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Sample records for black-pigmented corynebacterium aurimucosum

  1. Complete genome sequence and lifestyle of black-pigmented Corynebacterium aurimucosum ATCC 700975 (formerly C. nigricans CN-1 isolated from a vaginal swab of a woman with spontaneous abortion

    Directory of Open Access Journals (Sweden)

    Gartemann Karl-Heinz

    2010-02-01

    Full Text Available Abstract Background Corynebacterium aurimucosum is a slightly yellowish, non-lipophilic, facultative anaerobic member of the genus Corynebacterium and predominantly isolated from human clinical specimens. Unusual black-pigmented variants of C. aurimucosum (originally named as C. nigricans continue to be recovered from the female urogenital tract and they are associated with complications during pregnancy. C. aurimucosum ATCC 700975 (C. nigricans CN-1 was originally isolated from a vaginal swab of a 34-year-old woman who experienced a spontaneous abortion during month six of pregnancy. For a better understanding of the physiology and lifestyle of this potential urogenital pathogen, the complete genome sequence of C. aurimucosum ATCC 700975 was determined. Results Sequencing and assembly of the C. aurimucosum ATCC 700975 genome yielded a circular chromosome of 2,790,189 bp in size and the 29,037-bp plasmid pET44827. Specific gene sets associated with the central metabolism of C. aurimucosum apparently provide enhanced metabolic flexibility and adaptability in aerobic, anaerobic and low-pH environments, including gene clusters for the uptake and degradation of aromatic amines, L-histidine and L-tartrate as well as a gene region for the formation of selenocysteine and its incorporation into formate dehydrogenase. Plasmid pET44827 codes for a non-ribosomal peptide synthetase that plays the pivotal role in the synthesis of the characteristic black pigment of C. aurimucosum ATCC 700975. Conclusions The data obtained by the genome project suggest that C. aurimucosum could be both a resident of the human gut and possibly a pathogen in the female genital tract causing complications during pregnancy. Since hitherto all black-pigmented C. aurimucosum strains have been recovered from female genital source, biosynthesis of the pigment is apparently required for colonization by protecting the bacterial cells against the high hydrogen peroxide concentration in

  2. Black-pigmented sputum

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    Rafael Martínez-Girón

    2013-01-01

    Full Text Available Black-pigmented sputum, also called "melanoptysis," is a symptom that may be observed in certain pathologies such us coal workers′ pneumoconiosis (anthracosis. The cavitation and liquefaction of a fibrosed mass by an infectious process (tuberculosis, infections by anaerobes, etc. or by ischemic necrosis may cause expectoration of a blackish secretion. We report the case of a patient with labor precedents as a coal worker, from whom abundant black sputum was obtained in the course of an incidental expectoration. Cyto-histological findings are shown and a differential diagnosis is established.

  3. Black-pigmented Bacteroides spp. in the human oral cavity.

    OpenAIRE

    Zambon, J J; Reynolds, H S; Slots, J

    1981-01-01

    Five healthy children under 6 years of age, five healthy adults, and 10 adult periodontitis patients were examined for the prevalence and distribution of black-pigmented Bacteroides in the oral cavity. A total of 13 samples was obtained from each individual, including four supragingival and four subgingival dental plaques, dental occlusal surface, buccal mucosa, dorsal tongue, tonsil, and whole saliva. Black-pigmented Bacteroides were recovered from nine adult periodontitis patients. Healthy ...

  4. In vitro activity of tigecycline and 10 other antimicrobials against clinical isolates of the genus Corynebacterium.

    Science.gov (United States)

    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

  5. Characterization of ancient Chinese pottery decorated with a black pigment

    International Nuclear Information System (INIS)

    The Yangshao type pottery, made about 6000 yrs ago, was investigated by X-ray diffraction (XRD), and confirmed to be composed of quartz, feldspar, muscovite and calcite. A black pigment on it was assumed to be (Mn, Fe)3O4 from Particle Induced X-ray Emission (PIXE) and XRD experiments. Firing temperature of the pottery was assumed to be less than 600 deg. C from a heating experiment of the fragment of the pottery

  6. Long-wave UV light fluorescence for identification of black-pigmented Bacteroides spp.

    OpenAIRE

    Slots, J; Reynolds, H S

    1982-01-01

    Black-pigmented Bacteroides strains were grown on blood agar, and the colonies were evaluated for fluorescence from long-wave UV light. Most test strains of Bacteroides melaninogenicus subsp. intermedius exhibited a brilliant red fluorescence. B. melaninogenicus subsp. melaninogenicus fluoresced mostly red-orange. Bacteroides asaccharolyticus showed a yellow or red fluorescence. The intensity of the Bacteroides fluorescence weakened when the black pigment of the colonies developed. In contras...

  7. Synthesis and Application of Carbon–Iron Oxide Microspheres’ Black Pigments in Electrophoretic Displays

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    Meng Xianwei

    2010-01-01

    Full Text Available Abstract Carbon–iron oxide microspheres’ black pigments (CIOMBs had been prepared via ultrasonic spray pyrolysis of aqueous solutions containing ferrous chloride and glucose. Due to the presence of carbon, CIOMBs not only exhibited remarkably acid resistance, but also could be well dispersed in both polar solvents and nonpolar solvent. Finally, dispersions of hollow CIOMBs in tetrachloroethylene had successfully been applied in electrophoretic displays.

  8. The Functional Significance of Black-Pigmented Leaves: Photosynthesis, Photoprotection and Productivity in Ophiopogon planiscapus ‘Nigrescens’

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    Hatier, Jean-Hugues B.; Clearwater, Michael J.; Gould, Kevin S.

    2013-01-01

    Black pigmented leaves are common among horticultural cultivars, yet are extremely rare across natural plant populations. We hypothesised that black pigmentation would disadvantage a plant by reducing photosynthesis and therefore shoot productivity, but that this trait might also confer protective benefits by shielding chloroplasts against photo-oxidative stress. CO2 assimilation, chlorophyll a fluorescence, shoot biomass, and pigment concentrations were compared for near isogenic green- and ...

  9. Black pigments of rock art: identification of inorganic and organic components by combining analytical techniques

    International Nuclear Information System (INIS)

    Archaeological samples are complex in composition since they generally comprise a mixture of materials submitted to deterioration factors largely dependent on the environmental conditions. Therefore, the integration of analytical tools such as TXRF, FT-IR and GC-MS can maximize the amount of information provided by the sample. Recently, two black rock art samples of camelid figures at Alero Hornillos 2, an archaeological site located near the town of Susques (Jujuy Province, Argentina), were investigated. TXRF technique, selected for inorganic information, as well as FT-IR and GC-MS were employed in order to discover inorganic and organic composition of the black pigments. (author)

  10. Unlinked Mendelian inheritance of red and black pigmentation in snakes: Implications for Batesian mimicry.

    Science.gov (United States)

    Davis Rabosky, Alison R; Cox, Christian L; Rabosky, Daniel L

    2016-04-01

    Identifying the genetic basis of mimetic signals is critical to understanding both the origin and dynamics of mimicry over time. For species not amenable to large laboratory breeding studies, widespread color polymorphism across natural populations offers a powerful way to assess the relative likelihood of different genetic systems given observed phenotypic frequencies. We classified color phenotype for 2175 ground snakes (Sonora semiannulata) across the continental United States to analyze morph ratios and test among competing hypotheses about the genetic architecture underlying red and black coloration in coral snake mimics. We found strong support for a two-locus model under simple Mendelian inheritance, with red and black pigmentation being controlled by separate loci. We found no evidence of either linkage disequilibrium between loci or sex linkage. In contrast to Batesian mimicry systems such as butterflies in which all color signal components are linked into a single "supergene," our results suggest that the mimetic signal in colubrid snakes can be disrupted through simple recombination and that color evolution is likely to involve discrete gains and losses of each signal component. Both outcomes are likely to contribute to the exponential increase in rates of color evolution seen in snake mimicry systems over insect systems. PMID:26959901

  11. Promoters of Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Nešvera, Jan; Guyonvarch, A.; Reyes, O.; Leblon, G.

    2003-01-01

    Roč. 104, - (2003), s. 311-323. ISSN 0168-1656 R&D Projects: GA AV ČR IPP1050128; GA ČR GA525/01/0916 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * promoters * transcriptional regulation Subject RIV: EE - Microbiology, Virology Impact factor: 2.543, year: 2003

  12. Chemical investigation on black pigments in the carved decoration of sixteenth century alabaster tombs from Zaragoza (Spain).

