurer, Karl-Heinz; Bott, Michael; Freudl, Roland

    2013-01-01

    Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies. PMID:23163932

  5. Corynebacterium glutamicum methionine sulfoxide reductase A uses both mycoredoxin and thioredoxin for regeneration and oxidative stress resistance.

    PubMed

    Si, Meiru; Zhang, Lei; Chaudhry, Muhammad Tausif; Ding, Wei; Xu, Yixiang; Chen, Can; Akbar, Ali; Shen, Xihui; Liu, Shuang-Jiang

    2015-04-01

    Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under

  6. Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing.

    PubMed

    Marx, A; de Graaf, A A; Wiechert, W; Eggeling, L; Sahm, H

    1996-01-20

    To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase

  7. Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.

    PubMed

    Dong, Xunyan; Zhao, Yue; Zhao, Jianxun; Wang, Xiaoyuan

    2016-06-01

    Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering. PMID:27033538

  8. Heterologous expression of Escherichia coli fructose-1,6-bisphosphatase in Corynebacterium glutamicum and evaluating the effect on cell growth and L-lysine production.

    PubMed

    Xu, J Z; Zhang, J L; Guo, Y F; Jia, Q D; Zhang, W G

    2014-01-01

    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 heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC(T311I) by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A "funnel-cask" diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target. PMID:24397720

  9. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  10. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources.

    PubMed

    Becker, Judith; Klopprogge, Corinna; Zelder, Oskar; Heinzle, Elmar; Wittmann, Christoph

    2005-12-01

    The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfbr. The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP. PMID:16332851

  11. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum.

    PubMed

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport-NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885-were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  12. Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum.

    PubMed

    Hartmann, Michael; Tauch, Andreas; Eggeling, Lothar; Bathe, Brigitte; Möckel, Bettina; Pühler, Alfred; Kalinowski, Jörn

    2003-09-01

    The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains. PMID:12948639

  13. Genome-wide analysis of the role of global transcriptional regulator GntR1 in Corynebacterium glutamicum.

    PubMed

    Tanaka, Yuya; Takemoto, Norihiko; Ito, Terukazu; Teramoto, Haruhiko; Yukawa, Hideaki; Inui, Masayuki

    2014-09-01

    The transcriptional regulator GntR1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates ptsG, a gene encoding a major glucose transporter, and pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 on a genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include the upstream regions of carbon metabolism genes such as pyk, maeB, gapB, and icd, encoding pyruvate kinase, malic enzyme, glyceraldehyde 3-phosphate dehydrogenase B, and isocitrate dehydrogenase, respectively. Binding of GntR1 to the promoter region of these genes was confirmed by electrophoretic mobility shift assay. The activity of the icd, gapB, and maeB promoters was reduced by the mutation at the GntR1 binding site, in contrast to the pyk promoter activity, which was increased, indicating that GntR1 is a transcriptional activator of icd, gapB, and maeB and is a repressor of pyk. Thus, it is likely that GntR1 stimulates glucose uptake by inducing the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) gene while repressing pyk to increase PEP availability in the absence of gluconate. Repression of zwf and gnd may reduce the NADPH supply, which may be compensated by the induction of maeB and icd. Upregulation of icd, gapB, and maeB and downregulation of pyk by GntR1 probably support gluconeogenesis. PMID:24982307

  14. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example

    PubMed Central

    Taniguchi, Hironori; Wendisch, Volker F.

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. PMID:26257719

  15. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    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 varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production. PMID:23990041

  16. Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production--Strategies for the elimination of feedback inhibition.

    PubMed

    Kulis-Horn, Robert K; Persicke, Marcus; Kalinowski, Jörn

    2015-07-20

    L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog β-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches. PMID:25892668

  17. Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

    PubMed Central

    Baumgart, Meike; Unthan, Simon; Rückert, Christian; Sivalingam, Jasintha; Grünberger, Alexander; Kalinowski, Jörn; Bott, Michael; Noack, Stephan

    2013-01-01

    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 the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology. PMID:23892752

  18. Next-generation sequencing-based genome-wide mutation analysis of L-lysine-producing Corynebacterium glutamicum ATCC 21300 strain.

    PubMed

    Lee, Chang-Soo; Nam, Jae-Young; Son, Eun-Suk; Kwon, O-Chul; Han, Woorijarang; Cho, Jae-Yong; Park, Young-Jin

    2012-10-01

    In order to identify single nucleotide polymorphism and insertion/deletion mutations, we performed whole-genome re-sequencing of the enhanced L-lysine-producing Corynebacterium glutamicum ATCC 21300 strain. In total, 142 single nucleotide polymorphisms and 477 insertion/deletion mutations were identified in the ATCC 21300 strain when compared to 3,434 predicted genes of the wild-type C. glutamicum ATCC 13032 strain. Among them, 110 transitions and 29 transversions of single nucleotide polymorphisms were found from genes of the ATCC 21300 strain. In addition, 11 genes, involved in the L-lysine biosynthetic pathway and central carbohydrate metabolism, contained mutations including single nucleotide polymorphisms and insertions/deletions. Interestingly, RT-PCR analysis of these 11 genes indicated that they were normally expressed in the ATCC 21300 strain. This information of genome-wide gene-associated variations will be useful for genome breeding of C. glutamicum in order to develop an industrial amino acid-producing strain with minimal mutation. PMID:23124757

  19. Identification of an alpha(1-->6) mannopyranosyltransferase (MptA), involved in Corynebacterium glutamicum lipomanann biosynthesis, and identification of its orthologue in Mycobacterium tuberculosis.

    PubMed

    Mishra, Arun K; Alderwick, Luke J; Rittmann, Doris; Tatituri, Raju V V; Nigou, Jerome; Gilleron, Martine; Eggeling, Lothar; Besra, Gurdyal S

    2007-09-01

    Corynebacterium glutamicum and Mycobacterium tuberculosis share a similar cell wall architecture, and the availability of their genome sequences has enabled the utilization of C. glutamicum as a model for the identification and study of, otherwise essential, mycobacterial genes involved in lipomannan (LM) and lipoarabinomannan (LAM) biosynthesis. We selected the putative glycosyltransferase-Rv2174 from M. tuberculosis and deleted its orthologue NCgl2093 from C. glutamicum. This resulted in the formation of a novel truncated lipomannan (Cg-t-LM) and a complete ablation of LM/LAM biosynthesis. Purification and characterization of Cg-t-LM revealed an overall decrease in molecular mass, a reduction of alpha(1-->6) and alpha(1-->2) glycosidic linkages illustrating a reduced degree of branching compared with wild-type LM. The deletion mutant's biochemical phenotype was fully complemented by either NCgl2093 or Rv2174. Furthermore, the use of a synthetic neoglycolipid acceptor in an in vitro cell-free assay utilizing the sugar donor beta-D-mannopyranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C8 as a substrate, confirmed NCgl2093 and Rv2174 as an alpha(1-->6) mannopyranosyltransferase (MptA), involved in the latter stages of the biosynthesis of the alpha(1-->6) mannan core of LM. Altogether, these studies have identified a new mannosyltransferase, MptA, and they shed further light on the biosynthesis of LM/LAM in Corynebacterianeae. PMID:17714444

  20. Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Reprinted from Biotechnology and Bioengineering, Vol. 41, Pp 633-646 (1993).

    PubMed

    Vallino, J J; Stephanopoulos, G

    2000-03-20

    The two main contributions of this article are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support significant fluxes during growth and lysine overproduction, and that flux partitioning at the glucosa-6-phosphate branch point does not appear to limit lysine synthesis. PMID:10699864

  1. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

    PubMed Central

    Bott, Michael; van Ooyen, Jan

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  2. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts. PMID:24910972

  3. The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

    PubMed Central

    Schelder, Stephanie; Zaade, Daniela; Litsanov, Boris; Bott, Michael; Brocker, Melanie

    2011-01-01

    Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress. PMID:21799779

  4. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    PubMed

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  5. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

    PubMed

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J; Blombach, Bastian

    2013-09-01

    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 further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. PMID:23835179

  6. Efficient production of α-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy.

    PubMed

    Li, Yanjun; Sun, Lanchao; Feng, Jia; Wu, Ruifang; Xu, Qingyang; Zhang, Chenglin; Chen, Ning; Xie, Xixian

    2016-06-01

    Production of L-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH4OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to L-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an L-glutamate overproducing strain, to produce α-KG that is the direct precursor of L-glutamate. Based on the method of L-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH4OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively. PMID:26946492

  7. Functional characterization of a mycothiol peroxidase in Corynebacterium glutamicum that uses both mycoredoxin and thioredoxin reducing systems in the response to oxidative stress.

    PubMed

    Si, Meiru; Xu, Yixiang; Wang, Tietao; Long, Mingxiu; Ding, Wei; Chen, Can; Guan, Xinmeng; Liu, Yingbao; Wang, Yao; Shen, Xihui; Liu, Shuang-Jiang

    2015-07-01

    Previous studies have identified a putative mycothiol peroxidase (MPx) in Corynebacterium glutamicum that shared high sequence similarity to sulfur-containing Gpx (glutathione peroxidase; CysGPx). In the present study, we investigated the MPx function by examining its potential peroxidase activity using different proton donors. The MPx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of either the thioredoxin/Trx reductase (Trx/TrxR) or the mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) regeneration system. Mrx1 and Trx employ different mechanisms in reducing MPx. For the Mrx1 system, the catalytic cycle of MPx involves mycothiolation/demycothiolation on the Cys(36) sulfenic acid via the monothiol reaction mechanism. For the Trx system, the catalytic cycle of MPx involves formation of an intramolecular disulfide bond between Cys(36) and Cys(79) that is pivotal to the interaction with Trx. Both the Mrx1 pathway and the Trx pathway are operative in reducing MPx under stress conditions. Expression of mpx markedly enhanced the resistance to various peroxides and decreased protein carbonylation and intracellular reactive oxygen species (ROS) accumulation. The expression of mpx was directly activated by the stress-responsive extracytoplasmic function-σ (ECF-σ) factor [SigH]. Based on these findings, we propose that the C. glutamicum MPx represents a new type of GPx that uses both mycoredoxin and Trx systems for oxidative stress response. PMID:25891483

  8. The manganese-responsive regulator MntR represses transcription of a predicted ZIP family metal ion transporter in Corynebacterium glutamicum.

    PubMed

    Baumgart, Meike; Frunzke, Julia

    2015-01-01

    Manganese is an important trace element required as an enzyme cofactor and for protection against oxidative stress. In this study, we characterized the DtxR-type transcriptional regulator MntR (cg0741) of Corynebacterium glutamicum ATCC 13032 as a manganese-dependent repressor of the predicted ZIP family metal transporter Cg1623. Comparative transcriptome analysis of a ΔmntR strain and the wild type led to the identification of cg1623 as potential target gene of MntR which was about 50-fold upregulated when cells were grown in glucose minimal medium. Using electrophoretic mobility shift assays, a conserved 18 bp inverted repeat (TGTTCAATGCGTTGAACA) was identified as binding motif of MntR in the cg1623 promoter and confirmed by mutational analysis. Promoter fusion of Pcg1623 to eyfp confirmed that the MntR-dependent repression is only abolished in the absence of manganese. However, neither deletion of mntR nor cg1623 resulted in a significant growth phenotype in comparison to the wild type--strongly suggesting the presence of further manganese uptake and efflux systems in C. glutamicum. The control of cg1623 by the DtxR-type regulator MntR represents the first example of a predicted ZIP family protein that is regulated in a manganese-dependent manner in bacteria. PMID:25790484

  9. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase.

    PubMed

    Becker, Judith; Klopprogge, Corinna; Herold, Andrea; Zelder, Oskar; Bolten, Christoph J; Wittmann, Christoph

    2007-10-31

    In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme. PMID:17624457

  10. Impact of osmotic stress on volume regulation, cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B.

    PubMed

    Rönsch, Hendrik; Krämer, Reinhard; Morbach, Susanne

    2003-09-01

    The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield. PMID:12948632

  11. Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum.

    PubMed

    Xu, Meijuan; Rao, Zhiming; Dou, Wenfang; Jin, Jian; Xu, Zhenghong

    2012-02-01

    Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-L-glutamate (NAG) to N-acety-L-glutamy-L-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the L-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the L-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I₀.₅ (R) 50-60 fold, and the G287D and R209A mutants could increase the I₀.₅ (R) 30-40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition. PMID:22101454

  12. Monomeric Corynebacterium glutamicum N-acetyl glutamate kinase maintains sensitivity to L-arginine but has a lower intrinsic catalytic activity.

    PubMed

    Huang, Yuanyuan; Li, Cheng; Zhang, Hao; Liang, Shuli; Han, Shuangyan; Lin, Ying; Yang, Xiaorong; Zheng, Suiping

    2016-02-01

    N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine, and L-arginine-sensitive NAGK typically has hexameric architecture. Defining the relationship between this architecture and L-arginine inhibition can provide a foundation to identify the key amino acids involved in the allosteric regulation network of L-arginine. In the present study, the key amino acids in the N-terminal helix (N-helix) of Corynebacterium glutamicum (Cg) NAGK required for hexamer formation were determined using structural homology modeling and site-directed mutagenesis. It was also verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis, but that it is not the determinant of inhibition by L-arginine. Monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form, indicating that monomers contain an independent, sensitive signal transduction network of L-arginine to mediate allosteric regulation. Mutation studies of CgNAGKs also revealed that amino acid residues 18-23 of the N-helix are required for inhibition by L-arginine, and that E19 may be an essential amino acid influencing the apparent affinity of L-arginine. Collectively, these studies may illuminate the basic mechanism of metabolic homeostasis of C. glutamicum. PMID:26512006

  13. Cloning of the trp gene cluster from a tryptophan-hyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence.

    PubMed Central

    Heery, D M; Dunican, L K

    1993-01-01

    Corynebacterium glutamicum ATCC 21850 produces up to 5 g of extracellular L-tryptophan per liter in broth culture and displays resistance to several synthetic analogs of aromatic amino acids. Here we report the cloning of the tryptophan biosynthesis (trp) gene cluster of this strain on a 14.5-kb BamHI fragment. Subcloning and complementation of Escherichia coli trp auxotrophs revealed that as in Brevibacterium lactofermentum, the C. glutamicum trp genes are clustered in an operon in the order trpE, trpD, trpC, trpB, trpA. The cloned fragment also confers increased resistance to the analogs 5-methyltryptophan and 6-fluorotryptophan on E. coli. The sequence of the ATCC 21850 trpE gene revealed no significant changes when compared to the trpE sequence of a wild-type strain reported previously. However, analysis of the promoter-regulatory region revealed a nonsense (TGG-to-TGA) mutation in the third of three tandem Trp codons present within a trp leader gene. Polymerase chain reaction amplification and sequencing of the corresponding region confirmed the absence of this mutation in the wild-type strain. RNA secondary-structure predictions and sequence similarities to the E. coli trp attenuator suggest that this mutation results in a constitutive antitermination response. PMID:7683184

  14. Development of a potential stationary-phase specific gene expression system by engineering of SigB-dependent cg3141 promoter in Corynebacterium glutamicum.

    PubMed

    Kim, Min Jeong; Yim, Sung Sun; Choi, Jae Woong; Jeong, Ki Jun

    2016-05-01

    Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promoters is desired, which can be activated without expensive inducers and has significant advantages for industrial-scale use. Here, we developed a stationary-phase gene expression system by engineering a sigma factor B (SigB)-dependent promoter that can be activated during the transition phase between exponential and stationary growth phases in C. glutamicum. First, the inducibilities of three well-known SigB-dependent promoters were examined using super-folder green fluorescent protein as a reporter protein, and we found that promoter of cg3141 (P cg3141 ) exhibited the highest inducibility. Next, a synthetic promoter library was constructed by randomizing the flanking and space regions of P cg3141 , and the stationary-phase promoters exhibiting high strengths were isolated via FACS-based high-throughput screening. The isolated synthetic promoter (P4-N14) showed a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the use of the isolated P4-N14 for fed-batch cultivation was verified with the production of glutathione S-transferase as a model protein in a lab-scale (5-L) bioreactor. PMID:26782746

  15. RNase III mediated cleavage of the coding region of mraZ mRNA is required for efficient cell division in Corynebacterium glutamicum.

    PubMed

    Maeda, Tomoya; Tanaka, Yuya; Takemoto, Norihiko; Hamamoto, Nagisa; Inui, Masayuki

    2016-03-01

    The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3'-to-5' exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596. PMID:26713407

  16. Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

    PubMed

    Yanase, Masaki; Aikoh, Tohru; Sawada, Kazunori; Ogura, Kotaro; Hagiwara, Takuya; Imai, Keita; Wada, Masaru; Yokota, Atsushi

    2016-08-01

    Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum. PMID:26983943

  17. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

    PubMed

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-03-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698

  18. Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays.

    PubMed

    Jochmann, Nina; Kurze, Anna-Katharina; Czaja, Lisa F; Brinkrolf, Karina; Brune, Iris; Hüser, Andrea T; Hansmeier, Nicole; Pühler, Alfred; Borovok, Ilya; Tauch, Andreas

    2009-05-01

    The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter-operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression. PMID:19372162

  19. Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction

    PubMed Central

    Rückert, Christian; Koch, Daniel J; Rey, Daniel A; Albersmeier, Andreas; Mormann, Sascha; Pühler, Alfred; Kalinowski, Jörn

    2005-01-01

    Background Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria. Results The genome sequence of C. glutamicum was searched for genes involved in the assimilatory reduction of inorganic sulphur compounds. A cluster of eight candidate genes could be identified by combining sequence similarity searches with a subsequent synteny analysis between C. glutamicum and the closely related C. efficiens. Using mutational analysis, seven of the eight candidate genes, namely cysZ, cysY, cysN, cysD, cysH, cysX, and cysI, were demonstrated to be involved in the reduction of inorganic sulphur compounds. For three of the up to now unknown genes possible functions could be proposed: CysZ is likely to be the sulphate permease, while CysX and CysY are possibly involved in electron transfer and cofactor biosynthesis, respectively. Finally, the candidate gene designated fpr2 influences sulphur utilisation only weakly and might be involved in electron transport for the reduction of sulphite. Real-time RT-PCR experiments revealed that cysIXHDNYZ form an operon and that transcription of the extended cluster fpr2 cysIXHDNYZ is strongly influenced by the availability of inorganic sulphur, as well as L-cysteine. Mapping of the fpr2 and cysIXHDNYZ promoters using RACE-PCR indicated that both promoters overlap with binding-sites of the transcriptional repressor McbR, suggesting an involvement of McbR in the observed regulation. Comparative genomics revealed that large parts of the extended cluster are

  20. The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

    PubMed Central

    Clermont, Lina; Macha, Arthur; Müller, Laura M.; Derya, Sami M.; von Zaluskowski, Philipp; Eck, Alexander; Eikmanns, Bernhard J.