    Science.gov (United States)

    Pérez-Arantegui, Josefina; Ribechini, Erika; Pardos, Carlos; Colombini, Maria Perla

    2009-12-01

    An analytical protocol based on optical microscopy (OM), scanning electron microscopy (SEM) observation, energy-dispersive X-ray (EDX) analyses, analytical pyrolysis in the presence of hexamethyldisilazane followed by gas chromatographic/mass spectrometric analysis (Py-silylation-GC/MS) and gas chromatography/mass spectrometry (GC/MS) after alkaline hydrolysis, solvent extraction and trimethylsilylation was used to study the origin and nature of black pigments from the carved inscriptions of several panels of two alabaster tombs dated from the mid-sixteenth century. Optical microscopy and SEM observation showed the presence of an amorphous very dark-brown substance, from translucent to opaque. EDX analyses revealed that the samples were mainly made up of C and O, thus highlighting the organic nature of the material used in the inscriptions. Py-silylation-GC/MS and GC/MS analyses provided detailed molecular compositions, highlighting the presence of a wide range of compound classes including diterpenoid acids, tricyclic abietanes, mid- and long-chain monocarboxylic fatty acids, n-alkanols and nalkanes. The pyrograms, the chromatographic profiles and the presence of characteristic biomarkers indicated that a mixture of pine pitch and beeswax had been used to make the black inscriptions. PMID:19662388

  13. Spontaneous corynebacterium spondylodiskitis in an immunocompetent patient

    DEFF Research Database (Denmark)

    Sharifi, Guive; Hosseinzadeh Bakhtevari, Mehrdad; Nabizadeh, Naveed; Jabbari, Reza; Samadian, Mohammad; Rezaei, Omidvar

    2016-01-01

    Background Spontaneous Corynebacterium spondylodiskitis is an unusual diagnosis of spondylodiskitis, especially in healthy patients without any significant past medical history. Materials and methods We describe the case of a 78-year-old man with progressive low back pain for 3 months, irradiating...... Corynebacterium infection. Results One month after surgery, the pain diminished dramatically and the CRP titer diminished significantly. Conclusion Although cases are very rare, spontaneous Corynebacterium spondylodiskitis, with substantial invasion of the spine, may develop in patients lacking any history of...

  14. Skin Infections Due to Corynebacterium

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    Meltem Türkmen

    2010-03-01

    Full Text Available Corynebacteria are Gram-positive, non-sporulated, non-capsulated, aerobic diphtheroid bacteria accounting for nearly 50%of the natural skin biocene. This bacterial family is responsible for various skin diseases such as cutaneous diphteria, cromhydrosis, bromhydrosis but the most common of them are pitted keratolysis, trichobacteriosis and erythrasma. A warm and moist environment and poor hygiene are the predisposition factors for these three diseases. Although this skin diseases are seen more frequently, they usually mistaken for a mycotic infection by general practitioners, with subsequent antimycotic treatment. Here skin diseases compromised with Corynebacterium are presented with their demographic features and discussed on the basis of a literature review.

  15. Production of diphtheria toxin by selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis.

    OpenAIRE

    Wong, T P; Groman, N

    1984-01-01

    We determined the diphtheria toxin phenotype of specially selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (C. ovis). All produced proteins similar in size and immunological structure to diphtheria toxin. As with diphtheria toxin, they exhibited ADP-ribosylating activity, and their synthesis was regulated by iron.

  16. The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences

    OpenAIRE

    Kalinowski Jörn; Brinkrolf Karina; Brune Iris; Pühler Alfred; Tauch Andreas

    2005-01-01

    Abstract Background The genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is sca...

  17. Inhibition of Corynebacterium vaginale by metronidazole.

    Science.gov (United States)

    Smith, R F; Dunkelberg, W E

    1977-01-01

    Metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole], previously thought to only inhibit obligately anaerobic bacteria, was found in vitro, to inhibit the growth of 15 facultatively anaerobic strains of Corynebacterium vaginale (Haemophilus vaginalis) using agar disk diffusion and broth dilution methods. PMID:867202

  18. Sigma factors and promoters in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Nešvera, Jan

    2011-01-01

    Roč. 154, 2-3 (2011), s. 101-113. ISSN 0168-1656 R&D Projects: GA ČR GC204/09/J015 Institutional research plan: CEZ:AV0Z50200510 Keywords : Corynebacterium glutamicum * Sigma factors * Promoters Subject RIV: EE - Microbiology, Virology Impact factor: 3.045, year: 2011

  19. Promoters and Plasmid Vectors of Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Nešvera, Jan

    Vol. 23. Berlin,Heidelberg: Springer-Verlag, 2013 - (Yukawa, H.; Inui, M.), s. 51-88 ISBN 978-3-642-29856-1 R&D Projects: GA ČR GC204/09/J015 Institutional support: RVO:61388971 Keywords : gene expression * Corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  20. Construction of corynebacterium glutamicum strains overproducing valine

    Czech Academy of Sciences Publication Activity Database

    Elišáková, Veronika; Holátko, Jiří; Pátek, Miroslav

    2004, s. 119-120. [Meeting of Czech and Slovak Societies for Biochemistry and Molecular Biology /19./. Olomouc (CZ), 31.08.2004-03.09.2004] R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * valine Subject RIV: EE - Microbiology, Virology

  1. Corynebacterium haemolyticum infections in Sri Lanka.

    OpenAIRE

    Wickremesinghe, R. S.

    1981-01-01

    Corynebacterium haemolyticum infections are described for the first time in Sri Lanka. In a period of 2 years from 1978-80 C. haemolyticum was isolated from the pharynx of 9 patients wih tonsillitis and from local septic lesions in 7 other patients. Association with other pathogens was common. No patients had a rash. The properties of the isolates are described.

  2. The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences

    Directory of Open Access Journals (Sweden)

    Kalinowski Jörn

    2005-06-01

    Full Text Available Abstract Background The genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is scarce. Here, we apply a combination of bioinformatic tools and a comparative genomic approach to identify and characterize a set of conserved DNA-binding transcriptional regulators in the four corynebacterial genomes. Results A collection of 127 DNA-binding transcriptional regulators was identified in the C. glutamicum ATCC 13032 genome, whereas 103 regulators were detected in C. efficiens YS-314, 63 in C. diphtheriae NCTC 13129 and 55 in C. jeikeium K411. According to amino acid sequence similarities and protein structure predictions, the DNA-binding transcriptional regulators were grouped into 25 regulatory protein families. The common set of DNA-binding transcriptional regulators present in the four corynebacterial genomes consists of 28 proteins that are apparently involved in the regulation of cell division and septation, SOS and stress response, carbohydrate metabolism and macroelement and metal homeostasis. Conclusion This work describes characteristic features of a set of conserved DNA-binding transcriptional regulators present within the corynebacterial core genome. The knowledge on the physiological function of these proteins should not only contribute to our understanding of the regulation of gene expression but will also provide the basis for comprehensive modeling of transcriptional regulatory networks of these species.

  3. Corynebacterium ulcerans, an emerging human pathogen.

    Science.gov (United States)

    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

  4. Corynebacterium glutamicum promoters: a practical approach

    OpenAIRE

    Pátek, M. (Miroslav); Holátko, J. (Jiří); Busche, T.; Kalinowski, J; Nešvera, J. (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...

  5. Regulation of glutamate dehydrogenase in Corynebacterium glutamicum

    OpenAIRE

    Hänßler, Eva

    2008-01-01

    Die Glutamatdehydrogenase (GDH) aus Corynebacterium glutamicum, einem Actinomyceten mit herausragender biotechnologischer Bedeutung, befindet sich an einer wichtigen Position innerhalb des Stoffwechsels, da sie Stickstoffassimilation und den Zentralstoffwechsel verbindet. Unter Überschussbedingungen ist die GDH an der Ammoniumassimilation beteiligt und über das Substrat alpha-Ketoglutarat besteht eine direkte Verknüpfung zum Citrat Zyklus. Aufgrund der NADPH-Abhängigkeit kann weiterhin der po...

  6. Development and Validation of Corynebacterium DNA Microarrays

    OpenAIRE

    Loos, Andrea; Glanemann, Christoph; Willis, Laura B.; O'Brien, Xian M; Lessard, Philip A.; Gerstmeir, Robert; Guillouet, Stéphane; Sinskey, Anthony J.

    2001-01-01

    We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To es...

  7. Cloning vector system for Corynebacterium glutamicum.

    OpenAIRE

    Yoshihama, M; Higashiro, K; Rao, E A; Akedo, M; Shanabruch, W G; Follettie, M T; Walker, G C; Sinskey, A J

    1985-01-01

    A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. ...

  8. Engineering Corynebacterium glutamicum for isobutanol production

    OpenAIRE

    Smith, Kevin Michael; Cho, Kwang-Myung; Liao, James C

    2010-01-01

    The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of...

  9. Oxidation of acyclic terpenoids by Corynebacterium sp.

    OpenAIRE

    Yamada, Y; Seo, C W; Okada, H.

    1985-01-01

    Squalene analogs such as lycopersene, geranylfarnesyl, digeranyl, and 2-hydroxy-2,3-dihydrosqualene and terpene alcohol derivatives such as farnesyl benzyl ether, farnesyl pivalate, geranylgeranyl pivalate, geranyl pivalate, and geranyl benzyl ether were oxidized by Corynebacterium sp. strain SY-79, which was isolated from soil by using squalene as a carbon source. Lycopersene and geranylfarnesyl gave no major product. Digeranyl, geranyl benzyl ether, and geranyl pivalate gave terminal oxidat...

  10. Optimization of lysine metabolism in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Rytter, Jakob Vang

    Commercial pig and poultry production use the essential amino acid lysine as a feed additive with the purpose of optimizing the feed utilization. Lysine is produced by a fermentation process involving either Corynebacterium glutamicum or Escherichia coli. The global annual production is around 1......,000,000 tons. The aim of this project is to optimize the yield of lysine in C. glutamicum using metabolic engineering strategies. According to a genome scale model of C. glutamicum, theoretically there is much room for increasing the lysine yield (Kjeldsen and Nielsen 2009). Lysine synthesis requires NADPH...

  11. Lysine uptake and exchange in Corynebacterium glutamicum.

    OpenAIRE

    Bröer, S; Krämer, R

    1990-01-01

    Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in...

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

    OpenAIRE

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

    2010-01-01

    Crystallization of AcnR, a repressor of the aconitase gene in Corynebacterium glutamicum, is reported. Intentional manual scratching of the crystallization plates was applied to induce heterogeneous nucleation.

  13. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses

    OpenAIRE

    Fatma Mutlu Sariguzel; A. Nedret Koc; Gülhan Yagmur; Elife Berk

    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 wit...

  14. Implication ofCorynebacterium species in food’s contamination

    Institute of Scientific and Technical Information of China (English)

    Sana Alibi; Asma Ferjani; Jalel Boukadida

    2016-01-01

    Corynebacteriumspp. are part of the human microbiota. Recently, species of this genus are increasingly implicated in different types of infections especially in immunocompromized and hospitalized patients. The significance of the presence of the genusCorynebacterium in foods is not clearly established. These bacteria may be involved in spoilage or ripening of cheese and meats. This review focused on different researches concerning the implication of Corynebacterium species in food’s contamination.

  15. The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse.

    Science.gov (United States)

    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

  16. PRODUKSI L-LISIN OLEH GALUR MUTAN Corynebacterium glutamicum DENGAN MEMANFAATKAN MOLASE L-LYSINE PRODUCTION BY MUTANT STRAINS OF Corynebacterium glutamicum USING MOLASSES

    OpenAIRE

    Budiatman Satiawihardja; Erliza Noor*; Ahmad Haryo Oktamto 3

    2000-01-01

    The objective of this study was to obtain fermentation process for producing l-lysine using two mutans of Corynebacterium glutamicum. The process used a low cost natural raw material molasses as a carbon source which underwent a special treatment before its application for fermentation medium. Strain Corynebacterium glutamicum ATCC 21543 produced higher L-lysine as compared to Corynebacterium glutamicum ATCC 21513. ATCC strain 21543 produced using a 10.98 g/l L-lysine modified basal ...

  17. Synthetic promoter libraries for Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Rytter, Jakob Vang; Helmark, Søren; Chen, Jun;

    2014-01-01

    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 co...... 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....... 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...

  18. Antimicrobial resistance among Brazilian Corynebacterium diphtheriae strains

    Directory of Open Access Journals (Sweden)

    Gabriela Andrade Pereira

    2008-08-01

    Full Text Available The increasing problems with multidrug resistance in relation to Corynebacterium, including C. diphtheriae, are examples of challenges confronting many countries. For this reason, Brazilian C. diphtheriae strains were evaluated by the E-Test for their susceptibility to nine antibacterial drugs used in therapy. Resistance (MIC < 0.002; 0.38 µg/ml to penicillin G was found in 14.8% of the strains tested. Although erythromycin (MIC90 0.75 µg/ml and azithromycin (MIC90 0.064 µg/ml were active against C. diphtheriae in this study, 4.2% of the strains showed decreased susceptibility (MIC 1.0 µg/ml to erythromycin. Multiple resistance profiles were determined by the disk diffusion method using 31 antibiotics. Most C. diphtheriae strains (95.74% showed resistance to mupirocin, aztreonam, ceftazidime, and/or oxacillin, ampicillin, penicillin, tetracycline, clindamycin, lincomycin, and erythromycin. This study presents the antimicrobial susceptibility profiles of Brazilian C. diphtheriae isolates. The data are of value to practitioners, and suggest that some concern exists regarding the use of penicillin.

  19. Metabolic engineering Corynebacterium glutamicum to produce triacylglycerols.

    Science.gov (United States)

    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

  20. Tools for genetic manipulations in Corynebacterium glutamicum and their applications

    Czech Academy of Sciences Publication Activity Database

    Nešvera, Jan; Pátek, Miroslav

    2011-01-01

    Roč. 90, č. 5 (2011), s. 1641-1654. ISSN 0175-7598 R&D Projects: GA ČR GC204/09/J015 Institutional research plan: CEZ:AV0Z50200510 Keywords : Corynebacterium glutamicum * Plasmid vectors * Promoters Subject RIV: EE - Microbiology, Virology Impact factor: 3.425, year: 2011

  1. Influence of stringent response in Corynebacterium glutamicum on valine production

    Czech Academy of Sciences Publication Activity Database

    Prouza, Marek; Holátko, Jiří; Elišáková, Veronika; Nešvera, Jan; Pátek, Miroslav

    Praha: Verlag, 2006, s. 131-132. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * guanosine tetraphosphate synthetase Subject RIV: EE - Microbiology, Virology

  2. Screening of corynebacterium glutamicum promoters controlled by external stress factors

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Martina; Pátek, Miroslav; Nešvera, Jan