    2015-01-01

    ABSTRACT α-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial α-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the α-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial α-glucan phosphorylases. IMPORTANCE Bacterial α-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings

  1. Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

    PubMed Central

    Lange, Julian; Watzlawick, Hildegard; Kalinowski, Jörn; Altenbuchner, Josef

    2014-01-01

    Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10−6 s−1, with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator. PMID:25535273

  2. Involvement of Regulatory Interactions among Global Regulators GlxR, SugR, and RamA in Expression of ramA in Corynebacterium glutamicum

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Gunji, Wataru; Inui, Masayuki

    2013-01-01

    The central carbon metabolism genes in Corynebacterium glutamicum are under the control of a transcriptional regulatory network composed of several global regulators. It is known that the promoter region of ramA, encoding one of these regulators, interacts with its gene product, RamA, as well as with the two other regulators, GlxR and SugR, in vitro and/or in vivo. Although RamA has been confirmed to repress its own expression, the roles of GlxR and SugR in ramA expression have remained unclear. In this study, we examined the effects of GlxR binding site inactivation on expression of the ramA promoter-lacZ fusion in the genetic background of single and double deletion mutants of sugR and ramA. In the wild-type background, the ramA promoter activity was reduced to undetectable levels by the introduction of mutations into the GlxR binding site but increased by sugR deletion, indicating that GlxR and SugR function as the transcriptional activator and repressor, respectively. The marked repression of ramA promoter activity by the GlxR binding site mutations was largely compensated for by deletions of sugR and/or ramA. Furthermore, ramA promoter activity in the ramA-sugR double mutant was comparable to that in the ramA mutant but was significantly higher than that in the sugR mutant. Taken together, it is likely that the level of ramA expression is dynamically balanced by GlxR-dependent activation and repression by RamA along with SugR in response to perturbation of extracellular and/or intracellular conditions. These findings add multiple regulatory loops to the transcriptional regulatory network model in C. glutamicum. PMID:23396909

  3. Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two- and a novel three-compartment scale-down bioreactor.

    PubMed

    Lemoine, Anja; Maya Martίnez-Iturralde, Nina; Spann, Robert; Neubauer, Peter; Junne, Stefan

    2015-06-01

    The oscillatory conditions in substrate and oxygen supply that typically occur on a large (industrial) scale are usually simulated in two-compartment scale-down reactors. In this study, the performance of nutrient-limited fed-batch cultivations of Corynebacterium glutamicum in a standard two-compartment reactor (two-CR) is compared to the performance in a novel three-compartment reactor (three-CR). The three-CR is designed to mimic three distinct zones of an industrial scale bioreactor that occur if the feed addition is installed at the bottom of the fluid phase. Our findings show that lactate and succinate appear in concentrations two-fold higher in the three-CR cultivation than in the two-CR cultivation. Similar results are revealed for the amino acids glycine, threonine, glutamate, and glutamine. In contrast to the two-CR cultivation, no intracellular accumulation of pyruvate is observed in the three-CR cultivation, since the carbon fluxes are directed toward lactate. As previously reported, the expression of lactate dehydrogenase (LDH) is increased in the context of oxygen deprivation. Thus, C. glutamicum adapts to the oscillating environment in the three-CR. This successful adaptation is revealed by a flow cytometric analysis of BOX-stained cells and a series of electrooptical at line measurements of cell polarisability. Both methods indicate a higher polarisability of cells in the three-CR cultivation. PI-staining does not indicate any membrane damage or accelerated cell death in either system. However, although the strain shows robustness, the product yield of lysine is reduced in scale-down cultivations as compared to cultivations at homogeneous conditions, which underlines the relevance of process optimization. PMID:25728062

  4. Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

    PubMed

    Radoš, Dušica; Turner, David L; Fonseca, Luís L; Carvalho, Ana Lúcia; Blombach, Bastian; Eikmanns, Bernhard J; Neves, Ana Rute; Santos, Helena

    2014-05-01

    Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (~5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation. PMID:24610842

  5. Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum

    PubMed Central

    Radoš, Dušica; Turner, David L.; Fonseca, Luís L.; Carvalho, Ana Lúcia; Blombach, Bastian; Eikmanns, Bernhard J.; Neves, Ana Rute

    2014-01-01

    Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using 13C-labeled glucose and 13C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H+:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation. PMID:24610842

  6. Transcriptional regulation of the vanillate utilization genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor.

    PubMed

    Morabbi Heravi, Kambiz; Lange, Julian; Watzlawick, Hildegard; Kalinowski, Jörn; Altenbuchner, Josef

    2015-03-01

    Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10(-6) s(-1), with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator. PMID:25535273

  7. NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum dimerizes at high protein concentration and exclusively receives electrons from thioredoxin reductase.

    PubMed

    Van Laer, Koen; Dziewulska, Aleksandra M; Fislage, Marcus; Wahni, Khadija; Hbeddou, Abderahim; Collet, Jean-Francois; Versées, Wim; Mateos, Luis M; Tamu Dufe, Veronica; Messens, Joris

    2013-03-15

    NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10(6) and 5.6 × 10(6) M(-1) min(-1), respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pKa lower than 6.5 for nucleophilic attack, whereas the pKa of the C-terminal cysteine is ~10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum. PMID:23362277

  8. Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum.

    PubMed

    Ikeda, Masato; Mizuno, Yuta; Awane, Shin-ichi; Hayashi, Masahiro; Mitsuhashi, Satoshi; Takeno, Seiki

    2011-05-01

    Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157

  9. Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH.

    PubMed

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Flechoso, Fabio; Martín, Juan F

    2006-01-01

    Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7.0-9.0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9.0. Growth still occurred at pH 9.5 but at a reduced rate. The expression of the pH-regulated F0 F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7.5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9.0. The same occurred with a 1.2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0 F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0 F1 operon, was expressed at a lower level than the polycistronic 7.5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0 F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative sigma factor of C. glutamicum, whereas the -35 and -10 boxes of P-atp2 fitted the consensus sequence for sigma(H)-recognized Mycobacterium tuberculosis promoters C(C)/(G)GG(A)/(G)AC 17-22 nt (C)/(G)GTT(C)/(G), known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0 F1 operon is highly expressed at alkaline pH, probably using a sigma (H) RNA polymerase. PMID:16385111

  10. Improvement of cell growth and L-lysine production by genetically modified Corynebacterium glutamicum during growth on molasses.

    PubMed

    Xu, Jianzhong; Zhang, Junlan; Guo, Yanfeng; Zai, Yugui; Zhang, Weiguo

    2013-12-01

    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 broth, and heterologous expression of scrK prevents the fructose efflux. Heterologous expression of fbp and scrK not only increases significantly the activity of corresponding enzymes but also improves cell growth during growth on molasses. FBPase activities are increased tenfold by heterologous expression of fbp, whereas the FBPase activity is only increase fourfold during co-expression of scrK and fbp. Compared with glucose, the DCW of heterologous expression strains are higher on molasses except co-expression of fbp and scrK strain. In addition, heterologous expression of fbp and scrK can strongly increase the L-lysine production with molasses as the sole carbon source. The highest increase (88.4 %) was observed for C. glutamicum lysC (fbr) pDXW-8-fbp-scrK, but the increase was also significant for C. glutamicum lysC (fbr) pDXW-8-fbp (47.2 %) and C. glutamicum lysC (fbr) pDXW-8-scrK (36.8 %). By-products, such as glycerol and dihydroxyacetone, are decreased by heterologous expression of fbp and scrK, whereas trehalose is only slightly increased. The strategy for enhancing L-lysine production by regeneration of glucose-6-phosphate in PPP may provide a reference to enhance the production of other amino acids during growth on molasses or starch. PMID:24029876

  11. Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection.

    PubMed

    Liu, Long; Yang, Haiquan; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-11-01

    Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces. PMID:24068337

  12. A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2014-09-01

    Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. PMID:24953302

  13. CO₂ /HCO₃⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum.

    PubMed

    Buchholz, Jens; Graf, Michaela; Freund, Andreas; Busche, Tobias; Kalinowski, Jörn; Blombach, Bastian; Takors, Ralf

    2014-10-01

    The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.6 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.6 min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells. PMID:25139448

  14. Evaluation of two proteomics technologies used to screen the membrane proteomes of wild-type Corynebacterium glutamicum and an L-lysine-producing strain.

    PubMed

    Schluesener, Daniela; Rögner, Matthias; Poetsch, Ansgar

    2007-10-01

    The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques -- anion exchange chromatography AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE -- and the results were compared. Although both techniques allow for the fast screening of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation, it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected (the succinate dehydrogenase complex, an ABC-type cobalamin/Fe(3+) siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa(3) supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects. PMID:17221235

  15. A leuC mutation leading to increased L-lysine production and rel-independent global expression changes in Corynebacterium glutamicum.

    PubMed

    Hayashi, Mikiro; Mizoguchi, Hiroshi; Ohnishi, Junko; Mitsuhashi, Satoshi; Yonetani, Yoshiyuki; Hashimoto, Shin-ichi; Ikeda, Masato

    2006-10-01

    We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner. PMID:16944136

  16. Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R▿ †

    PubMed Central

    Tsuge, Yota; Ogino, Hidetaka; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2008-01-01

    In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (ΔcgR_1595 through cgR_1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum. PMID:18931118

  17. Numerical analysis of fatty and mycolic acid profiles of Corynebacterium urealyticum and other related corynebacteria.

    PubMed

    Herrera-Alcaraz, E; Valero-Guillén, P; Martín-Luengo, F; Canteras-Jordana, M

    1993-04-01

    The fatty and mycolic acid profiles of 52 strains of clinical origin belonging to Corynebacterium urealyticum were subjected to numerical analysis along with those of representative members of Corynebacterium ammoniagenes, Corynebacterium bovis, Corynebacterium glutamicum, Corynebacterium jèikeium, Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium xerosis, Corynebacterium renale, Corynebacterium cystitidis, "Corynebacterium ulcerans" and one strain of the Corynebacterium F1 group. Strains were divided into eight clusters at an amalgamation distance of 7.4. Corynebacterium urealyticum appeared as an homogeneous cluster clearly distant from others, that included several members of the genus Corynebacterium, and it was characterized by its content on unsaturated mycolic acids of mainly 28 (28:1) and 30 (30:3) carbon atoms. On the basis of these results the taxonomic "status" of Corynebacterium urealyticum, a new species within the genus Corynebacterium "sensu stricto", is further justified. PMID:8397966

  18. The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

    PubMed Central

    Townsend, Philip D.; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A.; Cole, Stewart T.; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J.; Pohl, Ehmke

    2014-01-01

    The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition. PMID:25469635

  19. AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

    PubMed Central

    Kuge, Takayuki; Teramoto, Haruhiko

    2015-01-01

    ABSTRACT In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSB mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BSE1 and/or BSE2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BSE1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on l-arabinose catabolism observed in the BSB mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by l-arabinose. IMPORTANCE Corynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar l-arabinose as a carbon source. However, genes for l-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the

  20. Crystal and Solution Studies Reveal That the Transcriptional Regulator AcnR of Corynebacterium glutamicum Is Regulated by Citrate-Mg2+ Binding to a Non-canonical Pocket

    PubMed Central

    García-Nafría, Javier; Baumgart, Meike; Turkenburg, Johan P.; Wilkinson, Anthony J.; Bott, Michael; Wilson, Keith S.

    2013-01-01

    Corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order Corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. Expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor AcnR, assigned by sequence identity to the TetR family. We report the structures of AcnR in two crystal forms together with ligand binding experiments and in vivo studies. First, there is a citrate-Mg2+ moiety bound in both forms, not in the canonical TetR ligand binding site but rather in a second pocket more distant from the DNA binding domain. Second, the citrate-Mg2+ binds with a KD of 6 mm, within the range of physiological significance. Third, citrate-Mg2+ lowers the affinity of AcnR for its target DNA in vitro. Fourth, analyses of several AcnR point mutations provide evidence for the possible involvement of the corresponding residues in ligand binding, DNA binding, and signal transfer. AcnR derivatives defective in citrate-Mg2+ binding severely inhibit growth of C. glutamicum on citrate. Finally, the structures do have a pocket corresponding to the canonical tetracycline site, and although we have not identified a ligand that binds there, comparison of the two crystal forms suggests differences in the region of the canonical pocket that may indicate a biological significance. PMID:23589369

  1. A propionate-inducible expression system based on the Corynebacterium glutamicum prpD2 promoter and PrpR activator and its application for the redirection of amino acid biosynthesis pathways.

    PubMed

    Plassmeier, Jens K; Busche, Tobias; Molck, Stella; Persicke, Marcus; Pühler, Alfred; Rückert, Christian; Kalinowski, Jörn

    2013-01-20

    A novel expression system for Corynebacterium glutamicum, based on the transcriptional activator of propionate metabolism genes PrpR and its target promoter/operator sequence, was developed and tested. The activator PrpR is co-activated by propionate added to the growth medium. In a minimal medium a propionate concentration of only 1 mg l⁻¹ was found to be sufficient for full induction (up to 120-fold) of its native target, the propionate metabolism operon prpDBC2. Then, an artificial transcription and translation reporter system, using the cat gene encoding chloramphenicol acetyl transferase was constructed and tested. The induction was found to be as fast and as high as in the natural system, reaching its maximal transcriptional induction rate within 2 min and a significant accumulation of Cat protein at around 30 min. The duration of the induced transcription was found to be controllable by the propionate concentration applied. The prpD2 promoter and PrpR activator based expression system revealed very similar characteristics in minimal and complex media, making it ideal for applications in large scale industrial fermentations. As a proof-of-principle, the expression system was employed for the propionate-inducible redirection of metabolites in a lysine-production C. glutamicum strain at the homoserine dehydrogenase (hom) branching point, which resulted in an up to 2.5-fold increase of the concentrations of the three amino acids (threonine, homoserine and isoleucine) in the supernatant. PMID:22982516

  2. Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

    PubMed Central

    Peter, Heidi; Weil, Brita; Burkovski, Andreas; Krämer, Reinhard; Morbach, Susanne

    1998-01-01

    Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km, 48 μM) to ectoine (Km, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km, 63 μM) to betaine (Km, 333 μM) and proline (Km, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP. PMID:9811661

  3. l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

    PubMed

    Takeno, Seiki; Hori, Kazumasa; Ohtani, Sachiko; Mimura, Akinori; Mitsuhashi, Satoshi; Ikeda, Masato

    2016-09-01

    We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the

  4. Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

    PubMed Central

    2012-01-01

    Background The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Results Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. Conclusions The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress

  5. 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.

    PubMed

    Klaffl, Simon; Brocker, Melanie; Kalinowski, Jörn; Eikmanns, Bernhard J; Bott, Michael

    2013-09-01

    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 irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5'-KGWCHTRACA-3') which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. PMID:23873914

  6. Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Becker, Judith; Wittmann, Christoph

    2013-01-01

    In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future. PMID:22871505

  7. The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

    PubMed Central

    Rückert, Christian; Milse, Johanna; Albersmeier, Andreas; Koch, Daniel J; Pühler, Alfred; Kalinowski, Jörn

    2008-01-01

    Background Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. Results Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. Conclusion CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules. PMID:18854009

  8. Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum

    PubMed Central

    Brunger, Axel T.; Das, Debanu; Deacon, Ashley M.; Grant, Joanna; Terwilliger, Thomas C.; Read, Randy J.; Adams, Paul D.; Levitt, Michael; Schröder, Gunnar F.

    2012-01-01

    Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence. PMID:22505259

  9. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    PubMed

    Podder, M S; Majumder, C B

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role. PMID:27289352

  10. A novel aceE mutation leading to a better growth profile and a higher L-serine production in a high-yield L-serine-producing Corynebacterium glutamicum strain.

    PubMed

    Guo, Wen; Chen, Ziwei; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

    2016-09-01

    A comparative genomic analysis was performed to study the genetic variations between the L-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced L-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as L-alanine and L-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the L-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher L-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest L-serine production to date. PMID:27344574

  11. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources.

    PubMed

    Wendisch, Volker F; Brito, Luciana Fernandes; Gil Lopez, Marina; Hennig, Guido; Pfeifenschneider, Johannes; Sgobba, Elvira; Veldmann, Kareen H

    2016-09-20

    Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given. PMID:27491712

  12. Transcriptional cross-regulation between Gram-negative and gram-positive bacteria, demonstrated using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum.

    PubMed

    Marbaniang, Carmelita N; Gowrishankar, J

    2012-10-01

    The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG. PMID:22904281

  13. Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

    PubMed Central

    Hodgson, A L; Tachedjian, M; Corner, L A; Radford, A J

    1994-01-01

    An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector. Images PMID:7960105

  14. Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.

    PubMed

    Krylov, Alexander A; Kolontaevsky, Egor E; Mashko, Sergey V

    2014-10-01

    Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods. PMID:25087479

  15. Corynebacterium Prosthetic Joint Infection

    PubMed Central

    Cazanave, Charles; Greenwood-Quaintance, Kerryl E.; Hanssen, Arlen D.

    2012-01-01

    Identification of Corynebacterium species may be challenging. Corynebacterium species are occasional causes of prosthetic joint infection (PJI), but few data are available on the subject. Based on the literature, C. amycolatum, C. aurimucosum, C. jeikeium, and C. striatum are the most common Corynebacterium species that cause PJI. We designed a rapid PCR assay to detect the most common human Corynebacterium species, with a specific focus on PJI. A polyphosphate kinase gene identified using whole-genome sequence was targeted. The assay differentiates the antibiotic-resistant species C. jeikeium and C. urealyticum from other species in a single assay. The assay was applied to a collection of human Corynebacterium isolates from multiple clinical sources, and clinically relevant species were detected. The assay was then tested on Corynebacterium isolates specifically associated with PJI; all were detected. We also describe the first case of C. simulans PJI. PMID:22337986

  16. Corynebacterium minutissimum infection.

    PubMed

    Golledge, C L; Phillips, G

    1991-07-01

    Two cases of infection due to Corynebacterium minutissimum are described. On the basis of biochemical tests the organisms were thought at first to be Corynebacterium jeikeium. Methods of distinguishing between these species and the role of C. minutissimum in the pathogenesis of erythrasma and other skin infections are discussed. PMID:1885917

  17. Corynebacterium appendicis sp. nov.

    PubMed

    Yassin, A F; Steiner, U; Ludwig, W

    2002-07-01

    A lipophilic, coryneform bacterium isolated from a human clinical specimen was characterized by phenotypic and molecular-taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium. The isolate could be distinguished from other members of the genus Corynebacterium by positive urease and catalase tests as well as its failure to produce acid from carbohydrates. Comparative 16S rRNA gene sequencing showed that this isolate constitutes a distinct subline within the genus Corynebacterium, displaying >3.0% sequence divergence from other known Corynebacterium species. Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium appendicis sp. nov., represented by strain IMMIB R-3491T (= DSM 44531T = NRRL B-24151T). PMID:12148623

  18. Quantitative proteomic overview on the Corynebacterium glutamicuml-lysine producing strain DM1730.

    PubMed

    Fränzel, Benjamin; Poetsch, Ansgar; Trötschel, Christian; Persicke, Marcus; Kalinowski, Jörn; Wolters, Dirk Andreas

    2010-11-10

    Corynebacterium glutamicum is one of the most important microorganisms because of its ability to produce and secrete glutamate, lysine and other amino acids. To optimize biotechnological amino acid synthesis it is therefore necessary to understand well how metabolic fluxes can be altered by studying the proteins directing these fluxes. In this work we give a comprehensive quantitative outline about the proteomic state of the l-lysine producing mutant strain DM1730 compared to wild type strain ATCC 13032 in the stationary phase of growth. This study comprises 1107 soluble and membrane proteins, of which 908 have been quantified. C. glutamicum DM1730 seems to produce a large amount of lysine even at the expense of various housekeeping functions. Generally, several proteins that are involved in stress response were found to be significantly more abundant, whereas many members of the protein expression machinery are less abundant as well as most proteins involved in cell growth and division and cell envelope synthesis. Extensive l-lysine production causes C. glutamicum to suffer from oxidative stress and iron limitation. Ultimately, a changed lipid composition of C. glutamicum's cell envelope seems to increase its fluidity, which might be related to altered physiology and membrane processes. PMID:20650338

  19. Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

    PubMed

    Kim, Ju-Sim; Holmes, Randall K

    2012-01-01

    Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Δcat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H(2)O(2). In C. diphtheriae C7(β), both catalase activity and cat transcription decreased ~2-fold during transition from exponential growth to early stationary phase. Prototypic OxyR in Escherichia coli senses oxidative stress and it activates katG transcription and catalase production in response to H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat. OxyR from C. diphtheriae and C. glutamicum have 52% similarity with E. coli OxyR and contain homologs of the two cysteine residues involved in H(2)O(2) sensing by E. coli OxyR. In-frame ΔoxyR deletion mutants of C. diphtheriae C7(β), C. diphtheriae NCTC13129, and C. glutamicum were much more resistant than their parental wild type strains to growth inhibition by H(2)O(2). In the C. diphtheriae C7(β) ΔoxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(β) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in ΔoxyR mutants of C. diphtheriae and C. glutamicum and decreased their H(2)O(2) resistance to the level of their parental strains. Gel-mobility shift, DNaseI footprint, and primer extension assays showed that purified OxyR from C. diphtheriae C7(β) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position -55 to -10 with respect to the +1 nucleotide in the cat ORF. These results demonstrate that OxyR from C. diphtheriae or C. glutamicum functions as a transcriptional repressor of the cat gene by a mechanism that is independent of oxidative stress induced by H(2)O(2). PMID:22438866

  20. Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum▿

    PubMed Central

    Youn, Jung-Won; Jolkver, Elena; Krämer, Reinhard; Marin, Kay; Wendisch, Volker F.

    2009-01-01

    Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C4-dicarboxylates, such as succinate or l-malate, in bacteria. A member of this family, dicarboxylate transporter A (DctA) from Corynebacterium glutamicum, was characterized to catalyze uptake of the C4-dicarboxylates succinate, fumarate, and l-malate, which was inhibited by oxaloacetate, 2-oxoglutarate, and glyoxylate. DctA activity was not affected by sodium availability but was dependent on the electrochemical proton potential. Efficient growth of C. glutamicum in minimal medium with succinate, fumarate, or l-malate as the sole carbon source required high dctA expression levels due either to a promoter-up mutation identified in a spontaneous mutant or to ectopic overexpression. Mutant analysis indicated that DctA and DccT, a C4-dicarboxylate divalent anion/sodium symporter-type transporter, are the only transporters for succinate, fumarate, and l-malate in C. glutamicum. PMID:19581365

  1. Corynebacterium simulans sp. nov., a non-lipophilic, fermentative Corynebacterium.

    PubMed

    Wattiau, P; Janssens, M; Wauters, G

    2000-01-01

    Three coryneform strains isolated from clinical samples were analysed. These strains fitted the biochemical profile of Corynebacterium striatum by conventional methods. However, according to recently described identification tests for fermenting corynebacteria, the strains behaved rather like Corynebacterium minutissimum. The three isolates could be distinguished from C. minutissimum by a positive nitrate and nitrite reductase test and by not fermenting maltose; from C. striatum by their inability to acidify ethylene glycol and to grow at 20 degrees C. Genetic studies based on 16S rRNA showed that the three strains were in fact different from C. minutissimum and C. striatum (96.9 and 98% similarity, respectively) and from other corynebacteria. They represent a new species for which the name Corynebacterium simulans sp. nov. is proposed. The type strain is DSM 44415T (= UCL 553T = Co 553T). PMID:10826822

  2. Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

    PubMed

    Käß, Friedrich; Hariskos, Ioanna; Michel, Andrea; Brandt, Hans-Jürgen; Spann, Robert; Junne, Stefan; Wiechert, Wolfgang; Neubauer, Peter; Oldiges, Marco

    2014-06-01

    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 performance, which is a subject of scale-down experiments. In this work, L-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C. glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application. PMID:24218302

  3. Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum▿

    PubMed Central

    Youn, Jung-Won; Jolkver, Elena; Krämer, Reinhard; Marin, Kay; Wendisch, Volker F.

    2008-01-01

    Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner. PMID:18658264

  4. Corynebacterium aurimucosum sp. nov. and emended description of Corynebacterium minutissimum Collins and Jones (1983).

    PubMed

    Yassin, A F; Steiner, U; Ludwig, W

    2002-05-01

    Two coryneform bacteria isolated from human clinical specimens were characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequence analysis showed that the two strains are genealogically highly related (99.8% sequence similarity) and constitute a new subline within the genus Corynebacterium, with Corynebacterium minutissimum as their nearest phylogenetic neighbours (98.8% sequence similarity). However, DNA-DNA hybridization experiments demonstrated unambiguously that the isolates are genealogically distinct from Corynebacterium minutissimum (42% homology). Biochemical testing indicated that the two isolates were hardly differentiated from Corynebacterium minutissimum. Based on both phenotypic and phylogenetic evidence it is proposed that these isolates be classified as a new species, Corynebacterium aurimucosum sp. nov. The type strain of Corynebacterium aurimucosum is represented by strain IMMIB D-1488T (= DSM 44532T = NRRL B-24143T). PMID:12054216

  5. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production.

    PubMed

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L(-1) L-arginine with yield up to 0.431 g L-arginine g(-1) glucose in fed-batch fermentation. PMID:27338253

  6. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    PubMed Central

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  7. Corynebacterium ulcerans from diseased wild boars.

    PubMed

    Contzen, M; Sting, R; Blazey, B; Rau, J

    2011-11-01

    Two Corynebacterium strains were isolated from lymph nodes of wild boars showing severe alterations caused by caseous lymphadenitis. The wild boars came from different districts in southern Germany; one was found dead, the other had been shot. The two Corynebacterium strains obtained were both positive for phospholipase D. Further analysis of biochemical profiles did not allow unambiguous differentiation between Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Fourier-transformed infrared spectroscopy as well as partial sequencing of the genes for 16S rRNA and RNA polymerase beta subunit (rpoB) clearly identified both strains as Corynebacterium ulcerans. The tox gene for diphtheria toxin (DT) could be detected in both porcine isolates by PCR. Partial DNA sequencing of this tox gene showed significant differences from sequences described for other Corynebacterium ulcerans strains and a higher degree of similarity to that of Corynebacterium diphtheria. Production of diphtheria toxin could not be detected. These results indicate that wild game could be a reservoir for zoonotic Corynebacterium ulcerans. PMID:21824349

  8. [Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum].

    PubMed

    Hao, Ning; Zhao, Zhi; Wang, Yu; Zhang, Ying-zi; Ding, Jiu-yuan

    2006-02-01

    N-Acetylglutamate kinase (EC 2.7.2.8;NAGK) genes from wild-type Corynebacterium crenatum AS 1.542 and a L-arginine-producing mutant C. crenatum 971.1 were cloned and sequenced. Analysis of argB sequences revealed that only one ORF existed, which used ATG as the initiation codon and coded a peptide of 317 amino acids with a calculated molecular weight of 33.6kDa. Only one nucleotide difference was found in the structure gene and the difference did not cause a change of amino acid by comparison of the gene sequences between the wild type C. crenatum AS 1.542 and the mutant 971.1. The ORF sequence of argB from C. crenatum AS 1.542 showed homologies of 99.89%, 76.62%, 37.94% to those from Corynebacterium glutamicum ATCC 13032, Corynebacterium efficient YS-314 and Escherichia coli k12. And the amino acid sequence deduced from ORF displayed homologies of 100%, 78.55%, 25.25% to those from microorganisms above, respectively. An internal promoter was found in the upstream of the argB gene from C. crenatum. The argB gene from C. crenatum AS 1.542 was expressed both in C. crenatum AS 1.542 and 971.1. The NAGK activity of transformed C. crenatum AS 1.542 was greatly increased by the induction of IPTG. The NAGK activity of transformed C. crenatum 971.1 was almost twice as much as that of C. crenatum 971.1 under the same induction. The amplification of the NAGK activity yielded 25% increase of L-arginine production in C. crenatum 971.1. PMID:16579472

  9. Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

    PubMed Central

    Norouzy, Amir; Schulz, Robert; Nau, Werner M.; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

    2013-01-01

    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 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. PMID:24116064

  10. Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis.

    PubMed

    Sagong, Hye-Young; Kim, Kyung-Jin

    2016-02-28

    Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP(+) and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme. PMID:26502738

  11. Identification and Characterization of a Bacterial Transport System for the Uptake of Pyruvate, Propionate, and Acetate in Corynebacterium glutamicum▿

    PubMed Central

    Jolkver, Elena; Emer, Denise; Ballan, Stefan; Krämer, Reinhard; Eikmanns, Bernhard J.; Marin, Kay

    2009-01-01

    The metabolism of monocarboxylic acids is of central importance for bacteria in their natural habitat as well as during biotechnological production. Although biosynthesis and degradation are well understood, the transport of such compounds is still a matter of discussion. Here we present the identification and characterization of a new transport system in Corynebacterium glutamicum with high affinity for acetate and propionate and with lower affinity for pyruvate. Biochemical analysis of this monocarboxylic acid transporter (MctC) revealed for the first time a quantitative discrimination of passive diffusion and active transport of acetate by bacterial cells. MctC is a secondary transporter and belongs to the class of sodium solute symporters, but it is driven by the electrochemical proton potential. The mctC gene is preceded by and cotranscribed with cg0952, a locus encoding a small membrane protein, and the transcription of the cg0952-mctC operon is under the control of the transcriptional regulators RamA and RamB. Both of these proteins directly bind to the promoter region of the operon; RamA is essential for expression and RamB exerts a slightly negative control on expression of the cg0952-mctC operon. mctC expression is induced in the presence of pyruvate and beneficial under substrate-limiting conditions for C. glutamicum. PMID:19028892

  12. Genome Sequence of Corynebacterium ulcerans Strain 210932

    PubMed Central

    Viana, Marcus Vinicius Canário; de Jesus Benevides, Leandro; Batista Mariano, Diego Cesar; de Souza Rocha, Flávia; Bagano Vilas Boas, Priscilla Carolinne; Folador, Edson Luiz; Pereira, Felipe Luiz; Alves Dorella, Fernanda; Gomes Leal, Carlos Augusto; Fiorini de Carvalho, Alex; Silva, Artur; de Castro Soares, Siomar; Pereira Figueiredo, Henrique Cesar; Guimarães, Luis Carlos

    2014-01-01

    In this work, we present the complete genome sequence of Corynebacterium ulcerans strain 210932, isolated from a human. The species is an emergent pathogen that infects a variety of wild and domesticated animals and humans. It is associated with a growing number of cases of a diphtheria-like disease around the world. PMID:25428977

  13. A Spontaneous Joint Infection with Corynebacterium striatum▿

    PubMed Central

    Scholle, David

    2007-01-01

    Corynebacterium striatum is a ubiquitous saprophyte with the potential to cause bacteremia in immunocompromised patients. Until now, spontaneous infection of a natural joint has not been reported. When phenotyping failed, gene sequencing was used to identify the species. The isolate demonstrated high-level resistance to most antibiotics. PMID:17151206

  14. Recurrent breast abscesses caused by Corynebacterium minutissimum.

    PubMed

    Berger, S A; Gorea, A; Stadler, J; Dan, M; Zilberman, M

    1984-12-01

    A 42-year-old woman developed severe, recurrent breast abscesses caused by Corynebacterium minutissimum. Prior reports of C. minutissimum infection have been limited to erythrasma, a minor dermatosis. The microbiological and clinical features of this species were reviewed. PMID:6520230

  15. Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum▿†

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-01-01

    Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant. PMID:21665967

  16. Corynebacterium frankenforstense sp. nov. and Corynebacterium lactis sp. nov., isolated from raw cow milk.

    PubMed

    Wiertz, Raika; Schulz, Stefanie Christine; Müller, Ute; Kämpfer, Peter; Lipski, André

    2013-12-01

    Two groups of Gram-stain positive, aerobic bacterial strains were isolated from raw cow's milk, from a milking machine and from bulk tank milk. Based on their 16S rRNA gene sequences these isolates formed two distinct groups within the genus Corynebacterium. The sequence similarities of the isolates to the type strains of species of the genus Corynebacterium were below 98.4%. The presence of menaquinones MK-8(H2) and MK-9(H2), the predominant fatty acid 18:1 cis 9 and a polar lipid pattern with several phospholipids but without aminolipids was in accord with the characteristics of this genus. The results of DNA-DNA hybridization, biochemical tests and chemotaxonomic properties allowed genotypic and phenotypic differentiation of the strains from all known species of the genus Corynebacterium. Therefore, the isolates were assigned to two novel species of this genus for which the names Corynebacterium frankenforstense sp. nov. (type strain ST18(T) =DSM 45800(T) = CCUG 63371(T)), and Corynebacterium lactis sp. nov. (type strain RW2-5(T) = DSM 45799(T) = CCUG 63372(T)) are proposed, respectively. PMID:23907219

  17. Corynebacterium ulcerans, an emerging human pathogen.

    PubMed

    Hacker, Elena; Antunes, Camila A; Mattos-Guaraldi, Ana L; Burkovski, Andreas; Tauch, Andreas

    2016-09-01

    While formerly known infections of Corynebacterium ulcerans are rare and mainly associated with contact to infected cattle, C. ulcerans has become an emerging pathogen today. In Western Europe, cases of respiratory diphtheria caused by C. ulcerans have been reported more often than infections by Corynebacterium diphtheria, while systemic infections are also increasingly reported. Little is known about factors that contribute to host colonization and virulence of this zoonotic pathogen. Research in this field has received new impetus by the publication of several C. ulcerans genome sequences in the past years. This review gives a comprehensive overview of the basic knowledge of C. ulcerans, as well as the recent advances made in the analysis of putative virulence factors. PMID:27545005

  18. Corynebacterium minutissimum infecting pseudomeningocele: a rare case.

    PubMed

    Eshwara, Vandana Kalwaje; Munim, Frenil; Shetty, Arjun; Gupta, Anurag; Nitin, Jagadhane; Mukhopadhyay, Chiranjay

    2014-04-01

    Invasive infections by Corynebacterium minutissimum are rarely documented. The significance of laboratory isolation of this bacterium from a sterile specimen such as cerebrospinal fluid is difficult to determine as it usually colonizes the skin. However, repeated isolation in a clinical setting should be treated appropriately. Here we report a first case of infected pseudomeningocele by C. minutissimum in an adult woman operated on for falcotentorial psammomatous meningioma. The patient was treated successfully with linezolid. PMID:23010538

  19. [Purulent meningitis, caused by Corynebacterium xerosis, after spinal anesthesia].

    PubMed

    Vukmirovits, G; Todorova, R; Arányi, Z; Káli, G

    1991-12-30

    A previously healthy 25 year old sportsman is reported who developed Corynebacterium xerosis meningitis with coma and seizures after spinal anaesthesia. The adequate therapy (dexamethason, penicillin, ampicillin, mannitol, intensive care, hyperventillation) resulted in a complete recovery. To the authors' knowledge this is the first case of Corynebacterium xerosis meningitis and the first bacterial meningitis reported after spinal anaesthesia in Hungary. PMID:1766661

  20. Corynebacterium minutissimum bacteremia in an immunocompetent host with cellulitis.

    PubMed

    Granok, Alexander B; Benjamin, Patti; Garrett, Lee S

    2002-08-15

    Since its original description in 1961, Corynebacterium minutissimum, the causative agent of erythrasma, has rarely been associated with extracutaneous disease. We report a case of cellulitis and bacteremia due to C. minutissimum. We discuss the treatment of C. minutissimum infection and describe the clinical settings in which isolation of Corynebacterium species from blood cultures should be considered significant. PMID:12145741

  1. Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes.

    PubMed

    Koizumi, S; Yonetani, Y; Maruyama, A; Teshiba, S

    2000-06-01

    Improved strains for the production of riboflavin (vitamin B2) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5'-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l(-1) for 72 h in a 5-l jar fermentor without any end product inhibition. PMID:10919325

  2. Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis.