    Stará Lesná, 2002, s. -. [Biochemical Congress /18./. Stará Lesná (SK), 10.09.2002-13.09.2002] R&D Projects: GA ČR GA204/01/0998 Keywords : corynebacterium * glutamicum Subject RIV: EE - Microbiology, Virology

  3. Analysis of Corynebacterium glutamicum Promoters and Their Applications

    Czech Academy of Sciences Publication Activity Database

    Nešvera, Jan; Holátko, Jiří; Pátek, Miroslav

    Dordrecht: Springer Science + Business Media Dordrecht, 2012 - (Wang, X.; Chen, J.; Quinn, P.), s. 203-221 ISBN 978-94-007-5054-8 R&D Projects: GA ČR GC204/09/J015; GA ČR GPP302/12/P633 Institutional support: RVO:61388971 Keywords : RNA polymerase * Corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  4. 5. Regulation of Gene Expression.Handbook of Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav

    Boca Raton, FL: CRC Press, Taylor and Francis Group, 2005, s. 81-98. ISBN 0-8493-1821-1 R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z5020903 Keywords : gene expression * corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  5. Chromosomally encoded small antisense RNA in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Martina; Kadeřábková, Pavla; Pátek, Miroslav; Knoppová, Monika; Šilar, Radoslav; Nešvera, Jan

    2008-01-01

    Roč. 279, č. 2 (2008), s. 195-201. ISSN 0378-1097 R&D Projects: GA ČR GC204/07/J012 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * srna * antisense rna Subject RIV: EE - Microbiology, Virology Impact factor: 2.021, year: 2008

  6. Experimental transmission of Corynebacterium pseudotuberculosis in horses by house flies

    Science.gov (United States)

    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...

  7. Genome sequence of the marine bacterium Corynebacterium maris type strain Coryn-1T (= DSM 45190T)

    OpenAIRE

    Schaffert, Lena; Albersmeier, Andreas; Bednarz, Hanna; Niehaus, Karsten; Kalinowski, Jörn; Rückert, Christian

    2013-01-01

    Corynebacterium maris Coryn-1T 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 com...

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

    OpenAIRE

    KIM, KYUNG-JIN; Kim, Sujin; Lee, Sujin; Kang, Beom Sik; Lee, Heung-Soo; Oh, Tae-Kwang; Kim, Myung Hee

    2006-01-01

    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.

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

    International Nuclear Information System (INIS)

    Crystallization of AcnR, a repressor of the aconitase gene in Corynebacterium glutamicum, is reported. Intentional manual scratching of the crystallization plates was applied to induce heterogeneous nucleation. 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

  10. Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

    OpenAIRE

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

    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 s...

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

    OpenAIRE

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

    2013-01-01

    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...

  12. "THE FREQUENCY OF CORYNEBACTERIUM UREALYTICUM IN PATIENTS WITH RENAL STONES"

    OpenAIRE

    A. Bordji; Etemadi, H; D. Mehraban M.I. Qureshi

    2004-01-01

    Urinary stones are prevalent and Corynebacterium urealyticum, a urease-positive bacteria, is a frequent urinary tract pathogen. However, the association of this organism with urinary stones is not sufficiently studied in Iran. This study was carried out to determine the prevalence of C. urealyticum in patients with urinary stones referred to the urology sections of Tehran University of Medical Sciences. This study was performed using case-control method. A total of 150 urine samples from pati...

  13. Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

    OpenAIRE

    Ikeda, Masato; Miyamoto, Aya; Mutoh, Sumire; Kitano, Yuko; Tajima, Mei; Shirakura, Daisuke; Takasaki, Manami; Mitsuhashi, Satoshi; Takeno, Seiki

    2013-01-01

    To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid,...

  14. A functionally split pathway for lysine synthesis in Corynebacterium glutamicium.

    OpenAIRE

    Schrumpf, B; Schwarzer, A; Kalinowski, J.; Pühler, A; Eggeling, L; Sahm, H

    1991-01-01

    Three different pathways of D,L-diaminopimelate and L-lysine synthesis are known in procaryotes. Determinations of the corresponding enzyme activities in Escherichia coli, Bacillus subtilis, and Bacillus sphaericus verified the fact that in each of these bacteria only one of the possible pathways operates. However, in Corynebacterium glutamicum activities are present which allow in principle the use of the dehydrogenase variant and succinylase variant of lysine synthesis together. Applying ge...

  15. Anaerobes Wachstum von Corynebacterium glutamicum durch gemischte Säurefermentation

    OpenAIRE

    Michel, Andrea

    2014-01-01

    Corynebacterium glutamicum is able to grow under anaerobic conditions with nitrate aselectron acceptor, but only very low cell densities are attainable because of the toxic nitritethat cannot be efficiently metabolized by C. glutamicum. Furthermore it is known thatC. glutamicum is metabolically active under oxygen-free conditions and produces lactate,succinate, and acetate from glucose. However, fermentative anaerobic growth ofC. glutamicum has not been described, yet. In this work, the abili...

  16. Metabolic engineering of Corynebacterium glutamicum for production of glutamate derivatives

    OpenAIRE

    Vold Korgaard Jensen, Jaide

    2016-01-01

    The identification of Corynebacterium glutamicum as a glutamate producer in the 1950’s was the start of its career as an amino acid producer. C. glutamicum has now been employed as cell factory for industrial amino acid production for over five decades and has a market size to reach $20 billion by 2020. As C. glutamicum was isolated for its natural ability to produce glutamate it makes it an excellent chassis for engineering it to produce its derivatives ornithine, proline, put...

  17. Untranslated RNA of Corynebacterium glutamicum induced by heat shock

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Martina; Pátek, Miroslav; Nešvera, Jan

    Prague: Institute of Microbiology ASCR, Prague, Czech Republic, 2006, s. 93-93. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Grant ostatní: XE(XE) ESF project No. CZ.04.3.07/4.2.01.1/0039 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * rna Subject RIV: EE - Microbiology, Virology

  18. Multiple promoters of stress-responding genes of Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Nešvera, Jan; Šilar, Radoslav; Kadeřábková, Pavla; Zemanová, Martina; Pátek, Miroslav

    Badajoz: Formatex Research Center, 2009. s. 599-599. [BioMicroWorld 2009. International Conference on Environmental, Industrial and Applied Microbiology, Fostering Cross-disciplinary Applied Research in Microbiology and Microbial Biotechnology /3./. 02.12.2009-04.12.2009, Lisabon] R&D Projects: GA ČR GC204/07/J012 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  19. Large-Scale Engineering of the Corynebacterium glutamicum Genome

    OpenAIRE

    Suzuki, Nobuaki; Okayama, Satoshi; Nonaka, Hiroshi; Tsuge, Yota; Inui, Masayuki; Yukawa, Hideaki

    2005-01-01

    The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a to...

  20. The effect of hydrodynamic conditions in Corynebacterium glutamicum growth

    OpenAIRE

    J.A. Oliveira; Braga, Adelaide; Rocha, I.; Faria, Nuno Filipe Monteiro

    2015-01-01

    [Excerpt] Corynebacterium glutamicum is a facultative anaerobic, gram-positive bacterium with a GRAS status that grows fast and achieves high cell densities. C. glutamicum is commonly used in amino acids production, and is also able to convert sugars in organic acids (OA) and alcohols in specific conditions: anaerobic and limited-oxygen environments. In these conditions, the carbon metabolism is modified, namely the flux shifts from the pentose phosphate pathway to glycolysis and the TCA cycl...

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

    OpenAIRE

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

    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 re...

  2. PRODUKSI L-LISIN OLEH GALUR MUTAN Corynebacterium glutamicum DENGAN MEMANFAATKAN MOLASE L-LYSINE PRODUCTION BY MUTANT STRAINS OF Corynebacterium glutamicum USING MOLASSES

    Directory of Open Access Journals (Sweden)

    Budiatman Satiawihardja 1

    2000-04-01

    Full Text Available The objective of this study was to obtain fermentation process for producing l-lysine using two mutans of Corynebacterium glutamicum. The process used a low cost natural raw material molasses as a carbon source which underwent a special treatment before its application for fermentation medium. Strain Corynebacterium glutamicum ATCC 21543 produced higher L-lysine as compared to Corynebacterium glutamicum ATCC 21513. ATCC strain 21543 produced using a 10.98 g/l L-lysine modified basal B medium containing molasses which was equivalent to 20 % of original molasses in terms of its sugar content.