    PubMed Central

    Wilson, M J; Brandon, M R; Walker, J

    1995-01-01

    A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40). PMID:7806359

  3. A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient L-lysine production.

    PubMed

    Ikeda, Masato; Ohnishi, Junko; Hayashi, Mikiro; Mitsuhashi, Satoshi

    2006-07-01

    Based on the progress in genomics, we have developed a novel approach that employs genomic information to generate an efficient amino acid producer. A comparative genomic analysis of an industrial L-lysine producer with its natural ancestor identified a variety of mutations in genes associated with L-lysine biosynthesis. Among these mutations, we identified two mutations in the relevant terminal pathways as key mutations for L-lysine production, and three mutations in central metabolism that resulted in increased titers. These five mutations when assembled in the wild-type genome led to a significant increase in both the rate of production and final L-lysine titer. Further investigations incorporated with transcriptome analysis suggested that other as yet unidentified mutations are necessary to support the L-lysine titers observed by the original production strain. Here we describe the essence of our approach for strain reconstruction, and also discuss mechanisms of L-lysine hyperproduction unraveled by combining genomics with classical strain improvement. PMID:16506038

  4. Postoperative Abdominal Infection Caused by Corynebacterium minutissimum

    PubMed Central

    Shin, Ji Young; Lee, Woon Kee; Seo, Yiel-Hea

    2014-01-01

    Corynebacterium minutissimum is a non-spore forming, gram-positive, aerobic or facultative anaerobic bacillus. It is the causative organism of erythrasma, a common superficial infection of skin, which typically presents as reddish-brown macular patches. To date, it has rarely been found to cause invasive disease, although other non-diphtheria corynebacteria are becoming increasingly common as opportunistic pathogens. We report on a rare case of abdominal infection due to C. minutissimum in an immunocompetent adult who was successfully treated with intravenous amoxicillin/sulbactam. PMID:25566407

  5. Embolic retinopathy due to Corynebacterium minutissimum endocarditis.

    PubMed

    Herschorn, B J; Brucker, A J

    1985-01-01

    Infective embolic retinopathy as a sequela of bacterial endocarditis is described in a 31-year-old woman with mitral valve prolapse. The infective organism, Corynebacterium minutissimum, has not been previously found to cause ocular or multisystem diseases. It is a common mucocutaneous inhabitant which causes erythrasma. In our case report both ocular involvement and septicaemia were present. The infection was confirmed by positive serial blood cultures. Mitral valve prolapse was confirmed by echocardiography. On clinical examination the retinopathy consisted of white intraretinal lesions which resolved with antibiotic therapy. By fluorescein angiography focal areas of hypofluorescence corresponding to the white fundus lesions were present. Optic disc oedema was also seen. PMID:3965026

  6. Postoperative Abdominal Infection Caused by Corynebacterium minutissimum.

    PubMed

    Shin, Ji Young; Lee, Woon Kee; Seo, Yiel-Hea; Park, Yoon Soo

    2014-12-01

    Corynebacterium minutissimum is a non-spore forming, gram-positive, aerobic or facultative anaerobic bacillus. It is the causative organism of erythrasma, a common superficial infection of skin, which typically presents as reddish-brown macular patches. To date, it has rarely been found to cause invasive disease, although other non-diphtheria corynebacteria are becoming increasingly common as opportunistic pathogens. We report on a rare case of abdominal infection due to C. minutissimum in an immunocompetent adult who was successfully treated with intravenous amoxicillin/sulbactam. PMID:25566407

  7. Antimicrobial Treatment Options for Granulomatous Mastitis Caused by Corynebacterium Species

    PubMed Central

    Dobinson, Hazel C.; Anderson, Trevor P.; Chambers, Stephen T.; Doogue, Matthew P.; Seaward, Lois

    2015-01-01

    Corynebacterium species are increasingly recognized as important pathogens in granulomatous mastitis. Currently, there are no published treatment protocols for Corynebacterium breast infections. This study describes antimicrobial treatment options in the context of other management strategies used for granulomatous mastitis. Corynebacterium spp. isolated from breast tissue and aspirate samples stored from 2002 to 2013 were identified and determined to the species level using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), 16S RNA sequencing, and rpoB gene targets. The MICs for 12 antimicrobials were performed using Etest for each isolate. Correlations of these with antimicrobial characteristics, choice of antimicrobial, and disease outcome were evaluated. Corynebacterium spp. from breast tissue and aspirate samples were confirmed in 17 isolates from 16 patients. Based on EUCAST breakpoints, Corynebacterium kroppenstedtii isolates (n = 11) were susceptible to seven antibiotic classes but resistant to β-lactam antibiotics. Corynebacterium tuberculostearicum isolates (n = 4) were multidrug resistant. Two nonlipophilic species were isolated, Corynebacterium glucuronolyticum and Corynebacterium freneyi, both of which have various susceptibilities to antimicrobial agents. Short-course antimicrobial therapy was common (median, 6 courses per subject; range, 1 to 9 courses). Patients with C. kroppenstedtii presented with a hot painful breast mass and underwent multiple surgical procedures (median, 4 procedures; range, 2 to 6 procedures). The management of Corynebacterium breast infections requires a multidisciplinary approach and includes culture and appropriate sensitivity testing to guide antimicrobial therapy. Established infections have a poor outcome, possibly because adequate concentrations of some drugs will be difficult to achieve in lipophilic granulomata. Lipophilic antimicrobial therapy may offer a therapeutic

  8. [Breast cancer treated by antibiotherapy? Granulomatous mastitis with Corynebacterium].

    PubMed

    Buhler, J; Grignon, Y; Gallon, F

    2015-09-01

    Granulomatous mastitis is a rare disease, often associated with Corynebacterium infection. It raises the problem of diagnosis of breast tumor with a fast evolution and inflammatory character. We report two cases of granulomatous mastitis with Corynebacterium. It concerns the clinical and radiological description, followed by the therapeutic alternatives and future of the patients. The clinical presentation is variable. The treatment consists in a surgical procedure of resection. The medical treatment based of corticosteroids also proves efficient. The association between Corynebacterium presence and this pathology seems frequent and needs a specific bacteriological search. PMID:25721346

  9. Corynebacterium minutissimum endocarditis: a case report and review.

    PubMed

    Aperis, George; Moyssakis, Ioannis

    2007-02-01

    We present a rare case of infectious endocarditis in a 40-year old male with native valve, caused by Corynebacterium minutissimum. The diagnosis was established with transesophageal echocardiogram. The patient was managed successfully with antibiotic therapy. PMID:16757031

  10. [The Corynebacterium pyogenes infection of cattle. 2. Tenacity of Corynebacterium pyogenes].

    PubMed

    Nattermann, H; Horsch, F

    1977-01-01

    Some common agents were tested for their effectiveness against Corynebacterium pyogenes. The pathogen proved most susceptable to Wofasteril. All germs were killed within ten minutes by a 0.005% solution. Equally good action was recorded from all the other tested agents as well (lactic acid, Lugol's solution, formalin, cupric sulphate, alcohol, and aethacridine. Other studies were conducted with the view to testing the survival capacity of Corynebacterium pyogenes in different media and storage conditions. The pathogen survived three months in routine media and mastitis secretion at room temperature. Regrowth of 38 in 50 strains took place after nine months of refrigerator storage in slanting blood agar tubes with paraffin plugs. Germs sampled from mastitis secretion and stored in a refrigerator were cultivable even after one year had elapsed. The detectability rate of Corynebacterium pyogenes did not change over months by storage of wound infection material at 12 degrees C below zero. The pathogen remained detectable five days from artificial contamination of cattle skin. PMID:336001

  11. Phenotypic and Phylogenetic Characterization of a New Corynebacterium Species from Dogs: Description of Corynebacterium auriscanis sp. nov.

    PubMed Central

    Collins, Matthew D.; Hoyles, Lesley; Lawson, Paul A.; Falsen, Enevold; Robson, Robert L.; Foster, Geoffrey

    1999-01-01

    Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus Corynebacterium; this subline is close to but distinct from C. falsenii, C. jeikeium, and C. urealyticum. The unknown bacterium from dogs was distinguished from all currently validated Corynebacterium species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species, Corynebacterium auriscanis. The type strain of C. auriscanis is CCUG 39938T. PMID:10523531

  12. In vitro activity of tigecycline and 10 other antimicrobials against clinical isolates of the genus Corynebacterium.

    PubMed

    Fernandez-Roblas, R; Adames, H; Martín-de-Hijas, N Z; Almeida, D García; Gadea, I; Esteban, J

    2009-05-01

    We studied the in vitro activity of tigecycline and 10 other commonly used antibiotics against 135 clinical isolates of non-diphtheria Corynebacterium spp. using the Etest system. Tigecycline minimum inhibitory concentrations for 50% and 90% of the organisms (MIC(50) and MIC(90) values, respectively, in mg/L) were: Corynebacterium urealyticum, 0.094 and 0.125; Corynebacterium amycolatum, 0.125 and 2; Corynebacterium jeikeium, 0.094 and 0.75; Corynebacterium coyleae, 0.064 and 0.064; Corynebacterium striatum, 0.064 and 1; Corynebacterium aurimucosum, 0.094 and 0.125; and Corynebacterium afermentans, 0.064 and 0.094. The activities of all other antimicrobials were variable, with good activity of glycopeptides, linezolid, quinupristin/dalfopristin and daptomycin and with resistance to macrolides in a high number of isolates. Tigecycline is a good alternative for the therapy of infections caused by non-diphtheria corynebacteria. PMID:19153032

  13. The killing of macrophages by Corynebacterium ulcerans.

    PubMed

    Hacker, Elena; Ott, Lisa; Schulze-Luehrmann, Jan; Lührmann, Anja; Wiesmann, Veit; Wittenberg, Thomas; Burkovski, Andreas

    2016-01-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen. PMID:26632348

  14. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  15. Complete genome sequence, lifestyle, and multi-drug resistance of the human pathogen Corynebacterium resistens DSM 45100 isolated from blood samples of a leukemia patient

    PubMed Central

    2012-01-01

    Background Corynebacterium resistens was initially recovered from human infections and recognized as a new coryneform species that is highly resistant to antimicrobial agents. Bacteremia associated with this organism in immunocompromised patients was rapidly fatal as standard minocycline therapies failed. C. resistens DSM 45100 was isolated from a blood culture of samples taken from a patient with acute myelocytic leukemia. The complete genome sequence of C. resistens DSM 45100 was determined by pyrosequencing to identify genes contributing to multi-drug resistance, virulence, and the lipophilic lifestyle of this newly described human pathogen. Results The genome of C. resistens DSM 45100 consists of a circular chromosome of 2,601,311 bp in size and the 28,312-bp plasmid pJA144188. Metabolic analysis showed that the genome of C. resistens DSM 45100 lacks genes for typical sugar uptake systems, anaplerotic functions, and a fatty acid synthase, explaining the strict lipophilic lifestyle of this species. The genome encodes a broad spectrum of enzymes ensuring the availability of exogenous fatty acids for growth, including predicted virulence factors that probably contribute to fatty acid metabolism by damaging host tissue. C. resistens DSM 45100 is able to use external L-histidine as a combined carbon and nitrogen source, presumably as a result of adaptation to the hitherto unknown habitat on the human skin. Plasmid pJA144188 harbors several genes contributing to antibiotic resistance of C. resistens DSM 45100, including a tetracycline resistance region of the Tet W type known from Lactobacillus reuteri and Streptococcus suis. The tet(W) gene of pJA144188 was cloned in Corynebacterium glutamicum and was shown to confer high levels of resistance to tetracycline, doxycycline, and minocycline in vitro. Conclusions The detected gene repertoire of C. resistens DSM 45100 provides insights into the lipophilic lifestyle and virulence functions of this newly recognized

  16. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species.

    PubMed

    Ramsey, Matthew M; Freire, Marcelo O; Gabrilska, Rebecca A; Rumbaugh, Kendra P; Lemon, Katherine P

    2016-01-01

    Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species. PMID:27582729

  17. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species

    PubMed Central

    Ramsey, Matthew M.; Freire, Marcelo O.; Gabrilska, Rebecca A.; Rumbaugh, Kendra P.; Lemon, Katherine P.

    2016-01-01

    Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe–microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species. PMID:27582729

  18. Seminal Corynebacterium strains in infertile men with and without leucocytospermia.

    PubMed

    Mashaly, M; Masallat, D T; Elkholy, A A; Abdel-Hamid, I A; Mostafa, T

    2016-04-01

    This study aimed to identify seminal Corynebacterium strains in infertile men with and without leucocytospermia. Semen samples from 60 infertile men were allocated into two equal groups: semen samples with leucocytospermia and semen samples without leucocytospermia. Semen culture for Corynebacterium species was carried out on Columbia agar medium confirmed by Gram-stained film and biochemical tests followed by analytical profile index biotyping and antibiotic susceptibility. Bacterial isolates were detected in 20/60 semen cultures (33.3%) as Corynebacteria, Staphylococci, Alpha haemolytic streptococci and E. coli. In all, 12/60 (20%) had Corynebacterium positive semen culture, whereas C. seminal was the major isolated species followed by C. amycolatum, C. jekium and C. urealyticum. There was nonsignificant difference between patients with/without Corynebacterium positive culture regarding sperm concentration and normal sperm morphology; however, in positive cultures sperm motility was significantly lower compared with negative cultures. Antimicrobial sensitivity among Corynebacteria strains was highest for vancomycin, rifampicin then imipenem, ampicillin + sulbactam, ciprofloxacin. It is concluded that positive semen cultures for different Corynebacteria species were demonstrated in infertile men, whereas Corynebacterium seminale was the most common isolated species. Vancomycin, rifampicin then imipenem and ampicillin + sulbactam are recommended as sensitive antibiotics. PMID:26228802

  19. Corynebacterium nigricans sp. nov.: Proposed Name for a Black-Pigmented Corynebacterium Species Recovered from the Human Female Urogenital Tract

    PubMed Central

    Shukla, Sanjay K.; Bernard, Kathryn A.; Harney, Mary; Frank, Daniel N.; Reed, Kurt D.

    2003-01-01

    Six independent isolates of an unusual black-pigmented Corynebacterium species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract. Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation. One isolate was recovered from a vaginal ulcer. All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35°C. The dry, adherent colonies pitted the agar surface. The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic. Only five of six isolates were available for characterization. Biochemical and chemotaxonomic studies revealed that the strains belong to the genus Corynebacterium but differ from known corynebacterial species. Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus Corynebacterium. We propose the name Corynebacterium nigricans sp. nov. for this group of coryneforms. The type strain of Corynebacterium nigricans is CN-1. It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346). PMID:12958268

  20. Genome sequence and description of Corynebacterium ihumii sp. nov.

    PubMed Central

    Padmanabhan, Roshan; Dubourg, Grégory; Lagier, Jean-Christophe; Couderc, Carine; Michelle, Caroline; Raoult, Didier; Fournier, Pierre-Edouard

    2014-01-01

    Corynebacterium ihumii strain GD7T sp. nov. is proposed as the type strain of a new species, which belongs to the family Corynebacteriaceae of the class Actinobacteria. This strain was isolated from the fecal flora of a 62 year-old male patient, as a part of the culturomics study. Corynebacterium ihumii is a Gram positive, facultativly anaerobic, nonsporulating bacillus. Here, we describe the features of this organism, together with the high quality draft genome sequence, annotation and the comparison with other member of the genus Corynebacteria. C. ihumii genome is 2,232,265 bp long (one chromosome but no plasmid) containing 2,125 protein-coding and 53 RNA genes, including 4 rRNA genes. The whole-genome shotgun sequence of Corynebacterium ihumii strain GD7T sp. nov has been deposited in EMBL under accession number GCA_000403725. PMID:25197488

  1. Corynebacterium singulare sp. nov., a new species for urease-positive strains related to Corynebacterium minutissimum.

    PubMed

    Riegel, P; Ruimy, R; Renaud, F N; Freney, J; Prevost, G; Jehl, F; Christen, R; Monteil, H

    1997-10-01

    We studied two coryneform strains from clinical specimens. These strains had type IV and corynemycolic acids in their cell walls and also had phenotypic characteristics, such as urease activity and fermentation of glucose and sucrose but not trehalose, which did not permit assignment to any previously recognized taxon. According to DNA-DNA hybridization data, these two strains are members of the same species (level of DNA similarity, 86%). Phylogenetic analysis based on comparisons of almost complete small-subunit ribosomal DNA sequences revealed that these strains are closely related to Corynebacterium minutissimum, but DNA relatedness experiments clearly showed that they constitute a distinct new species with a level of DNA relatedness to the C. minutissimum type strain of less than 40%. This new species can be differentiated from C. minutissimum strains by its enzymatic activities and carbon source utilization, and the name Corynebacterium singulare is proposed for it. The type strain is strain IBS B52218 (= CCUG 37330), which was isolated from a semen specimen. PMID:9336912

  2. Cutaneous abscess caused by Corynebacterium lactis in a companion dog.

    PubMed

    Antunes, João Marcelo Azevedo de Paula; Ribeiro, Márcio Garcia; Demoner, Larissa de Castro; Ramos, Juliana Nunes; Baio, Paulo Victor Pereira; Simpson-Louredo, Liliane; Santos, Cíntia Silva; Hirata, Raphael; Ferioli, Raquel Beneton; Romera, Adriana Resmond Cruz; Vieira, Verônica Viana; Mattos-Guaraldi, Ana Luíza

    2015-07-01

    Many new, emerging and re-emerging diseases of humans are caused by pathogens which originate from animals or products of animal origin. Corynebacterium lactis, a recently described species of the genus Corynebacterium, was first isolated from milk of asymptomatic cows. In the present study a cutaneous abscess caused by C. lactis in a dog was recognized by cytologic and histologic examination in addition to 16S rRNA gene analysis of the microorganism. Therefore, C. lactis should be included among other bacterial species recognized as emerging pathogens for companion animals. PMID:25937144

  3. Corynebacterium endocarditis species-specific risk factors and outcomes

    PubMed Central

    Belmares, Jaime; Detterline, Stephanie; Pak, Janet B; Parada, Jorge P

    2007-01-01

    Background Corynebacterium species are recognized as uncommon agents of endocarditis, but little is known regarding species-specific risk factors and outcomes in Corynebacterium endocarditis. Methods Case report and Medline search of English language journals for cases of Corynebacterium endocarditis. Inclusion criteria required that cases be identified as endocarditis, having persistent Corynebacterium bacteremia, murmurs described by the authors as identifying the affected valve, or vegetations found by echocardiography or in surgical or autopsy specimens. Cases also required patient-specific information on risk factors and outcomes (age, gender, prior prosthetic valve, other prior nosocomial risk factors (infected valve, involvement of native versus prosthetic valve, need for valve replacement, and death) to be included in the analysis. Publications of Corynebacterium endocarditis which reported aggregate data were excluded. Univariate analysis was conducted with chi-square and t-tests, as appropriate, with p = 0.05 considered significant. Results 129 cases of Corynebacterium endocarditis involving nine species met inclusion criteria. Corynebacterium endocarditis typically infects the left heart of adult males and nearly one third of patients have underlying valvular disease. One quarter of patients required valve replacement and one half of patients died. Toxigenic C. diphtheriae is associated with pediatric infections (p < 0.001). Only C. amycolatum has a predilection for women (p = 0.024), while C. pseudodiphtheriticum infections are most frequent in men (p = 0.023). C. striatum, C. jeikeium and C. hemolyticum are associated with nosocomial risk factors (p < 0.001, 0.028, and 0.024, respectively). No species was found to have a predilection for any particular heart valve. C. pseudodiphtheriticum is associated with a previous prosthetic valve replacement (p = 0.004). C. jeikeium infections are more likely to require valve replacement (p = 0.026). Infections

  4. Corynebacterium minutissimum vascular graft infection: case report and review of 281 cases of prosthetic device-related Corynebacterium infection.

    PubMed

    Reece, Rebecca M; Cunha, Cheston B; Rich, Josiah D

    2014-09-01

    Corynebacterium spp. have proven their pathogenic potential in causing infections, particularly in the setting of immunosuppression and prosthetic devices. We conducted a PubMed literature review of all cases of Corynebacterium prosthetic device infections published in the English language through December 2013. The majority of cases involved peritoneal dialysis and central venous catheters, but prosthetic joints and central nervous system shunts/drains were also involved. The management of these cases in terms of retention or removal of the device was not uniform; however, the overall mortality remained the same among both groups. All of these prosthetic device infections pose potential problems in management when the device cannot be removed safely for the patient, especially with the lack of data on the pathogenicity of Corynebacterium species. However with better identification of species and sensitivities, successful treatment is possible even with retention of the device. PMID:24934988

  5. Corynebacterium pseudotuberculosis liver abscess in a mature alpaca (Lama pacos)

    PubMed Central

    Sprake, Philippa; Gold, Jenifer R.

    2012-01-01

    A mature female alpaca was evaluated for weight loss and a 10-day history of anorexia, diarrhea, abdominal distension, and ventral edema. Ultrasonography revealed a hepatic mass, culture of which identified Corynebacterium pseudotuberculosis. This is the first reported case of an internal caseous lymphadenitis lesion resulting in clinical disease in a camelid. PMID:23024384

  6. Central venous catheter-related Corynebacterium minutissimum bacteremia.

    PubMed

    Rupp, M E; Stiles, K G; Tarantolo, S; Goering, R V

    1998-10-01

    Although Corynebacterium minutissimum is well-known as the cause of erythrasma, it is noted as the etiologic agent of nondermatologic disease only rarely. We document this organism as a cause of central venous catheter-associated bacteremia and report the use of pulsed-field gel electrophoresis to characterize its molecular epidemiology. PMID:9801290

  7. Experimental transmission of Corynebacterium pseudotuberculosis in horses by house flies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The route of infection of pigeon fever remains undetermined. The purpose of this study was to investigate house flies (Musca domestica L.) as vectors of Corynebacterium pseudotuberculosis in horses. Eight ponies were used in a randomized, controlled, blinded experimental study. Ten wounds were creat...

  8. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  9. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  10. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  11. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  12. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  13. Evolution, epidemiology and diversity of Corynebacterium diphtheriae: New perspectives on an old foe.

    PubMed

    Sangal, Vartul; Hoskisson, Paul A

    2016-09-01

    Diphtheria is a debilitating disease caused by toxigenic Corynebacterium diphtheriae strains and has been effectively controlled by the toxoid vaccine, yet several recent outbreaks have been reported across the globe. Moreover, non-toxigenic C. diphtheriae strains are emerging as a major global health concern by causing severe pharyngitis and tonsillitis, endocarditis, septic arthritis and osteomyelitis. Molecular epidemiological investigations suggest the existence of outbreak-associated clones with multiple genotypes circulating around the world. Evolution and pathogenesis appears to be driven by recombination as major virulence factors, including the tox gene and pilus gene clusters, are found within genomic islands that appear to be mobile between strains. The number of pilus gene clusters and variation introduced by gain or loss of gene function correlate with the variable adhesive and invasive properties of C. diphtheriae strains. Genomic variation does not support the separation of C. diphtheriae strains into biovars which correlates well with findings of studies based on multilocus sequence typing. Genomic analyses of a relatively small number of strains also revealed a recombination driven diversification of strains within a sequence type and indicate a wider diversity among C. diphtheriae strains than previously appreciated. This suggests that there is a need for increased effort from the scientific community to study C. diphtheriae to help understand the genomic diversity and pathogenicity within the population of this important human pathogen. PMID:27291708

  14. Corynebacterium tapiri sp. nov. and Corynebacterium nasicanis sp. nov., isolated from a tapir and a dog, respectively.

    PubMed

    Baumgardt, Sandra; Loncaric, Igor; Kämpfer, Peter; Busse, Hans-Jürgen

    2015-11-01

    Two Gram-stain-positive bacterial isolates, strain 2385/12T and strain 2673/12T were isolated from a tapir and a dog's nose, respectively. The two strains were rod to coccoid-shaped, catalase-positive and oxidase-negative. The highest 16S rRNA gene sequence similarity identified Corynebacterium singulare CCUG 37330T (96.3% similarity) as the nearest relative of strain 2385/12T and suggested the isolate represented a novel species. Corynebacterium humireducens DSM 45392T (98.7% 16S rRNA gene sequence similarity) was identified as the nearest relative of strain 2673/12T. Results from DNA-DNA hybridization with the type strain of C. humireducens demonstrated that strain 2673/12T also represented a novel species. Strain 2385/12T showed a quinone system consisting predominantly of menaquinones MK-8(H2) and MK-9(H2) whereas strain 2673/12T contained only MK-8(H2) as predominant quinone. The polar lipid profiles of the two strains showed the major compounds phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. Phosphatidylinositol was identified as another major lipid in 2673/12T whereas it was only found in moderate amounts in strain 2385/12T. Furthermore, moderate to minor amounts of phosphatidylinositol-mannoside, β-gentiobiosyl diacylglycerol and variable counts of several unidentified lipids were detected in the two strains. Both strains contained corynemycolic acids. The polyamine patterns were characterized by the major compound putrescine in strain 2385/12T and spermidine in strain 2673/12T. In the fatty acid profiles, predominantly C18:1ω9c and C16:0 were detected. The two strains are distinguishable from each other and the nearest related established species of the genus Corynebacterium phylogenetically and phenotypically. In conclusion, two novel species of the genus Corynebacterium are proposed, namely Corynebacterium tapiri sp. nov. (type strain, 2385/12T = CCUG 65456T = LMG 28165T) and Corynebacterium nasicanis sp. nov. (type

  15. Application of granular activated carbon/MnFe2O4 composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-01-01

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG0, ΔH0 and ΔS0 revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

  16. Application of granular activated carbon/MnFe₂O₄ composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies.

    PubMed

    Podder, M S; Majumder, C B

    2016-01-15

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG(0), ΔH(0) and ΔS(0) revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions. PMID:26322840

  17. Genome Sequence of Corynebacterium ulcerans Strain FRC11

    PubMed Central

    Benevides, Leandro de Jesus; Viana, Marcus Vinicius Canário; Mariano, Diego César Batista; Rocha, Flávia de Souza; Bagano, Priscilla Carolinne; Folador, Edson Luiz; Pereira, Felipe Luiz; Dorella, Fernanda Alves; Leal, Carlos Augusto Gomes; Carvalho, Alex Fiorini; Soares, Siomar de Castro; Carneiro, Adriana; Ramos, Rommel; Badell-Ocando, Edgar; Guiso, Nicole; Silva, Artur; Figueiredo, Henrique; Guimarães, Luis Carlos

    2015-01-01

    Here, we present the genome sequence of Corynebacterium ulcerans strain FRC11. The genome includes one circular chromosome of 2,442,826 bp (53.35% G+C content), and 2,210 genes were predicted, 2,146 of which are putative protein-coding genes, with 12 rRNAs and 51 tRNAs; 1 pseudogene was also identified. PMID:25767241

  18. Corynebacterium mucifaciens in an immunocompetent patient with cavitary pneumonia

    PubMed Central

    2010-01-01

    Background Corynebacterium mucifaciens has been mainly isolated from skin, blood and from other normally-sterile body fluids. It has rarely been described as a human pathogen since its description. Case presentation We herein report the first case of cavitary pneumonia due to C. mucifaciens in an immunocompetent man returning from Maghreb. Conclusion C. mucifaciens should be considered as important human pathogen in patients with severe illness and compatible history of exposure even in individuals with no clearly identified immunosuppression. PMID:21162757

  19. Nosocomial Endocarditis Caused by Corynebacterium amycolatum and Other Nondiphtheriae Corynebacteria

    PubMed Central

    Holmes, Alison H.

    2002-01-01

    The nondiphtheriae corynebacteria are uncommon but increasingly recognized as important agents of community-acquired endocarditis in patients with underlying structural heart disease, as well as of prosthetic-valve endocarditis. We describe three cases of nondiphtheriae corynebacterial endocarditis, including the first reported case of endocarditis caused by Corynebacterium amycolatum, occurring over an 18-month period, all in association with indwelling intravascular devices. PMID:11749760

  20. Nosocomial valve endocarditis due to corynebacterium striatum: a case report

    PubMed Central

    Marull, Jorge; Casares, Pablo A

    2008-01-01

    Staphylococcus aureus, Coagulase-negative staphylococci, and Enterococci sp. are the usual pathogens involved in nosocomial bacterial endocarditis. Corynebacterium species isolation in blood specimens is usually considered to be a contaminant. We present an interesting case of native mitral valve endocarditis in a 73 year old African American female that was diagnosed days after she was discharged from our institution. The infection was cleared with medical therapy alone. PMID:19077258

  1. Heterogeneity within Corynebacterium minutissimum strains is explained by misidentified Corynebacterium amycolatum strains.

    PubMed

    Zinkernagel, A S; von Graevenitz, A; Funke, G

    1996-09-01

    Forty-eight clinical strains that were tentatively identified as Corynebacterium minutissimum on the basis of standard biochemical reactions (Hollis-Weaver tables) as well as by the use of the API (RAPID) Coryne system were examined further. Two different groups of strains were observed. The first group (including the type strain of C minutissimum) contained 27 strains showing creamy colonies. These strains grew homogeneously in 6.5% NaCl broth, exhibited DNase activity, were susceptible to the vibriocidal compound O/129, produced succinic acid, and contained mycolic acids. The second group comprised 21 strains with dry colonies. They grew in clumps at the surface of 6.5% NaCl broth, DNase activity was not detected, they were resistant against O/129, produced large amounts of propionic acid, and mycolic acids were not detected. In combination with quantitative DNA-DNA hybridizations, it was demonstrated that strains of the second cluster belonged, in fact, to C amycolatum. Furthermore, it was observed that a few C minutissimum strains may also ferment mannitol. These data indicate that the clinical microbiologist must be careful not to misidentify C amycolatum strains as C minutissimum. PMID:8816598

  2. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses

    PubMed Central

    Sariguzel, Fatma Mutlu; Koc, A. Nedret; Yagmur, Gülhan; Berk, Elife

    2014-01-01

    Interdigital foot infections are mostly caused initially by dermatophytes, yeasts and less frequently by bacteria. Erythrasma caused by Corynebacterium minutissimum can be confused with superficial mycoses. The aim of the study was to determine the prevalence of the etiologic agents of superficial mycoses and the frequency of Corynebacterium minutissimum in interdigital foot infections. All the samples obtained from the 121 patients with interdigital foot infections were examined directly with the use of 20% potassium hydroxide mounts and Gram stain under the microscope and cultured on Sabouraud’s dextrose agar plates. In identification of superficial mycoses, the rate was found to be 14% with the cultural method and 14% with direct microscopic examination. Using a combination of direct microscopic examination and culture, a 33.8% ratio was achieved. In the culture of these samples, the most isolated factor was Trichophyton rubrum (33.7%). In 24 of the patients (19.8%) Corynebacterium minutissimum was detected by Gram staining, in 6 of these patients Trichophyton rubrum was found, Trichophyton mentagrophytes was found in 2 and Trichosporon spp. was found in 1. The examination of interdigital foot lesions in the laboratory, the coexistence of erythrasma with dermatophytes and yeast should be considered. PMID:25477907

  3. rpoB Gene Sequencing for Identification of Corynebacterium Species

    PubMed Central

    Khamis, Atieh; Raoult, Didier; La Scola, Bernard

    2004-01-01

    The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species. PMID:15364970

  4. A pyrolysis-mass spectrometry study of Corynebacterium spp.

    PubMed

    Hindmarch, J M; Magee, J T; Hadfield, M A; Duerden, B I

    1990-02-01

    Clinical (66) and collection (38) strains of Corynebacterium spp., including C. jeikeium and CDC group D2, and of Listeria monocytogenes were examined. Conventional characters used in species identification were assessed by a microbiochemical method, and pyrolysis-mass spectrometry (Py-MS) was performed with a Horizon Instruments PYMS 200X. Classification based on Py-MS data yielded clusters that corresponded with species identification and classification groups from conventional data. One small group of clinical strains, homogeneous in conventional tests and Py-MS, comprised isolates from sputum samples from patients undergoing ventilation; they were similar to collection strains of C. renale and C. striatum; the latter species has been implicated in chest infection. Another group, similar to C. minutissimum in both systems, comprised clinical strains isolated from urogenital specimens. L. monocytogenes strains were clearly distinct from Corynebacterium spp. Groups comprising CDC D2 strains and C. jeikeium were resolved, and were similar to other Corynebacterium spp. Two collection strains of C. xerosis were distinct in conventional tests and Py-MS. PMID:2106034

  5. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses.

    PubMed

    Sariguzel, Fatma Mutlu; Koc, A Nedret; Yagmur, Gülhan; Berk, Elife

    2014-01-01

    Interdigital foot infections are mostly caused initially by dermatophytes, yeasts and less frequently by bacteria. Erythrasma caused by Corynebacterium minutissimum can be confused with superficial mycoses. The aim of the study was to determine the prevalence of the etiologic agents of superficial mycoses and the frequency of Corynebacterium minutissimum in interdigital foot infections. All the samples obtained from the 121 patients with interdigital foot infections were examined directly with the use of 20% potassium hydroxide mounts and Gram stain under the microscope and cultured on Sabouraud's dextrose agar plates. In identification of superficial mycoses, the rate was found to be 14% with the cultural method and 14% with direct microscopic examination. Using a combination of direct microscopic examination and culture, a 33.8% ratio was achieved. In the culture of these samples, the most isolated factor was Trichophyton rubrum (33.7%). In 24 of the patients (19.8%) Corynebacterium minutissimum was detected by Gram staining, in 6 of these patients Trichophyton rubrum was found, Trichophyton mentagrophytes was found in 2 and Trichosporon spp. was found in 1. The examination of interdigital foot lesions in the laboratory, the coexistence of erythrasma with dermatophytes and yeast should be considered. PMID:25477907

  6. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  7. Corynebacterium minutissimum bacteremia in a patient with chronic myeloid leukemia in blast crisis.

    PubMed

    Guarderas, J; Karnad, A; Alvarez, S; Berk, S L

    1986-11-01

    Serious infections and sepsis due to nondiphtheria Corynebacteria have been well described. A patient with chronic myeloid leukemia in blast crisis, who developed Corynebacterium minutissimum bacteremia, is described in this report. Corynebacterium minutissimum is the causative agent of erythrasma and to our knowledge, this is the first published report of septicemia due to this organism. PMID:3465494

  8. Corynebacterium accolens Isolated from Breast Abscess: Possible Association with Granulomatous Mastitis▿

    PubMed Central

    Ang, Lei M. N.; Brown, Hamish

    2007-01-01

    Corynebacterium accolens is rarely isolated as a human pathogen. We describe here a case of C. accolens isolated from a breast abscess in a patient previously diagnosed with granulomatous mastitis. The possible association of Corynebacterium accolens and granulomatous mastitis in this patient is discussed. PMID:17344355

  9. In silico identification of Corynebacterium pseudotuberculosis antigenic targets and application in immunodiagnosis.

    PubMed

    Rezende, Andrea de Fátima Silva; Brum, Alexandre Antunes; Reis, Carlos Guilherme; Angelo, Henrique Ramos; Leal, Karen Silva; Silva, Mara Thais de Oliveira; Simionatto, Simone; Azevedo, Vasco; Santos, Anderson; Portela, Ricardo Wagner; Dellagostin, Odir; Borsuk, Sibele

    2016-06-01

    Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis. It affects mainly small ruminants and causes significant economic losses worldwide. Because symptoms are not immediately noticeable, CLA clinical diagnosis is not effective. Numerous serological tests are being developed to detect the disease in asymptomatic animals, but currently available immunoassays have problems with sensitivity. Current ELISA formats use native bacterial antigens, and recombinant proteins could be useful for improving the immunoassay parameters. The C. pseudotuberculosis proteins CP0126a, CP0369 and CP1957 were identified from 2097 candidate proteins by mature epitope density (MED) analysis, expressed in Escherichia coli and evaluated in an indirect immunoenzymic system. The CP0126a, CP0369 and CP1957 ELISAs showed 77.5 %, 92.5 % and 92.5 % specificity and 95 %, 90 % and 85 % sensitivity, respectively. Receiver operating characteristic (ROC) curve analysis showed an area under the curve of 0.874, 0.951 and 0.881, respectively. The proteins identified in silico were recognized by antibodies in the sera from infected animals without being recognized in negative samples. The ELISA assay using the rCP0369 protein as antigen had the greatest specificity and sensitivity values, followed by rCP1957. This is an interesting strategy for seroepidemiological investigations in sheep flocks due to its significant specificity and sensitivity. PMID:27071381

  10. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  11. Corynebacterium ulcerans in humans and cattle in North Devon.

    PubMed Central

    Hart, R. J.