  3. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses

    Science.gov (United States)

    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

  4. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses

    Directory of Open Access Journals (Sweden)

    Fatma Mutlu Sariguzel

    2014-09-01

    Full Text Available 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.

  5. Genome Sequence of Corynebacterium glutamicum S9114, a Strain for Industrial Production of Glutamate

    OpenAIRE

    Lv, Yangyong; Wu, Zhanhong; Han, Shuangyan; Lin, Ying; Zheng, Suiping

    2011-01-01

    Here we report the genome sequence of Corynebacterium glutamicum S9114, an industrial producer widely used in production of glutamate in China. Preliminary comparison with the sequences of the Corynebacterium glutamicum strains ATCC 13032 and R revealed some notable mutagenesis that might be related to the high yield of glutamate.

  6. Corynebacterium accolens Isolated from Breast Abscess: Possible Association with Granulomatous Mastitis▿

    OpenAIRE

    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.

  7. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    OpenAIRE

    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 towar...

  8. Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

    OpenAIRE

    Billman-Jacobe, H; Wang, L.; Kortt, A; Stewart, D; Radford, A

    1995-01-01

    Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding s...

  9. Analysis of the Corynebacterium glutamicum dapA Promoter

    OpenAIRE

    Vašicová, Pavla; Pátek, Miroslav; Nešvera, Jan; Sahm, Hermann; Eikmanns, Bernhard

    1999-01-01

    Deletion and mutational analysis of the promoter P-dapA from Corynebacterium glutamicum was performed to identify regions and particular nucleotides important for its function. An extended −10 region and a stretch of six T’s at positions −55 to −50 were found to be the most important elements in the promoter function. The results of mutational analysis of P-dapA are consistent with the conclusions of statistical computer-aided analysis of 44 C. glutamicum promoter sequences.

  10. Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses

    OpenAIRE

    Pablo H.C.G. de Sá; Veras, Adonney A. O.; Carneiro, Adriana R.; Baraúna, Rafael A.; Luís C. Guimarães; Pinheiro, Kenny C.; Pinto, Anne C.; Soares, Siomar C.; Schneider, Maria P. C.; Vasco Azevedo; Artur Silva; Ramos, Rommel T. J.

    2015-01-01

    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...

  11. A microbiological and clinical review on Corynebacterium kroppenstedtii.

    Science.gov (United States)

    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

  12. Genome sequence of the marine bacterium Corynebacterium maris type strain Coryn-1(T) (= DSM 45190(T)).

    Science.gov (United States)

    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

  13. Experimental inoculation of house flies Musca domestica with Corynebacterium pseudotuberculosis serovar equi

    Science.gov (United States)

    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...

  14. Corynebacterium ulcerans in an Immunocompromised Patient with Diphtheria and Her Dog

    OpenAIRE

    Lartigue, Marie-Frédérique; Monnet, Xavier; Le Flèche, Anne; Grimont, Patrick A. D.; Benet, Jean-Jacques; Durrbach, Antoine; Fabre, Monique; Nordmann, Patrice

    2005-01-01

    Corynebacterium ulcerans causes zoonotic infections, such as diphtheria and extrapharyngeal infections. We report here the first case of a diphtheria-like illness caused by C. ulcerans in France and transmitted likely by a dog to an immunocompromised woman.

  15. Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies

    Science.gov (United States)

    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...

  16. Complete genome sequence of Corynebacterium pseudotuberculosis Cp31, isolated from an Egyptian buffalo

    DEFF Research Database (Denmark)

    Silva, Artur; Ramos, Rommel Thiago Jucá; Ribeiro Carneiro, Adriana; Cybelle Pinto, Anne; de Castro Soares, Siomar; Rodrigues Santos, Anderson; Silva Almeida, Sintia; Guimarães, Luis Carlos; Figueira Aburjaile, Flávia; Barbosa, Eudes; Alves Dorella, Fernanda; Souza Rocha, Flávia; Souza Lopes, Thiago; Kawasaki, Regiane; Gomes Sá, Pablo; da Rocha Coimbra, Nilson Antônio; Teixeira Cerdeira, Louise; Silvanira Barbosa, Maria; Cruz Schneider, Maria Paula; Miyoshi, Anderson; Selim, Salah Abdel Karim; Moawad, Mohamed Salah; Azevedo, Vasco

    2012-01-01

    Corynebacterium pseudotuberculosis is of major veterinary importance because it affects many animal species, causing economically significant livestock diseases and losses. Therefore, the genomic sequencing of various lines of this organism, isolated from different hosts, will aid in the developm......Corynebacterium pseudotuberculosis is of major veterinary importance because it affects many animal species, causing economically significant livestock diseases and losses. Therefore, the genomic sequencing of various lines of this organism, isolated from different hosts, will aid in the...

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

    Science.gov (United States)

    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

  18. Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses.

    Science.gov (United States)

    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

  19. Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses

    Directory of Open Access Journals (Sweden)

    Pablo H.C.G. de Sá

    2015-12-01

    Full Text Available 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 [2–4]. 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.

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

    Science.gov (United States)

    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

  1. Úlceras leishmanióticas cutâneas com presença de Corynebacterium diphtheriae Cutaneous leishmaniotic ulcers with Corynebacterium diphtheriae

    Directory of Open Access Journals (Sweden)

    Luis Angel Vera

    2002-08-01

    Full Text Available Em um estudo prospectivo para avaliar a influência da infecção bacteriana secundária na evolução da leishmaniose cutânea, em Corte de Pedra (Bahia, obteve-se o isolamento de Corynebacterium diphtheriae em 7(8,3% de 84 pacientes portadores de úlceras, avaliados. Devido ao pequeno número de pacientes com a presença da bactéria na úlcera, não foi possível concluir se Corynebacterium diphtheriae comporta-se apenas como colonizante, nem sobre a sua influência no processo de cicatrização da úlcera leishmaniótica.In a prospective study to evaluate the influence of secondary bacterial infection on the evaluation of cutaneous leishmaniasis, in Corte de Pedra (Bahia, we isolated Corynebacterium diphtheriae in 7 (8.3% out of 84 patients with ulcers studied. Due to the small number of patients with the presence of the bacteria in the ulcer, we could not conclude whether Corynebacterium diphtheriae behaves only as a colonizer nor its influence on the healing of the leishmaniotic ulcer.

  2. Diagnostic and experimental study of Corynebacterium renale isolated from urinary tract infection of cattle

    Directory of Open Access Journals (Sweden)

    S. A. Hussein

    2011-01-01

    Full Text Available The study includes isolation and identification of Corynebacterium renale from urine of cow apparently suffering from urinary tract infection. C. renale represent highest isolate 49. 99% followed by Corynebacterium pyogenes 24.24% from the total number of Corynebacterium 74.23%. on the other hand Staphylococcus saprophyticus also isolated from urine samples 25.75%. Since C. renale was isolated at highest rate we studied its pathogenesis via inoculation of isolate intraperitoneally into white Swiss mice. Results showed that C. renale type I has ability to produce kidney damage after 48 hr. post inoculation revealed embolic glomeruler nephritis with less number of C. renale, also there is infiltration of polymorphnuclear inflammatory cell and nephrosis, in addition to vacular degeneration, coagulative necrosis with blood vessel congestion in liver tissue.