    1984-01-01

    A case of Corynebacterium ulcerans sore throat in a community that drank raw milk from its own farm led to the discovery of another symptomless human infection. Eight cows in the herd were found to be infected and the intermittent pattern of excretion was demonstrated in another cow followed through its lactation. Further evidence of milk infected by C. ulcerans was found by examining all raw milk samples submitted to the laboratory. Two other human cases were diagnosed in Devon during the period of this investigation. PMID:6707468

  12. Corynebacterium urealyticum: a comprehensive review of an understated organism

    PubMed Central

    Salem, Nagla; Salem, Lamyaa; Saber, Sally; Ismail, Ghada; Bluth, Martin H

    2015-01-01

    Corynebacterium urealyticum is a Gram positive, slow-growing, lipophilic, multi-drug resistant, urease positive micro-organism with diphtheroid morphology. It has been reported as an opportunistic nosocomial pathogen and as the cause of a variety of diseases including but not limited to cystitis, pyelonephritis, and bacteremia among others. This review serves to describe C. urealyticum with respect to its history, identification, laboratory investigation, relationship to disease and treatment in order to allow increased familiarity with this organism in clinical disease. PMID:26056481

  13. A microbiological and clinical review on Corynebacterium kroppenstedtii.

    PubMed

    Tauch, Andreas; Fernández-Natal, Isabel; Soriano, Francisco

    2016-07-01

    The genus Corynebacterium represents a taxon of Gram-positive bacteria with a high G+C content in the genomic DNA. Corynebacterium kroppenstedtii is an unusual member of this taxon as it lacks the characteristic mycolic acids in the cell envelope. Genome sequence analysis of the C. kroppenstedtii type strain has revealed a lipophilic (lipid-requiring) lifestyle and a remarkable repertoire of carbohydrate uptake and utilization systems. Clinical isolates of C. kroppenstedtii have been obtained almost exclusively from female patients and mainly from breast abscesses and cases of granulomatous mastitis. However, the role of C. kroppenstedtii in breast pathologies remains unclear. This review provides a comprehensive overview of the taxonomy, microbiology, and microbiological identification of C. kroppenstedtii, including polyphasic phenotypic approaches, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the use of 16S rRNA gene sequencing. A clinical review presents reported cases, various antimicrobial treatments, antibiotic susceptibility assays, and antibiotic resistance genes detected during genome sequencing. C. kroppenstedtii must be considered a potential opportunistic human pathogen and should be identified accurately in clinical laboratories. PMID:27155209

  14. Corynebacterium CDC Group G Native and Prosthetic Valve Endocarditis

    PubMed Central

    Sattar, Adil; Yu, Siegfried; Koirala, Janak

    2015-01-01

    We report the first case of native and recurrent prosthetic valve endocarditis with Corynebacterium CDC group G, a rarely reported cause of infective endocarditis (IE). Previously, there have been only two cases reported for prosthetic valve IE caused by these organisms. A 69-year-old female with a known history of mitral valve regurgitation presented with a 3-day history of high-grade fever, pleuritic chest pain and cough. Echocardiography confirmed findings of mitral valve thickening consistent with endocarditis, which subsequently progressed to become large and mobile vegetations. Both sets of blood cultures taken on admission were positive for Corynebacterium CDC group G. Despite removal of a long-term venous access port, the patient’s presumed source of line associated bacteremia, mitral valve replacement, and aggressive antibiotic therapy, the patient had recurrence of vegetations on the prosthetic valve. She underwent replacement of her prosthetic mitral valve in the subsequent 2 weeks, before she progressed to disseminated intravascular coagulation and expired. Although they are typically considered contaminants, corynebacteria, in the appropriate clinical setting, should be recognized, identified, and treated as potentially life-threatening infections, particularly in the case of line-associated bacteremias, and native and prosthetic valve endocarditis. PMID:26500737

  15. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  16. Genome sequence of the marine bacterium Corynebacterium maris type strain Coryn-1(T) (= DSM 45190(T)).

    PubMed

    Schaffert, Lena; Albersmeier, Andreas; Bednarz, Hanna; Niehaus, Karsten; Kalinowski, Jörn; Rückert, Christian

    2013-07-30

    Corynebacterium maris Coryn-1(T) Ben-Dov et al. 2009 is a member of the genus Corynebacterium which contains Gram-positive, non-spore forming bacteria with a high G+C content. C. maris was isolated from the mucus of the Scleractinian coral Fungia granulosa and belongs to the aerobic and non-haemolytic corynebacteria. It displays tolerance to salts (up to 10%) and is related to the soil bacterium Corynebacterium halotolerans. As this is a type strain in a subgroup of Corynebacterium without complete genome sequences, this project, describing the 2.78 Mbp long chromosome and the 45.97 kbp plasmid pCmaris1, with their 2,584 protein-coding and 67 RNA genes, will aid the G enomic E ncyclopedia of Bacteria and Archaea project. PMID:24501635

  17. Experimental inoculation of house flies Musca domestica with Corynebacterium pseudotuberculosis serovar equi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corynebacterium pseudotuberculosis (Actinomycetales: Corynebacteriaceae) infection in horses causes external abscesses, infection of internal organs and ulcerative lymphangitis. The exact mechanism of infection remains unknown, but fly transmission is suspected. Scientists at Auburn University and U...

  18. Corynebacterium riegelii sp. nov., an Unusual Species Isolated from Female Patients with Urinary Tract Infections

    PubMed Central

    Funke, Guido; Lawson, Paul A.; Collins, Matthew D.

    1998-01-01

    Four strains of an unknown coryneform bacterium were isolated in pure culture from females with urinary tract infections. Strong urease activity and the ability to slowly ferment maltose but not glucose were the most significant phenotypic features of this catalase-positive, nonmotile, nonlipophilic, rod-shaped bacterium which served to distinguish it from all other presently defined coryneform bacteria. Chemotaxonomic investigations demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis revealed that the isolates were genealogically identical and represented a new subline within the genus Corynebacterium, for which the designation Corynebacterium riegelii sp. nov. is proposed. The type strain of Corynebacterium riegelii is CCUG 38180 (DSM 44326, CIP 105310). PMID:9508284

  19. Corynebacterium mucifaciens sp. nov., an unusual species from human clinical material.

    PubMed

    Funke, G; Lawson, P A; Collins, M D

    1997-10-01

    Eight strains of a previously undescribed coryneform bacterium had been isolated from human clinical material over a 5-year period. Colonies of the unknown coryneform bacterium had an unusual appearance as they were slightly yellowish and very mucoid. Biochemical and chemotaxonomic characterization revealed that the unknown coryneform bacterium belonged to the genus Corynebacterium. It could be readily differentiated from all previously described Corynebacterium species. Electron microscopy demonstrated the production of an extracellular substance causing connecting filaments between cells as a morphological correlate to the mucoid colonies. Comparative 16S rRNA gene sequence analysis revealed that the unknown coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium mucifaciens sp. nov. is proposed. The type strain is CCUG 36878 (= DSM 44265 = CIP 105129). PMID:9336892

  20. Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The route of Corynebacterium pseudotuberculosis infection in horses remains undetermined, but transmission by insects is suspected. Scientists from CMAVE and Auburn University investigated house flies (Musca domestica L.) as possible vectors. Three ponies were directly inoculated with C. pseudotuber...

  1. Experimental inoculation of house flies, Musca domestica L., with Corynebacterium pseudotuberculosis serovar equi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corynebacterium pseudotuberculosis (Actinomycetales: Corynebacteriaceae) infection in horses causes three different disease syndromes: external abscesses, infection of internal organs and ulcerative lymphangitis. The route of infection in horses remains undetermined, but transmission by insect vecto...

  2. Antimicrobial susceptibilities of Corynebacterium species and other non-spore-forming gram-positive bacilli to 18 antimicrobial agents.

    PubMed

    Soriano, F; Zapardiel, J; Nieto, E

    1995-01-01

    The susceptibilities of 265 strains of Corynebacterium species and other non-spore-forming gram-positive bacilli to 18 antimicrobial agents were tested. Most strains were susceptible to vancomycin, doxycycline, and fusidic acid. Corynebacterium jeikeium and Corynebacterium urealyticum were the most resistant organisms tested. Resistance to beta-lactams, clindamycin, erythromycin, azythromycin, ciprofloxacin and gentamicin was common among strains of Corynebacterium xerosis and Corynebacterium minutissimum. Ampicillin resistance among Listeria monocytogenes was more prevalent than previously reported. Optochin, fosfomycin, and nitrofurantoin showed very little activity against most organisms tested, but the use of nitrofurantoin as a selective agent in culture medium may prevent the recovery of some isolates. Except for the unvarying activity of vancomycin against Corynebacterium species, the antimicrobial susceptibilities of the latter to other antibiotics are usually unpredictable, such that susceptibility tests are necessary for selecting the best antimicrobial treatment. PMID:7695308

  3. Three cases of opportunistic infection caused by propionic acid producing Corynebacterium minutissimum.

    PubMed

    Van Bosterhaut, B; Cuvelier, R; Serruys, E; Pouthier, F; Wauters, G

    1992-07-01

    Propionic acid producing strains of Corynebacterium minutissimum were isolated from three patients with opportunistic infections. One neutropenic patient was undergoing chemotherapy for prolymphocytic leukemia; the other two patients were undergoing hemodialysis and peritoneal dialysis respectively. An unusual feature of these three strains was their resistance to several antibiotics, which is seldom seen in diphtheroids other than Corynebacterium jeikeium and CDC group D2. PMID:1396773

  4. Corynebacterium minutissimum endophthalmitis: management with antibiotic irrigation of the capsular bag.

    PubMed

    Arsan, A K; Sizmaz, S; Ozkan, S B; Duman, S

    Chronic endophthalmitis, due to Corynebacterium minutissimum, developed in a patient following uncomplicated extracapsular cataract extraction and posterior chamber intraocular lens (PC-IOL) implantation. In this case, which to our knowledge is the first reported case of Corynebacterium minutissimum endophthalmitis, chronic inflammation persisted for 8 months with fluctuations in the inflammation. The specimens from the capsular bag yielded positive culture, but the vitreous culture was negative. The case was successfully treated by a capsular bag irrigation with vancomycin. PMID:8864817

  5. Identification of Corynebacterium amycolatum and other nonlipophilic fermentative corynebacteria of human origin.

    PubMed

    Wauters, G; Van Bosterhaut, B; Janssens, M; Verhaegen, J

    1998-05-01

    Four identification tests, proposed in addition to conventional methods, were evaluated with 320 fermentative nonlipophilic Corynebacterium strains: growth at 20 degrees C, glucose fermentation at 42 degrees C, alkalinization of sodium formate, and acid production from ethylene glycol. These tests were highly discriminant. Corynebacterium amycolatum displayed a unique profile, allowing it to be distinguished from similar species, such as C. xerosis, C. striatum, and C. minutissimum. PMID:9574722

  6. Identification of n-Decane Oxidation Products in Corynebacterium Cultures by Combined Gas Chromatography-Mass Spectrometry

    PubMed Central

    Bacchin, Paolo; Robertiello, Andrea; Viglia, Aurelio

    1974-01-01

    The gas chromatography-mass spectrometry technique was employed to characterize n-decane oxidation products of Corynebacterium strains 7E1C and 269 (SNAM Progetti collection) after 73 h of incubation at 35 C. Corynebacterium 7E1C accumulated consistent amounts of esters of long chain acids with long chain alcohols, mainly decyldecanoate as well as products with mono- and diterminal carboxylic functions. Corynebacterium 269 yielded 1-decanol and 1-10 decanediol as principal oxidation products. PMID:4441062

  7. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

    PubMed

    Dou, Wenfang; Xu, Meijuan; Cai, Dongmei; Zhang, Xiaomei; Rao, Zhiming; Xu, Zhenghong

    2011-10-01

    Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the L-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and β-subunits. When the argJ regions corresponding to the α- and β-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on L: -arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of L: -arginine significantly (16.8%). PMID:21785983

  8. Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1

    SciTech Connect

    Omori, Toshio; Monna, L.; Saiki, Yuko; Kodama, Tohru )

    1992-03-01

    Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS{sub 2}, FeS{sub 2}, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.

  9. Corynebacterium diphtheriae infections currently and in the past.

    PubMed

    Zasada, Aleksandra Anna

    2015-01-01

    Along with the introduction of common obligatory vaccinations against diphtheria, the disease has been limited in developed countries. However, diphtheria is still endemic in developing countries. Due to a growing popularity of visiting these countries, there is a risk of importation of the disease to Europe. Studies revealed that over 60% of persons aged >40 years in the Polish population do not have a protective level of antibodies against diphtheria. Furthermore, an access to diphtheria antitoxin, which is essential in diphtheria treatment, is now hardly accessible in Europe. On the other hand, in many countries, including Poland, new infections caused by non-toxigenic Corynebacterium diphtheriae have been emerged. Such infections are frequently manifested by bacteraemia and endocarditis with a high fatality rate, amounting even to 41%. PMID:26519837

  10. Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses.

    PubMed

    de Sá, Pablo H C G; Veras, Adonney A O; Carneiro, Adriana R; Barúna, Rafael A; Guimarães, Luís C; Pinheiro, Kenny C; Pinto, Anne C; Soares, Siomar C; Schneider, Maria P C; Azevedo, Vasco; Silva, Artur; Ramos, Rommel T J

    2015-12-01

    Corynebacterium pseudotuberculosis causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death (Ruiz et al., 2011) [1]. This bacterium was grown under osmotic (2 M), acid (pH) and heat (50 °C) stress and under control (Normal-BHI brain heart infusion) conditions, which simulate the conditions faced by the bacteria during the infectious process. Subsequently, cDNA of each condition was sequenced by the SOLiD3 Plus platform using the RNA-Seq technique [2], [3], [4]. The data produced was processed to evaluate the differential gene expression, which is helpful to understand the adaptation mechanisms during the infection in the host. The sequencing data of all conditions are available in the European Bioinformatics Institute (EBI) repository under accession number E-MTAB-2017. PMID:26702428

  11. [Corynebacterium ulcerans infection in roe deer (Capreolus capreolus)].

    PubMed

    Rau, Jörg; Blazey, Birgit; Contzen, Matthias; Sting, Reinhard

    2012-01-01

    This is the first report of a Corynebacterium (C) ulcerans-infection in European roe deer (Capreolus capreolus). The bacterium was isolated from a grapefruit sized abscess of an animal that had been shot. In addition to biochemical tests, the isolate was identified by Fourier transform infrared spectroscopy (FT-IR) and partial sequencing of the rpoB gene. The isolated bacteria showed phospholipase D activity that could be demonstrated by reverse CAMP-test. A tox-gene could be detected by PCR but the Elek-test specific for diphtheria toxin failed.The isolate was compared to two C. ulcerans-strains isolated from wild boar (Sus scrofa) from the state of Baden-Wuerttemberg described recently. PMID:22515035

  12. Outbreak of Corynebacterium pseudodiphtheriticum infection in cystic fibrosis patients, France.

    PubMed

    Bittar, Fadi; Cassagne, Carole; Bosdure, Emmanuelle; Stremler, Nathalie; Dubus, Jean Christophe; Sarles, Jacques; Reynaud-Gaubert, Martine; Raoult, Didier; Rolain, Jean-Marc

    2010-08-01

    An increasing body of evidence indicates that nondiphtheria corynebacteria may be responsible for respiratory tract infections. We report an outbreak of Corynebacterium pseudodiphtheriticum infection in children with cystic fibrosis (CF). To identify 18 C. pseudodiphtheriticum strains isolated from 13 French children with CF, we used molecular methods (partial rpoB gene sequencing) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Clinical symptoms were exhibited by 10 children (76.9%), including cough, rhinitis, and lung exacerbations. The results of MALDI-TOF identification matched perfectly with those obtained from molecular identification. Retrospective analysis of sputum specimens by using specific real-time PCR showed that approximately 20% of children with CF were colonized with these bacteria, whereas children who did not have CF had negative test results. Our study reemphasizes the conclusion that correctly identifying bacteria at the species level facilitates detection of an outbreak of new or emerging infections in humans. PMID:20678316

  13. Outbreak of Corynebacterium pseudodiphtheriticum Infection in Cystic Fibrosis Patients, France

    PubMed Central

    Bittar, Fadi; Cassagne, Carole; Bosdure, Emmanuelle; Stremler, Nathalie; Dubus, Jean-Christophe; Sarles, Jacques; Reynaud-Gaubert, Martine; Raoult, Didier

    2010-01-01

    An increasing body of evidence indicates that nondiphtheria corynebacteria may be responsible for respiratory tract infections. We report an outbreak of Corynebacterium pseudodiphtheriticum infection in children with cystic fibrosis (CF). To identify 18 C. pseudodiphtheriticum strains isolated from 13 French children with CF, we used molecular methods (partial rpoB gene sequencing) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Clinical symptoms were exhibited by 10 children (76.9%), including cough, rhinitis, and lung exacerbations. The results of MALDI-TOF identification matched perfectly with those obtained from molecular identification. Retrospective analysis of sputum specimens by using specific real-time PCR showed that ≈20% of children with CF were colonized with these bacteria, whereas children who did not have CF had negative test results. Our study reemphasizes the conclusion that correctly identifying bacteria at the species level facilitates detection of an outbreak of new or emerging infections in humans. PMID:20678316

  14. Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice

    PubMed Central

    2011-01-01

    Background Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants. Findings To construct the DNA vaccine, the hsp60 gene of C. pseudotuberculosis was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/hsp60). Conclusion This vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity. PMID:21774825

  15. High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

    PubMed Central

    De Briel, D; Couderc, F; Riegel, P; Jehl, F; Minck, R

    1992-01-01

    A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory. PMID:1624556

  16. High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

    PubMed

    De Briel, D; Couderc, F; Riegel, P; Jehl, F; Minck, R

    1992-06-01

    A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory. PMID:1624556

  17. Corynebacterium Diphtheriae Endocarditis with Multifocal Septic Emboli: Can Prompt Diagnosis Help Avoid Surgery?

    PubMed Central

    Patris, Vasileios; Argiriou, Orestis; Konstantinou, Charalampos; Lama, Niki; Georgiou, Haris; Katsanevakis, Emmanouil; Argiriou, Mihalis; Charitos, Christos

    2014-01-01

    Patient: Male, 23 Final Diagnosis: Corynebacterium diphtheriae endocarditis Symptoms: Abdominal pain • cachexia • diarrhea • fever • vomiting Medication: — Clinical Procedure: Mitral valve replacement Specialty: Surgery Objective: Rare disease Background: Although Corynebacterium diphtheriae is well known for causing diphtheria and other respiratory tract infections, in very rare cases it can lead to severe systemic disease. Case Report: This is a case of a previously healthy young man (no prosthetic valve in situ or other known congenital defect), presenting with a Corynebacterium diphtheriae infection leading to endocarditis. The patient reported no I.V. drug use, so it can be assumed that no risk factors for infective endocarditis were present. Conclusions: This report aims to raise suspicion for this specific infection in order to proceed with the right treatment as soon as possible. PMID:25153519

  18. Characteristics of Rare or Recently Described Corynebacterium Species Recovered from Human Clinical Material in Canada

    PubMed Central

    Bernard, K. A.; Munro, C.; Wiebe, D.; Ongsansoy, E.

    2002-01-01

    Nineteen new Corynebacterium species or taxa described since 1995 have been associated with human disease. We report the characteristics of 72 strains identified as or most closely resembling 14 of these newer, medically relevant Corynebacterium species or taxa, as well as describe in brief an isolate of Corynebacterium bovis, a rare pathogen for humans. The bacteria studied in this report were nearly all derived from human clinical specimens and were identified by a polyphasic approach. Most were characterized by nearly full 16S rRNA gene sequence analysis. Some isolates were recovered from previously unreported sources and exhibited unusual phenotypes or represented the first isolates found outside Europe. Products of fermentation, with emphasis on the presence or absence of propionic acid, were also studied in order to provide an additional characteristic with which to differentiate among phenotypically similar species. PMID:12409436

  19. Isolation and Characterization of a Black-Pigmented Corynebacterium sp. from a Woman with Spontaneous Abortion

    PubMed Central

    Shukla, Sanjay K.; Vevea, Dirk N.; Frank, Daniel N.; Pace, Norman R.; Reed, Kurt D.

    2001-01-01

    An unusual black-pigmented coryneform bacterium was isolated from the urogenital tract of a woman who experienced a spontaneous abortion during month 6 of pregnancy. Biochemical and chemotaxonomic analyses demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Phylogenetic analysis based on 16S rRNA sequences (GenBank accession no. AF220220) revealed that the organism was a member of a distinct subline which includes uncultured Corynebacterium MTcory 1P (GenBank accession no. AF115934), derived from prostatic fluid, and Corynebacterium CDC B8037 (GenBank accession no. AF033314), an uncharacterized black-pigmented coryneform bacterium. On the basis of chemotaxonomic and phylogenetic evidence, this organism probably represents a new species and is most closely related to the uncharacterized Centers for Disease Control and Prevention group 4 coryneforms. Our strain is designated CN-1 (ATCC 700975). PMID:11230435

  20. Corynebacterium pelargi sp. nov., isolated from the trachea of white stork nestlings.

    PubMed

    Kämpfer, Peter; Jerzak, Leszek; Bochenski, Marcin; Kasprzak, Mariusz; Wilharm, Gottfried; Golke, Jan; Busse, Hans-Jürgen; Glaeser, Stefanie P

    2015-05-01

    A Gram-stain-positive, pleomorphic, oxidase-negative, non-motile isolate from the trachea of a white stork from Poland, designated strain 136/3(T), was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to Corynebacterium mustelae , Corynebacterium pseudotuberculosis , Corynebacterium vitaeruminis and Corynebacterium ulcerans (96.0-96.3%). The quinone system consisted of major amounts of MK-8(H2), minor amounts of MK-9(H2) and traces of MK-8 and MK-9. The polar lipid profile of strain 136/3(T) contained phosphatidylinositol and phosphatidylinositol-mannoside as major lipids and phosphatidylglycerol and an acidic glycolipid in moderate amounts. In addition small amounts of diphosphatidylglycerol, a phospholipid, an aminolipid and two lipids of unknown group affiliation were found. The polyamine pattern was composed of the major components spermidine and spermine. Putrescine, 1,3-diaminopropane, cadaverine, sym-homospermidine and tyramine were found in minor or trace amounts. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. In the fatty acid profile straight-chain, saturated and mono-unsaturated fatty acids predominated (C(18 : 1)ω9c, C(16 : 1)ω7c, C16 : 0, C(18  : 0)). Corynemycolic acids were detected. Physiological traits as well as unique traits of the polar lipid profile and the fatty acid pattern distinguished strain 136/3(T) from the most closely related species. All these results indicate that strain 136/3(T) represents a novel species of the genus Corynebacterium for which we propose the name Corynebacterium pelargi sp. nov. The type strain is 136/3(T) ( =CIP 110778(T) =CCM 8517(T) =LMG 28174(T)). PMID:25678678

  1. Structure of a DsbF homologue from Corynebacterium diphtheriae

    PubMed Central

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-01-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein. PMID:25195886

  2. Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis?

    PubMed

    dos Santos, Louisy Sanches; Antunes, Camila Azevedo; de Oliveira, Daniel Martins; Sant'Anna, Lincoln de Oliveira; Pereira, José Augusto Adler; Hirata Júnior, Raphael; Burkovski, Andreas; Mattos-Guaraldi, Ana Luíza

    2015-11-01

    Corynebacterium diphtheriae strains continue to circulate worldwide causing diphtheria and invasive diseases, such as endocarditis, osteomyelitis, pneumonia and catheter-related infections. Presumptive C. diphtheriae infections diagnosis in a clinical microbiology laboratory requires a primary isolation consisting of a bacterial culture on blood agar and agar containing tellurite (TeO3(2-)). In this study, nine genome sequenced and four unsequenced strains of C. diphtheriae from different sources, including three samples from a recent outbreak in Brazil, were characterized with respect to their growth properties on tellurite-containing agar. Levels of tellurite-resistance (Te(R)) were evaluated by determining the minimum inhibitory concentrations of potassium tellurite (K2TeO3) and by a viability reduction test in solid culture medium with K2TeO3. Significant differences in Te(R) levels of C. diphtheriae strains were observed independent of origin, biovar or presence of the tox gene. Data indicated that the standard initial screening with TeO3(2-)-selective medium for diphtheria bacilli identification may lead to false-negative results in C. diphtheriae diagnosis laboratories. PMID:26459339

  3. Fatal case of bacteremia caused by an atypical strain of Corynebacterium mucifaciens.

    PubMed

    Cantarelli, Vlademir Vicente; Brodt, Teresa Cristina Z; Secchi, Carina; Inamine, Everton; Pereira, Fabiana de Souza; Pilger, Diogo Andre

    2006-12-01

    Corynebacterium species have often been considered normal skin flora or contaminants; however, in recent years they have been increasingly implicated in serious infections. Moreover, many new species have been discovered and old species renamed, especially after molecular biology techniques were introduced. Corynebacterium mucifaciens is mainly isolated from blood and from other normally-sterile body fluids; it forms slightly yellow, mucoid colonies on blood agar. We report a fatal case of bacteremia due to an atypical strain of C. mucifaciens. This strain had atypical colony morphology; analysis of the 16S rRNA gene was used to define the species. PMID:17420918

  4. Non-contiguous finished genome sequence of Corynebacterium timonense type strain 5401744T

    PubMed Central

    Robert, Catherine; Raoult, Didier

    2014-01-01

    Corynebacterium timonense strain 5401744T is a member of the genus Corynebacterium which contains Gram-positive bacteria with a high G+C content. It was isolated from the blood of a patient with endocarditis. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 2,553,575 bp long genome contains 2,401 protein-coding genes and 55 RNA genes, including between 5 and 6 rRNA operons. PMID:25197476

  5. Studies of an outbreak of Corynebacterium equi pneumonia in foals.

    PubMed

    Smith, B P; Robinson, R C

    1981-10-01

    Five out of 6 foals between 2 and 4 months old, on a ranch in northern California, developed pneumonia within a 3 week period in June and July 1978. Corynebacterium equi was recovered from each of the 5 foals by transtracheal aspiration. Clinical signs were variable but included increased respiratory rate, fever, cough, nasal discharge, harsh airway sounds over middle sized airways and wheezing over small airways. Cyanosis was present in the most severely affected foal. Radiographic findings included diffusely increased interstitial and peribronchial densities, areas of consolidation and, in 3 cases, dense focal areas indicating abscessation. Foals were treated with several different antimicrobial agents. Most were treated with penicillin and gentamicin. Four of the 5 affected foals recovered within 2 to 3 weeks but the first foal to be affected died 2 days after first receiving veterinary attention. At postmortem examination, pulmonary changes considered typical of C equi pneumonia were found, including wet, heavy dark red lungs which failed to collapse and numerous 1 to 7 cm thin-walled abscesses throughout the parenchyma, containing inspissated exudate. C equi was cultured from the exudate. Samples of soil and dust from 9 of 20 areas inhabited by infected foals yielded C equi of the same serological group as found in the foals. Eight paddocks in which foals had not been kept were negative for C equi. The organism was recovered from cobwebs in the stalls occupied by infected foals. Aerosol infection via dust was considered to be the route of infection. Pharyngeal, vaginal and faecal cultures from the dams of 3 affected foals were negative for C equi. Early diagnosis by transtracheal aspiration and appropriate therapy are considered to be extremely important in the successful treatment of C equi pneumonia. Preventive therapy should include control of environmental dust. PMID:7318800

  6. Corynebacterium pseudotuberculosis Pneumonia in a Veterinary Student Infected During Laboratory Work

    PubMed Central

    Heggelund, Lars; Gaustad, Peter; Håvelsrud, Othilde Elise; Blom, Jochen; Borgen, Lars; Sundset, Arve; Sørum, Henning; Frøland, Stig Sophus

    2015-01-01

    We present a case of Corynebacterium pseudotuberculosis pneumonia in a veterinary student, with molecular genetic evidence of acquisition during laboratory work, an observation relevant for laboratory personnel working with C pseudotuberculosis isolates. The patient was clinically cured with 14 months trimethoprim/sulfamethoxazole and rifampicin combination treatment. PMID:26380345

  7. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 03-8664 Isolated from a Human Throat.

    PubMed

    Guimarães, Luis C; Viana, Marcus V C; Benevides, Leandro J; Mariano, Diego C B; Veras, Adonney A O; Sá, Pablo H C; Rocha, Flávia S; Vilas Boas, Priscilla C B; Soares, Siomar C; Barbosa, Maria S; Guiso, Nicole; Badell, Edgar; Azevedo, Vasco; Ramos, Rommel T J; Silva, Artur

    2016-01-01

    Corynebacterium ulcerans is an emergent pathogen infecting wild and domesticated animals worldwide that may serve as reservoirs for zoonotic infections. In this study, we present the draft genome of C. ulcerans strain 03-8664. The draft genome has 2,428,683 bp, 2,262 coding sequences, and 12 rRNA genes. PMID:27469956

  8. Draft Genome Sequence of Corynebacterium ulcerans Strain 04-3911, Isolated from Humans

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adooney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Carneiro, Adriana R.; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Corynebacterium ulcerans is a pathogenic bacterium infecting wild and domesticated animals; some infection cases in humans have increased throughout the world. The current study describes the draft genome of strain 04-3911, isolated from humans. The draft genome has 2,492,680 bp, 2,143 coding sequences, 12 rRNA genes, and 50 tRNA genes. PMID:27034486

  9. Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil

    PubMed Central

    Encinas, Fernando; Marin, Michel A; Ramos, Juliana N; Vieira, Verônica V; Mattos-Guaraldi, Ana Luiza; Vicente, Ana Carolina P

    2015-01-01

    We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains. PMID:26517665

  10. Nosocomial Outbreak of Corynebacterium striatum Infection in Patients with Chronic Obstructive Pulmonary Disease▿

    PubMed Central

    Renom, Feliu; Garau, Margarita; Rubí, Mateu; Ramis, Ferran; Galmés, Antònia; Soriano, Joan B.

    2007-01-01

    We describe an unusual cluster of Corynebacterium striatum infections in 21 patients with chronic obstructive pulmonary disease (COPD) admitted to a medium-size respiratory unit. Eleven isolates from eight patients occurred simultaneously within a month. C. striatum is a potentially pathogenic microorganism with the ability to produce nosocomial infectious outbreaks and respiratory colonization in patients with advanced COPD. PMID:17409213

  11. Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon

    PubMed Central

    Muge, Gabriel R. S.; Veras, Adonney A. O.; de Sá, Pablo H. C. G.; Cavalcante, Ana Lídia Queiroz; Alves, Jorianne Thyeska Castro; Morais, Ezequiel; Silva, André G. M.; Azevedo, Vasco; Folador, Adriana Ribeiro Carneiro; Silva, Artur

    2016-01-01

    In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes. PMID:27516524

  12. Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon.

    PubMed

    Muge, Gabriel R S; Veras, Adonney A O; de Sá, Pablo H C G; Cavalcante, Ana Lídia Queiroz; Alves, Jorianne Thyeska Castro; Morais, Ezequiel; Silva, André G M; Guimarães, Luís C; Azevedo, Vasco; Folador, Adriana Ribeiro Carneiro; Silva, Artur; Ramos, Rommel T J

    2016-01-01

    In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes. PMID:27516524

  13. Lack of effect of immunotherapy with BCG and Corynebacterium parvum on hepatic drug hydroxylation in man.

    PubMed Central

    Wan, H. H.; Thatcher, N.; Mullen, P. W.; Smith, G. N.; Wilkinson, P. M.

    1979-01-01

    Serial serum diphenylhydantoin and urinary 5-(p-hydroxphenyl)-5-phenylhydantoin concentrations were determined in 8 patients with malignant disease and 4 healthy volunteers on 2 separate occasions after an oral dose of diphenylhydantoin (500 mg). No significant difference was observed between metabolism before and 10 days after immunization with BCG or Corynebacterium parvum. Volunteers without intervening immunization similarly showed no difference. PMID:444399

  14. Nontoxigenic tox-bearing Corynebacterium ulcerans Infection among Game Animals, Germany

    PubMed Central

    Kutzer, Peter; Peters, Martin; Sing, Andreas; Contzen, Matthias; Rau, Jörg

    2014-01-01

    Corynebacterium ulcerans may cause diphtheria in humans and caseous lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from 13 game animals in Germany. Our results indicate a role for game animals as reservoirs for zoonotic C. ulcerans. PMID:24572455

  15. Successful treatment of Corynebacterium urealyticum encrusting cystitis with systemic and intravesical antimicrobial therapy

    PubMed Central

    Raab, Oriana; Béraud, Romain; Tefft, Karen M.; Muckle, C. Anne

    2015-01-01

    A 6-year-old Saint Bernard dog was diagnosed with encrusting cystitis caused by Corynebacterium urealyticum. The infection persisted despite the prolonged use of antimicrobials and surgical debridement of the urinary bladder. Resolution occurred following intravenous vancomycin, urine acidification, and intravesical gentamicin. The challenges involved in the treatment of encrusting cystitis are described. PMID:25969578

  16. Comparison of three techniques for isolation of Rhodococcus (Corynebacterium) equi from contaminated sources.

    PubMed

    Barton, M D; Hughes, K L

    1981-01-01

    Inoculation of a liquid medium comprised of Trypticase soy broth (BBL Microbiology Systems), cycloheximide, nalidixic acid, penicillin, and potassium tellurite and subcultured onto M3 medium plus potassium tellurite was highly successful for the isolation of Rhodococcus (Corynebacterium) equi from soil. PMID:7007424

  17. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 03-8664 Isolated from a Human Throat

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adonney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Corynebacterium ulcerans is an emergent pathogen infecting wild and domesticated animals worldwide that may serve as reservoirs for zoonotic infections. In this study, we present the draft genome of C. ulcerans strain 03-8664. The draft genome has 2,428,683 bp, 2,262 coding sequences, and 12 rRNA genes. PMID:27469956

  18. Microbial Transformation of Squalene: Terminal Methyl Group Oxidation by Corynebacterium sp

    PubMed Central

    Seo, Chull Won; Yamada, Yasuhiro; Takada, Nobuo; Okada, Hirosuke

    1983-01-01

    Corynebacterium sp. strain SY-79 was isolated from soil, using squalene as a carbon source. Microbiological properties of this bacterium are described. The metabolic product of this bacterium from squalene was identified as 2,6,10,15,19, 23-hexamethyl-2,6,10,14,18,22-tetracosahexaenedioic acid (squalenedioic acid). PMID:16346201

  19. An unusual etiological agent of implantable cardioverter device endocarditis: Corynebacterium mucifaciens.

    PubMed

    Kaya, Adnan; Tekkesin, Ahmet Ilker; Kalenderoglu, Koray; Alper, Ahmet Taha

    2016-01-01

    Cardiac pacing devices and implantable cardioverter defibrillator (ICD) are becoming the mainstay of therapy in cardiology and infective endocarditis (IE) and pocket infection; however, these devices require careful monitoring. Here, we describe a case of a 68-year-old female with an ICD presenting with a previously unknown etiological agent of IE, Corynebacterium mucifaciens. PMID:27133333

  20. Complete Genome Sequence of Corynebacterium minutissimum, an Opportunistic Pathogen and the Causative Agent of Erythrasma.

    PubMed

    Penton, Patricia K; Tyagi, Eishita; Humrighouse, Ben W; McQuiston, John R

    2015-01-01

    Corynebacterium minutissimum was first isolated in 1961 from infection sites of patients presenting with erythrasma, a common cutaneous infection characterized by a rash. Since its discovery, C. minutissimum has been identified as an opportunistic pathogen in immunosuppressed cancer and HIV patients. Here, we report the whole-genome sequence of C. minutissimum. PMID:25792058

  1. Complete Genome Sequence of Corynebacterium minutissimum, an Opportunistic Pathogen and the Causative Agent of Erythrasma

    PubMed Central

    Tyagi, Eishita; Humrighouse, Ben W.; McQuiston, John R.

    2015-01-01

    Corynebacterium minutissimum was first isolated in 1961 from infection sites of patients presenting with erythrasma, a common cutaneous infection characterized by a rash. Since its discovery, C. minutissimum has been identified as an opportunistic pathogen in immunosuppressed cancer and HIV patients. Here, we report the whole-genome sequence of C. minutissimum. PMID:25792058

  2. A case of costochondral abscess due to Corynebacterium minutissimum in an HIV-infected patient.

    PubMed

    Bandera, A; Gori, A; Rossi, M C; Degli Esposti, A; Ferrario, G; Marchetti, G; Tocalli, L; Franzetti, F

    2000-07-01

    Corynebacterium minutissimum, known as the causative agent of erythrasma, has recently been reported as a clinically significant pathogen in the immunocompromised host. We report for the first time the possible involvement of a multidrug-resistant C. minutissimum strain in a costochondral abscess occurring in an HIV-infected patient. PMID:11041706

  3. Corynebacterium minutissimum pyelonephritis with associated bacteraemia: a case report and review of literature.

    PubMed

    Ahmad, Nasir M; Ahmad, Khalid M

    2005-12-01

    We report a case of Corynebacterium minutissimum pyelonephritis with associated bacteraemia in an immunocompetent adult. The patient was successfully treated with a 14-day course of intravenous vancomycin. We review the clinical features of all the reported cases of invasive C. minutissimum infections with bacteraemia. PMID:16321644

  4. Pancreatic panniculitis complicated by infection with Corynebacterium tuberculostearicum: A case report

    PubMed Central

    Omland, S.H.; Ekenberg, C.; Henrik-Nielsen, R.; Friis-Møller, A.