  3. A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

    Directory of Open Access Journals (Sweden)

    Scrivens James H

    2011-01-01

    Full Text Available Abstract Background Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA in sheep and goats. Results An optimized protocol of three-phase partitioning (TPP was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93 of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  8. 3-Methyl-1-butanol Biosynthesis in an Engineered Corynebacterium glutamicum.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  12. Isolamento de Corynebacterium diphtheriae de líquido espermático Isolation of Corynebacterium diphtheriae from sperm

    Directory of Open Access Journals (Sweden)

    Thaís Lisbôa Machado

    1989-06-01

    Full Text Available Descrevemos o isolamento de Corynebacterium diphtheriae toxígeno de espermocultura. O microrganismo foi identificado pelo teste de fluorescência sob luz ultravioleta, pesquisa da enzima pirazina-carboxilamidase (Pyz, testes de virulência in vitro e in vivo (imunodifusão radial simples, cultura de células e teste intradérmico em cobaio. A amostra foi inicialmente considerada atoxígena pelo teste de imunodifusão radial simples, mas sua virulência foi observada posteriormente quando os testes acima foram aplicados. Sem adecuada especificação, a amostra poderia ter sido considerada como um "difteróide".The isolation of tosigenic Corynebacterum diphtheriae from sperm is reported. The organism was identified through the investigation of fluorescence under the UV light, the presence of pirazinecarboxilamidase enzyme (Pyz, in vitro and in vivo and virulence methods (single radial immunodiffusion, cell culture, guiena pig intradermic test. The strain was initially cosnsidered montoxinogenic by single radial immunodiffusion, but its virulence was observed afterwards, when we applied the tests already mentioned. The strain could be considered a "Diphtheroid" without adequate specification.

  13. Úlceras leishmanióticas cutâneas com presença de Corynebacterium diphtheriae Cutaneous leishmaniotic ulcers with Corynebacterium diphtheriae

    OpenAIRE

    Luis Angel Vera; Vanize de Oliveira Macêdo; Albino Verçosa Magalhães; Isolina Allen Ciuffo; Conceição Guerra Santos; João Barberino Santos

    2002-01-01

    Em um estudo prospectivo para avaliar a influência da infecção bacteriana secundária na evolução da leishmaniose cutânea, em Corte de Pedra (Bahia), obteve-se o isolamento de Corynebacterium diphtheriae em 7(8,3%) de 84 pacientes portadores de úlceras, avaliados. Devido ao pequeno número de pacientes com a presença da bactéria na úlcera, não foi possível concluir se Corynebacterium diphtheriae comporta-se apenas como colonizante, nem sobre a sua influência no processo de cicatrização da úlcer...

  14. Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

    OpenAIRE

    Gubler, M; Jetten, M.; Lee, S. H.; Sinskey, A J

    1994-01-01

    The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on mini...

  15. Detection of Differences in the Nucleotide and Amino Acid Sequences of Diphtheria Toxin from Corynebacterium diphtheriae and Corynebacterium ulcerans Causing Extrapharyngeal Infections

    OpenAIRE

    Sing, Andreas; Hogardt, Michael; Bierschenk, Suse; Heesemann, Jürgen

    2003-01-01

    While Corynebacterium ulcerans can mimic classical diphtheria, extrapharyngeal infections are extremely rare. Sequencing of the diphtheria toxin (DT)-encoding tox gene of two C. ulcerans isolates from extrapharyngeal infections revealed differences from C. diphtheriae DT sequences, mainly in the translocation and receptor-binding domains. C. ulcerans supernatants were much less potent than supernatant from C. diphtheriae. A C. ulcerans DT-specific PCR is described below.

  16. METODE CEPAT EKSTRAKSI DNA Corynebacterium diphtheriae UNTUK PEMERIKSAAN PCR

    Directory of Open Access Journals (Sweden)

    Sunarno Sunarno

    2014-10-01

    Full Text Available AbstractDiagnosis of diphtheria caused byCorynebacterium diphtheriaeshould be done immediately since delay of therapy may cause 20-fold increase rate of death. One method of rapid diagnostic to identify diphtheria is by using polymerase chain reaction (PCR. The fundamental issue of this method depends on the DNA, either its quality or quantity. The simple DNA extraction method, which is using mechanical/physical principles with a little of chemical reagents (such as boiling method and the use of sodium hydroxide (NAOH, will have some benefits, such as easy to be performed, low cost, fast, and environmentally friendly. This study aimed to evaluate effectivity and efficiency of boiling method with NaOH to extract DNA of C. diphtheriae compared to the use of a commercial diagnostic kit for PCR assay. We used C. diphtheriae toxygenic(NCTC 10648 isolates, which are grown in blood agar plates. We then prepared the suspensions of cell/colony in aquadest with several dilutions. Each dilution was extracted using boiling method, NaOH and controlled with the use of a commercial diagnostic kit (QiAmp DNA Minikit. The results were evaluated quantitatively with spectrophotometer and qualitatively with gel electrophoresis. The results showed that the extracted DNA from boiling method with NaOH has an adequate quality and quantity for PCR assay (up to 9 CFU/uL cell/reaction. Therefore, it can be summarized that boiling method with NaOH is effective and efficient to be applied in PCR assay forC. diphtheriae.Key words: boiling extraction method, NaOH, C.diphtheriae, PCRAbstrakKematian kasus difteri yang disebabkan oleh Corynebacterium diphtheriaedapat meningkat 20 kali lipat karena keterlambatan pengobatan sehingga penegakan diagnosis harus dilakukan sesegera mungkin. Salah satu metode diagnostik yang cukup cepat untuk mendeteksi penyakit difteri adalah pemeriksaan polymerase chain reaction(PCR. Keberhasilan pemeriksaan PCR dipengaruhi oleh kualitas dan kuantitas

  17. Regulons of global transcription factors in Corynebacterium glutamicum.

    Science.gov (United States)

    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

  18. Activity of disinfectants and biofilm production of Corynebacterium pseudotuberculosis

    Directory of Open Access Journals (Sweden)

    Maria da C.A. Sá

    2013-11-01

    Full Text Available To verify the occurrence of caseous lymphadenitis in sheep and goats on farms of Pernambuco, Brazil, and in animals slaughtered in two Brazilian cities (Petrolina/PE and Juazeiro/BA, and to characterize the susceptibility profile of Corynebacterium pseudotuberculosis to disinfectants and antimicrobials, and its relationship with biofilm production were the objectives of this study. 398 samples were tested for sensitivity to antimicrobial drugs, disinfectants, and biofilm production. Among the 108 samples collected on the properties, 75% were positive for C. pseudotuberculosis. Slaughterhouse samples indicated an occurrence of caseous lymphadenitis in 15.66% and 6.31% for animals slaughtered in Petrolina and Juazeiro respectively. With respect to antimicrobials, the sensitivity obtained was 100% for florfenicol and tetracycline; 99.25% for enrofloxacin, ciprofloxacin and lincomycin; 98.99% for cephalothin; 98.74% for norfloxacin and sulfazotrim; 97.74% for gentamicin; 94.22% for ampicillin; 91.71% for amoxicillin; 91.21% for penicillin G; 89.19% for neomycin and 0% for novobiocin. In analyzes with disinfectants, the efficiency for chlorhexidine was 100%, 97.20% for quaternary ammonium, 87.40% for chlorine and 84.40% for iodine. 75% of the isolates were weak or non-biofilm producers. For the consolidated biofilm, found that iodine decreased biofilm formation in 13 isolates and quaternary ammonia in 11 isolates. The reduction of the biofilm formation was observed for iodine and quaternary ammonium in consolidated biofilm formation in 33% and 28% of the isolates, respectively. The results of this study highlight the importance of establishing measures to prevent and control the disease.

  19. The use of SDS-polyacrylamide gel electrophoresis in epidemiological studies of Corynebacterium diphtheriae.

    OpenAIRE

    Hallas, G.

    1988-01-01

    Polyacrylamide gel electrophoresis of cell proteins was investigated as a possible typing method for Corynebacterium diphtheriae. A method was developed using stock strains which were representatives of the five gravis serotypes described by Robinson & Peeney (1936). This technique was then applied to recent isolates sent to our laboratory for identification.