    2014-01-01

    We present a case of pancreatic panniculitis in a patient with alcohol abuse where Corynebacterium tuberculostearicum was isolated from a pannicular nodule on the crus. The patient was started on linezolid treatment leading to regression of the patient's symptoms. Upon discontinuation of linezolid treatment progression of the skin symptoms progressed. PMID:26955524

  5. Contact Lens-Related Infectious Keratitis with White Plaque Formation Caused by Corynebacterium propinquum

    PubMed Central

    Eguchi, Hiroshi; Yamada, Norihiro; Sodeyama, Hirotake; Hosoya, Ryuichi; Kishi, Shoji

    2015-01-01

    We report the first case of Corynebacterium propinquum keratitis in the compromised cornea of a diabetic patient wearing therapeutic contact lenses. The strain was identified to the species level based on sequencing of the 16S rRNA gene and RNA polymerase β-subunit-encoding gene (rpoB). Ophthalmologists should be aware of nondiphtherial corynebacterial infection of compromised corneas. PMID:26179302

  6. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain PA01, Isolated from Sheep in Pará, Brazil

    PubMed Central

    Alves, Jorianne T. C.; Veras, Adonney A. O.; Cavalcante, Ana Lídia Q.; de Sá, Pablo H. C. G.; Dias, Larissa M.; Guimarães, Luis C.; Morais, Ezequiel; Silva, André G. M.; Azevedo, Vasco; Ramos, Rommel T. J.; Silva, Artur

    2016-01-01

    Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis disease. In this work, we present the first complete genome sequence of Corynebacterium pseudotuberculosis strain PA01, isolated in northern Brazil from an infected sheep. The genome length is 2,337,920 bp, and 2,003 coding sequences (CDS), 12 rRNAs, and 49 tRNAs were predicted. PMID:26823595

  7. Identification of Non-diphtheriae Corynebacterium by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

    PubMed Central

    Alatoom, Adnan A.; Cazanave, Charles J.; Cunningham, Scott A.; Ihde, Sherry M.

    2012-01-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum. PMID:22075579

  8. Identification of non-diphtheriae corynebacterium by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Alatoom, Adnan A; Cazanave, Charles J; Cunningham, Scott A; Ihde, Sherry M; Patel, Robin

    2012-01-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum. PMID:22075579

  9. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  10. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  11. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  12. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  13. Exudative pharyngitis possibly due to Corynebacterium pseudodiphtheriticum, a new challenge in the differential diagnosis of diphtheria.

    PubMed Central

    Izurieta, H. S.; Strebel, P. M.; Youngblood, T.; Hollis, D. G.; Popovic, T.

    1997-01-01

    Corynebacterium pseudodiphtheriticum has rarely been reported to cause disease in humans, despite its common presence in the flora of the upper respiratory tract. We report here a case of exudative pharyngitis with pseudomembrane possibly caused by C. pseudodiphtheriticum in a 4-year-old girl. The case initially triggered clinical and laboratory suspicion of diphtheria. Because C. pseudodiphtheriticum can be easily confused with Corynebacterium diphtheriae in Gram stain, clarification of its role in the pathogenesis of exudative pharyngitis in otherwise healthy persons is of public health importance. Simple and rapid screening tests to differentiate C. pseudodiphtheriticum from C. diphtheriae should be performed to prevent unnecessary concern in the community and unnecessary outbreak control measures. PMID:9126447

  14. Isolation of Corynebacterium tuscaniae sp. nov. from Blood Cultures of a Patient with Endocarditis

    PubMed Central

    Riegel, Philippe; Creti, Roberta; Mattei, Romano; Nieri, Alfredo; von Hunolstein, Christina

    2006-01-01

    A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genus Corynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars. Acid production from glucose and maltose, pyrazinamidase and alkaline phoshatase activities, and hippurate hydrolysis were the most characteristic phenotypic features of the bacterium. On the basis of both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium tuscaniae sp. nov. The type strain, ISS-5309, has been deposited in the American Type Culture Collection (ATCC BAA-1141) and in the Culture Collection of the University of Göteborg (CCUG 51321). PMID:16455875

  15. [Pitted keratolysis of hyperkeratotic form and isolation of the etiologic agent: Corynebacterium sp].

    PubMed

    Conti Díaz, I A; Cestau de Peluffo, I; Civila, E; Calegari, L; Sanabria, D; Viegas, M C

    1987-01-01

    Two cases of "pitted keratolysis" with a very accentuated plantar hyperkeratosis, and the isolation on chocolate thelurite agar of the presumptive etiologic agent, Corynebacterium sp., is presented. In order to keep permanently in mind, for a proper diagnosis, the original description of the disease as "keratoma plantare sulcatum" (Castellani, 1910), we are proposing to distinguish two different clinical forms: The hyperkeratotic one and the common or usual form of "pitted keratolysis" with keratolysis as the main sign. The prosecution of our biochemical studies with a significant number of strains isolated from both "pitted keratolysis" and from classical erythrasma cases, will surely permit us to definitively determine if all of them should or not be assimilated to Corynebacterium minutissimum. PMID:3309502

  16. Corynebacterium faecale sp. nov., isolated from the faeces of Assamese macaque.

    PubMed

    Chen, Xiu; Li, Gui-Ding; Li, Qin-Yuan; Hu, Cai-Juan; Liu, Cheng-Bin; Jiang, Yi; Jiang, Cheng-Lin; Han, Li; Huang, Xue-Shi

    2016-07-01

    A Gram-stain-positive, facultatively anaerobic, short rod-shaped, oxidase-negative and non-motile novel strain, designated YIM 101505T, was isolated from the faeces of a primate, Assamese macaque, and was studied to determine its taxonomic position. The cell wall contained meso-diaminopimelic acid and short-chain mycolic acids. Whole cell sugars were mannose, galactose and arabinose as major components. The major fatty acids (>10 %) were C18 : 1ω9c, C16 : 0 and C17 : 1ω8c and the major menaquinone was MK-9(H2). The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, glycolipid and six unidentified lipids. The new isolate shared most of the typical chemotaxonomic characteristics of members of the genus Corynebacterium. The closest related species was Corynebacterium efficiens based on 16S rRNA gene (98.1 % similarity) and partial rpoB gene (91.4 % similarity) sequences. Similarities with other species of this genus were below 97 % based on the 16S rRNA gene. The DNA-DNA hybridization value between YIM 101505T and C. efficiens DSM 44549T was 47.7±3.6 %. Moreover, the physiological and biochemical characteristics of YIM 101505T and C. efficiens DSM 44549T were different. Thus, strain YIM 101505T is considered to represent a novel member of the genus Corynebacterium, for which the name Corynebacterium faecale sp. nov. is proposed. The type strain is YIM 101505T (=DSM 45971T=CCTCC AB 2013226T). PMID:27073837

  17. Complete Genome Sequence of the Attenuated Corynebacterium pseudotuberculosis Strain T1.

    PubMed

    Almeida, Sintia; Loureiro, Dan; Portela, Ricardo W; Mariano, Diego C B; Sousa, Thiago J; Pereira, Felipe L; Dorella, Fernanda A; Carvalho, Alex F; Moura-Costa, Lilia F; Leal, Carlos A G; Figueiredo, Henrique C; Meyer, Roberto; Azevedo, Vasco

    2016-01-01

    We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes. PMID:27609922

  18. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain E19, Isolated from a Horse in Chile

    PubMed Central

    Cavalcante, Ana Lídia Q.; Dias, Larissa M.; Alves, Jorianne T. C.; Veras, Adonney A. O.; Guimarães, Luis C.; Rocha, Flávia S.; Gala-García, Alfonso; Ramos, Rommel T. J.; Azevedo, Vasco; Silva, Artur

    2015-01-01

    Corynebacterium pseudotuberculosis is related to several diseases infecting horses and small ruminants, causing economic losses to agribusiness. Here, we present the genome sequence of C. pseudotuberculosis strain E19. The genome includes one circular chromosome 2,367,956 bp (52.1% G+C content), with 2,112 genes predicted, 12 rRNAs, and 48 tRNAs. PMID:26607893

  19. Identification of Some Charcoal-Black-Pigmented CDC Fermentative Coryneform Group 4 Isolates as Rothia dentocariosa and Some as Corynebacterium aurimucosum: Proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum

    PubMed Central

    Daneshvar, Maryam I.; Hollis, Dannie G.; Weyant, Robbin S.; Jordan, Jean G.; MacGregor, John P.; Morey, Roger E.; Whitney, Anne M.; Brenner, Don J.; Steigerwalt, Arnold G.; Helsel, Leta O.; Raney, Patti M.; Patel, Jean B.; Levett, Paul N.; Brown, June M.

    2004-01-01

    Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum. PMID

  20. Complete Genome Sequence and Annotation of Corynebacterium singulare DSM 44357, Isolated from a Human Semen Specimen

    PubMed Central

    Merten, Madlen; Brinkrolf, Karina; Albersmeier, Andreas; Kutter, Yvonne; Rückert, Christian

    2015-01-01

    Corynebacterium singulare DSM 44357 is a urease-positive microorganism isolated from human semen. The complete genome sequence of C. singulare DSM 44357 comprises 2,830,519 bp with a mean G+C content of 60.12% and 2,581 protein-coding genes. The deduced antibiotic resistance pattern of this strain includes macrolides, lincosamides, aminoglycosides, chloramphenicol, and tetracyline. PMID:25814602

  1. Whole-Genome Sequence of Corynebacterium pseudotuberculosis 262 Biovar equi Isolated from Cow Milk

    PubMed Central

    Araújo, Carlos Leonardo de A.; Dias, Larissa M.; Veras, Adonney A. O.; Alves, Jorianne T. C.; Cavalcante, Ana Lídia Q.; Dowson, Christopher G.; Azevedo, Vasco; Ramos, Rommel T. J.; Silva, Artur

    2016-01-01

    We report the complete genome sequence of Corynebacterium pseudotuberculosis 262, isolated from a bovine host. C. pseudotuberculosis is an etiological agent of diseases with medical and veterinary relevance. The genome contains 2,325,749 bp, 52.8% G+C content, 2,022 coding sequences (CDS), 50 pseudogenes, 48 tRNAs, and 12 rRNAs. PMID:27013052

  2. Pyrolysis patterns of 5 close Corynebacterium species analyzed by artificial neural networks.

    PubMed

    Voisin, Sébastien; Terreux, Raphaël; Renaud, François N R; Freney, Jean; Domard, Monique; Deruaz, Daniel

    2004-05-01

    In the present study, an artificial neural network was trained with the Stuttgart Neural Networks Simulator, in order to identify Corynebacterium species by analyzing their pyrolysis patterns. An earlier study described the combination of pyrolysis, gas chromatography and atomic emission detection we used on whole cell bacteria. Carbon, sulfur and nitrogen were detected in the pyrolysis compounds. Pyrolysis patterns were obtained from 52 Corynebacterium strains belonging to 5 close species. These data were previously analyzed by Euclidean distances calculation followed by Unweighted Pair Group Method of Averages, a clustering method. With this early method, strains from 3 of the 5 species (C. xerosis, C. freneyi and C. amycolatum) were correctly characterized even if the 29 strains of C. amycolatum were grouped into 2 subgroups. Strains from the 2 remaining species (C. minutissimum and C. striatum) cannot be separated. To build an artificial neural network, able to discriminate the 5 previous species, the pyrolysis data of 42 selected strains were used as learning set and the 10 remaining strains as testing set. The chosen learning algorithm was Back-Propagation with Momentum. Parameters used to train a correct network are described here, and the results analyzed. The obtained artificial neural network has the following cone-shaped structure: 144 nodes in input, 25 and 9 nodes in 2 successive hidden layers, and then 5 outputs. It could classify all the strains in their species group. This network completes a chemotaxonomic method for Corynebacterium identification. PMID:15028867

  3. Corynebacterium pseudogenitalium sp. nov. Commensals of the human male and female urogenital tracts.

    PubMed

    Furness, G; Sambury, S; Evangelista, A T

    1979-01-01

    Antisera to Corynebacterium genitalium Types C-1 to C-6 were prepared in rabbits and the titers of complement fixing antibodies to the homologous strains, to the heterologous strains, to C. genitalium Types I to V, and to the reference species Corynebacterium xerosis and Corynebacterium minutissimum ascertained. Five Types stimulated low levels of cross-reacting antibodies to all corynebacteria tested including Type C-3. In contrast the antiserum to Type C-3 had antibodies to only two heterologous strains suggesting that these corynebacteria usually shared more than one minor cell wall antigen. The biologic reactions and serotypes of C. genitalium Types C-1 to C-6 have been compared with those of Types I to V. It is considered that C. genitalium should be retained for corynebacteria having the properties of Types I to V whereas corynebacteria having the characteristics of Types C-1 to C-6 that are commensals of the male and female urogenital tracts should be incorporated in a new species Corynebacteria pseudogenitalium sp. nov. The differences in the biologic characteristics of the two species have been discussed and summarized. PMID:429123

  4. Virulence Factor Genes Detected in the Complete Genome Sequence of Corynebacterium uterequi DSM 45634, Isolated from the Uterus of a Maiden Mare.

    PubMed

    Rückert, Christian; Kriete, Martin; Jaenicke, Sebastian; Winkler, Anika; Tauch, Andreas

    2015-01-01

    The complete genome sequence of the type strain Corynebacterium uterequi DSM 45634 from an equine urogenital tract specimen comprises 2,419,437 bp and 2,163 protein-coding genes. Candidate virulence factors are homologs of DIP0733, DIP1281, and DIP1621 from Corynebacterium diphtheriae and of sialidase precursors from Trueperella pyogenes and Chlamydia trachomatis. PMID:26227590

  5. Complete Genome Sequence of Corynebacterium ureicelerivorans DSM 45051, a Lipophilic and Urea-Splitting Isolate from the Blood Culture of a Septicemia Patient

    PubMed Central

    Tippelt, Anna; Albersmeier, Andreas; Brinkrolf, Karina; Rückert, Christian; Fernández-Natal, Isabel; Soriano, Francisco

    2014-01-01

    Corynebacterium ureicelerivorans is an opportunistic pathogen with a lipophilic lifestyle and an exceptionally high urease activity. The genome sequence of the type strain revealed that lipophilism is caused by the lack of a fatty acid synthase gene. The ureABCEFGD genes are similar to the urease gene region of Corynebacterium glucuronolyticum. PMID:25414509

  6. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  7. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  8. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  9. Intraspecific Variation of Unusual Phospholipids from Corynebacterium spp. Containing a Novel Fatty Acid

    PubMed Central

    Niepel, Tanja; Meyer, Holger; Wray, Victor; Abraham, Wolf-Rainer

    1998-01-01

    The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132T and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132T and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches. PMID:9721308

  10. Metabolism of tetralin (1,2,3,4-tetrahydronaphthalene) in Corynebacterium sp. strain C125

    SciTech Connect

    Sikkema, J.; Bont, J.A.M. de )

    1993-02-01

    Tetralin, widely used as a solvent in the petrochemical industry and in paints and waxes, degrades slowly in mixed cultures of microorganisms or in the presence of cosubstrates. This study reports on the metabolism of tetralin in the o-xylene-isolated Corynebacterium sp. strain C125. The researchers found that this organism attacks tetralin by an initial oxidation of the aromatic nucleus at positions C-5 and C-6 and they propose a four step inducible degradation pathway for tetralin starting at that point. The presence of the pathway makes this bacteria an excellent catalyst for the specific production of special cis-dihydro diols.

  11. Corynebacterium minutissimum bacteremia and meningitis: a case report and review of literature.

    PubMed

    Dalal, Aman; Likhi, Rishi

    2008-01-01

    Corynebacterium minutissimum, the causative agent of erythrasma, is a gram-positive, non-spore forming, aerobic or facultative anaerobic bacillus. It has rarely been associated with extracutaneous disease, since its description in 1961. A computerized medline search for review of literature was performed. To our knowledge, there have been 18 cases of C. minutissimum infections that caused conditions other than erythrasma. These include reports of cases of abscess formation, intravascular catheter-related bacteremias, ophthalmologic involvement, endocarditis, peritonitis, cutaneous granulomas, pyelonephritis in an infant and primary bacteremia with underlying hematologic malignancy. We report a rare case of bacteremia and meningitis due to C. minutissimum successfully treated with intravenous ampicillin. PMID:18036665

  12. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  13. Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

    PubMed

    Schmitt, M P

    1997-02-01

    Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases. PMID:9006041

  14. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  15. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 04-7514, Isolated from a Dog in France

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adooney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Carneiro, Adriana R.; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Here, we present the draft genome of toxigenic Corynebacterium ulcerans strain 04-7514. The draft genome has 2,497,845 bp, 2,059 coding sequences, 12 rRNA genes, 46 tRNA genes, 150 pseudogenes, 1 clustered regularly interspaced short palindromic repeat (CRISPR) array, and a G+C content of 53.50%. PMID:27034487

  17. Draft Genome Sequence of Corynebacterium variabile Mu292, Isolated from Munster, a French Smear-Ripened Cheese.

    PubMed

    Dugat-Bony, Eric; Sarthou, Anne-Sophie; Loux, Valentin; Vidal, Marie; Bonnarme, Pascal; Irlinger, Françoise; Layec, Séverine

    2016-01-01

    Here, we report the draft genome sequence of Corynebacterium variabile Mu292, which was originally isolated from the surface of Munster, a French smear-ripened cheese. This genome investigation will improve our knowledge on the molecular determinants potentially involved in the adaptation of this strain during the Munster-type cheese manufacturing process. PMID:27445372

  18. In Vitro Susceptibility of Equine-Obtained Isolates of Corynebacterium pseudotuberculosis to Gallium Maltolate and 20 Other Antimicrobial Agents

    PubMed Central

    Batista, M.; Lawhon, S. D.; Zhang, S.; Kuskie, K. R.; Swinford, A. K.; Bernstein, L. R.; Cohen, N. D.

    2014-01-01

    This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials. PMID:24829243

  19. Complete Genome Sequence of Corynebacterium camporealensis DSM 44610, Isolated from the Milk of a Manchega Sheep with Subclinical Mastitis.

    PubMed

    Rückert, Christian; Albersmeier, Andreas; Winkler, Anika; Tauch, Andreas

    2015-01-01

    Corynebacterium camporealensis has been isolated in pure culture from milk samples of dairy sheep affected by subclinical mastitis. The complete genome sequence of the type strain DSM 44610, recovered from milk of a Manchega sheep, comprises 2,451,810 bp with a mean G+C content of 59.41% and 2,249 protein-coding genes. PMID:26021938

  20. Draft Genome Sequence of Corynebacterium variabile Mu292, Isolated from Munster, a French Smear-Ripened Cheese

    PubMed Central

    Sarthou, Anne-Sophie; Loux, Valentin; Vidal, Marie; Bonnarme, Pascal; Irlinger, Françoise

    2016-01-01

    Here, we report the draft genome sequence of Corynebacterium variabile Mu292, which was originally isolated from the surface of Munster, a French smear-ripened cheese. This genome investigation will improve our knowledge on the molecular determinants potentially involved in the adaptation of this strain during the Munster-type cheese manufacturing process. PMID:27445372