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

    OpenAIRE

    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.

  1. Draft Genome Sequence of Corynebacterium ulcerans Strain 04-3911, Isolated from Humans

    Science.gov (United States)

    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

  2. In Silico Genome-Scale Reconstruction and Validation of the Corynebacterium glutamicum Metabolic Network

    DEFF Research Database (Denmark)

    Kjeldsen, Kjeld Raunkjær; Nielsen, J.

    2009-01-01

    A genome-scale metabolic model of the Gram-positive bacteria Corynebacterium glutamicum ATCC 13032 was constructed comprising 446 reactions and 411 metabolite, based on the annotated genome and available biochemical information. The network was analyzed using constraint based methods. The model w...

  3. Pancreatic panniculitis complicated by infection with Corynebacterium tuberculostearicum: A case report

    Directory of Open Access Journals (Sweden)

    S.H. Omland

    2014-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  5. Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum▿

    OpenAIRE

    Becker, Judith; Klopprogge, Corinna; Schröder, Hartwig; Wittmann, Christoph

    2009-01-01

    In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.

  6. Analysis of Corynebacterium glutamicum promoters and their use for construction of amino acid-producing strains

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Holátko, Jiří; Šilar, Radoslav; Kadeřábková, Pavla; Nešvera, Jan

    Varanasi: Banaras Hindu University, 2009. s. 52-52. [International Conference on Emerging Trends in Biotechnology. 04.12.2009-06.12.2009, Varanasi] Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  7. Involvement of alternative sigma factors in expression of stress responding genes in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Šilar, Radoslav; Kadeřábková, Pavla; Zemanová, Martina; Pátek, Miroslav; Nešvera, Jan

    Sevilla: Formatex, 2007, s. 559-559. [BioMicroWorld – 2007. Sevilla (ES), 28.11.2007-01.12.2007] R&D Projects: GA ČR GC204/07/J012 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * sigma factors * promoters Subject RIV: EE - Microbiology, Virology

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

    OpenAIRE

    Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa; KIM, KYUNG-JIN

    2007-01-01

    PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH2PO4 and K2HPO4 as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline.

  9. Plasmid Vectors for Testing In Vivo Promoter Activities in Corynebacterium glutamicum and Rhodococcus erythropolis

    Czech Academy of Sciences Publication Activity Database

    Knoppová, Monika; Phensaijai, M.; Veselý, Martin; Zemanová, Martina; Nešvera, Jan; Pátek, Miroslav

    2007-01-01

    Roč. 55, - (2007), s. 234-239. ISSN 0343-8651 R&D Projects: GA ČR GA526/04/0542; GA ČR GA204/06/0330 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium * rhodoccoccus * promoter-probe vectors Subject RIV: EE - Microbiology, Virology Impact factor: 1.167, year: 2007

  10. Control of rep Gene Expression in Plasmid pGA1 from Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Venkova, Tatiana; Pátek, Miroslav; Nešvera, Jan

    2003-01-01

    Roč. 185, č. 8 (2003), s. 2402-2409. ISSN 0021-9193 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/01/0998 Institutional research plan: CEZ:AV0Z5020903 Keywords : plasmid pga1 * corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology Impact factor: 4.175, year: 2003

  11. The role of sigma factor SigH in transcriptional regulation in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Šilar, Radoslav; Busche, T.; Kadeřábková, Pavla; Pátek, Miroslav; Kalinowski, J.; Nešvera, Jan

    Bratislava: Československá spoločnosť mikrobiologická, 2010. s. 202-202. ISBN 970-80-970477-8-8 R&D Projects: GA ČR GC204/09/J015 Institutional research plan: CEZ:AV0Z50200510 Keywords : Corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

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

    Science.gov (United States)

    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

  13. Is the expression of the leua gene in corynebacterium glutamicum controlled by transcriptional attenuation?

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Hochmannová, Jitka; Dolečková, K.; Nešvera, Jan

    - (2002), s. 1-410. [Biochemický Zjazd /18./. Stará Lesná, 10.09.2002-13.09.2002] R&D Projects: GA ČR GA525/01/0916 Keywords : leua gene * corynebacterium * glutamicum Subject RIV: EE - Microbiology, Virology

  14. Searching for Corynebacterium glutamicum promoters regulated differently in various growth phases

    Czech Academy of Sciences Publication Activity Database

    Phensaijai, M.; Knoppová, Monika; Pátek, Miroslav; Nešvera, Jan

    Praha: Springer, 2006, s. 82-82. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Grant ostatní: XE(XE) CZ.04.3.07/4.2.01.1/0039 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * promoters Subject RIV: EE - Microbiology, Virology

  15. Genetic modifications in biosynthesis pathways of branched-chain-amino acids in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Holátko, Jiří; Elišáková, Veronika

    Praha: Verlag, 2006, s. 142-142. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * genetic modifications Subject RIV: EE - Microbiology, Virology

  16. Function of Corynebacterium glutamicum promoters in Eschrichia coli, Streptomyces lividans, and Baccillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Muth, G.; Wohlleben, W.

    2003-01-01

    Roč. 104, - (2003), s. 325-334. ISSN 0168-1656 R&D Projects: GA AV ČR IPP1050128; GA ČR GA525/01/0916 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * escherichia coli * promoters Subject RIV: EE - Microbiology, Virology Impact factor: 2.543, year: 2003

  17. Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Elišáková, Veronika; Prouza, Marek; Sobotka, Miroslav; Nešvera, Jan; Pátek, Miroslav

    2009-01-01

    Roč. 139, č. 3 (2009), s. 203-210. ISSN 0168-1656 R&D Projects: GA ČR GA204/06/0330 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * valine production * promoters Subject RIV: EE - Microbiology, Virology Impact factor: 2.881, year: 2009

  18. Negative regulatory function of the Rep rotein of the plasmid pGA1 from Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Elišáková, Veronika; Pátek, Miroslav; Nešvera, Jan

    Kanoni, 2004, s. 86-87. [Plasmid Biology 2004. Corfu (GR), 15.09.2004-21.09.2004] Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * rep protein Subject RIV: EE - Microbiology, Virology

  19. Negative regulatory function of the Rep protein of the plasmid pGA1 from Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Elišáková, Veronika; Pátek, Miroslav; Nešvera, Jan

    2004, s. 153. [Plasmid Biology 2004. Corfu (GR), 15.09.2004-21.09.2004] R&D Projects: GA ČR GA525/04/0548 Keywords : corynebacterium glutamicum * plasmid pga1 Subject RIV: EE - Microbiology, Virology

  20. Comparative Complete Genome Sequence Analysis of the Amino Acid Replacements Responsible for the Thermostability of Corynebacterium efficiens

    OpenAIRE

    Nishio, Yousuke; Nakamura, Yoji; Kawarabayasi, Yutaka; Usuda, Yoshihiro; Kimura, Eiichiro; Sugimoto, Shinichi; Matsui, Kazuhiko; Yamagishi, Akihiko; Kikuchi, Hisashi; Ikeo, Kazuho; Gojobori, Takashi

    2003-01-01

    Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40°C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our compar...

  1. The Zur regulon of Corynebacterium glutamicum ATCC 13032

    Directory of Open Access Journals (Sweden)

    Jochmann Nina

    2010-01-01

    Full Text Available Abstract 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

  2. Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

    OpenAIRE

    Luciene de Fátima Costa Torres; Dayana Ribeiro; Raphael Hirata Jr; Luis Gustavo Carvalho Pacheco; Monica Cristina de Souza; Louisy Sanches dos Santos; Cíntia Silva dos Santos; Mohammad Salah; Mateus Matiuzzi da Costa; Marcio Garcia Ribeiro; Selim, Salah A; Vasco Ariston de Carvalho Azevedo; Ana Luiza Mattos-Guaraldi

    2013-01-01

    Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultan...

  3. Screening for Corynebacterium diphtheriae and Corynebacterium ulcerans in patients with upper respiratory tract infections 2007-2008: a multicentre European study.

    LENUS (Irish Health Repository)

    Wagner, K S

    2011-04-01

    Diphtheria is now rare in most European countries but, when cases do arise, the case fatality rate is high (5-10%). Because few countries continue to routinely screen for the causative organisms of diphtheria, the extent to which they are circulating amongst different European populations is largely unknown. During 2007-2008, ten European countries each screened between 968 and 8551 throat swabs from patients with upper respiratory tract infections. Six toxigenic strains of Corynebacterium diphtheriae were identified: two from symptomatic patients in Latvia (the country with the highest reported incidence of diphtheria in the European Union) and four from Lithuania (two cases, two carriers); the last reported case of diphtheria in Lithuania was in 2002. Carriage rates of non-toxigenic organisms ranged from 0 (Bulgaria, Finland, Greece, Ireland, Italy) to 4.0 per 1000 (95% CI 2.0-7.1) in Turkey. A total of 28 non-toxigenic strains were identified during the study (26 C. diphtheriae, one Corynebacterium ulcerans, one Corynebacterium pseudotuberculosis). The non-toxigenic C. ulcerans strain was isolated from the UK, the country with the highest reported incidence of cases due to C. ulcerans. Of the eleven ribotypes detected, Cluj was seen most frequently in the non-toxigenic isolates and, amongst toxigenic isolates, the major epidemic clone, Sankt-Petersburg, is still in circulation. Isolation of toxigenic C. diphtheriae and non-toxigenic C. diphtheriae and C. ulcerans in highly-vaccinated populations highlights the need to maintain microbiological surveillance, laboratory expertise and an awareness of these organisms amongst public health specialists, microbiologists and clinicians.

  4. In vitro activity of 79 antimicrobial agents against Corynebacterium group D2.

    OpenAIRE

    García-Rodriguez, J A; García Sánchez, J E; Muñoz Bellido, J L; Nebreda Mayoral, T; García Sánchez, E; García García, I

    1991-01-01

    Corynebacterium group D2 (CGD2) is involved in urinary tract infections in patients with underlying predisposing factors. This microorganism is highly resistant to a number of antimicrobial agents. We tested the activities of 79 antimicrobial agents against CGD2. beta-Lactams, aminoglycosides, and macrolides were ineffective. Fluorinated quinolones showed irregular activities, ofloxacin being the most active one. Doxycycline, rifampin, and mainly glycopeptides (vancomycin and teicoplanin) wer...

  5. Fermentative production of the diamine putrescine: systems metabolic engineering of *Corynebacterium glutamicum*

    OpenAIRE

    Nguyen, Anh Q. D.; Jens Schneider; Gajendar Komati Reddy; 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 po...

  6. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    OpenAIRE

    Wittmann Christoph; Klopprogge Corinna; Becker Judith

    2008-01-01

    Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruv...

  7. Corynebacterium macginleyi: A cause of ventilator associated pneumonia in an immunocompromised patient

    OpenAIRE

    Jad Kebbe; Jeffery Mador, M

    2015-01-01

    Purpose and Importance: Corynebacterium macginleyi, a lipophilic diphtheroid from the genus Corynebacteria, is a known cause of conjunctivitis. It was recently reported as a cause of serious infections in immunocompromised individuals. It has never been reported as a cause of ventilator-associated pneumonia, that which carries a high burden and risk of mortality. Our report intends to increase awareness of a potentially lethal nosocomial bacterial infection. Observations: This case reports...

  8. Development of thiosine-resistant mutant of corynebacterium glutamicum and its potency for lysine production

    International Nuclear Information System (INIS)

    A new strain of corynebacterium glutamicum was isolated and treatment by UV (Ultraviolet) radiation for 30 second at 15 cm from UV germicidal lamp. As a result, thiosine-resistant mutants were isolated and screened for lysine production. The potent mutant produced 22 g/L L. lysine in glucose salt medium, 20 g/L L. lysine in molasses and 15 g/L L. lysine in starch hydrolyzates medium, in stirred tank fermenter at 30 degree centigrade after 4 days. (author)

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

    OpenAIRE

    Binder, Stephan; Siedler, S.; Marienhagen, J; Bott, M; Eggeling, L

    2013-01-01

    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-ce...

  10. Studies on polar cell wall growth and antibiotic susceptibility of Corynebacterium glutamicum

    OpenAIRE

    Sieger, Boris

    2015-01-01

    Corynebacterium glutamicum is a Gram positive soil bacterium with high industrial importance in ton scale production of amino acids. Apart from that, it becomes more and more important for medical studies, where it serves as model organism due to its close relation to bacteria causing several pathogens such as tuberculosis, diphtheria and leprosy. C. glutamicum, like Mycobacterium tuberculosis, has a distinct cell wall which is composed of a peptidoglycan layer (murein) with covalently bo...

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

    Science.gov (United States)

    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

  12. Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

    OpenAIRE

    Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

    2002-01-01

    Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overe...

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

    OpenAIRE

    Volker Fritz Wendisch; Steffen Nikolaus Lindner; Ahmed Zahoor

    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 t...

  14. Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose

    OpenAIRE

    Kiefer, Patrick; Heinzle, Elmar; Zelder, Oskar; Wittmann, Christoph

    2004-01-01

    A comprehensive approach to 13C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drasti...

  15. Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production▿

    OpenAIRE

    Kabus, Armin; Niebisch, Axel; Bott, Michael

    2006-01-01

    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 ...

  16. Development of thiosine-resistant mutant of corynebacterium glutamicum and its potency for lysine production

    International Nuclear Information System (INIS)

    A new strain of corynebacterium glutamicum was isolated and treated by UV (Ultraviolet) radiation for 30 second at 15 cm from UV germicidal lamp. As a result, thiosine-resistant mutants were isolated and screened for lysine production. The potent mutant produced 22 g/L L. lysine in glucose salt medium, 20 g/L L. lysine in molasses and 15 g/L L. lysine in starch hydrolyzate medium, in stirred tank fermenter at 30 degree centigrade after 4 days. (author)

  17. Phosphotransferase System-Mediated Glucose Uptake Is Repressed in Phosphoglucoisomerase-Deficient Corynebacterium glutamicum Strains

    OpenAIRE

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

    2013-01-01

    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 product...

  18. Coevolutionary Analysis Enabled Rational Deregulation of Allosteric Enzyme Inhibition in Corynebacterium glutamicum for Lysine Production ▿

    OpenAIRE

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

    2011-01-01

    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 deregul...

  19. Thick Juice-Based Production of Amino Acids and Putrescine by *Corynebacterium glutamicum*

    OpenAIRE

    Meiswinkel, Tobias; Lindner, Steffen; Wendisch, Volker F.

    2014-01-01

    Thick juice (also regarded as syrup) is an intermediate product of sugar processing. It is cheaper than processed sugar and is mainly composed of sucrose. Sucrose is a preferred carbon source of Corynebacterium glutamicum, a workhorse of biotechnology used for million-ton-scale amino acid production. Here, it is shown for C. glutamicum that sugar beet thick juice led to higher growth rates and faster carbon source consumption than pure sucrose. Comparative DNA microarray analysis revealed dif...

  20. Phenotypic characterization of Corynebacterium glutamicum using elementary modes towards synthesis of amino acids

    OpenAIRE

    Radhakrishnan, Devesh; Rajvanshi, Meghna; Venkatesh, K. V.

    2010-01-01

    Elementary flux mode (EFM) analysis is a powerful tool to represent the metabolic network structure and can be further utilized for flux analysis. The method enables characterization and quantification of feasible phenotypes in microbes. EFM analysis was employed to characterize the phenotype of Corynebacterium glutamicum to yield various amino acids. The metabolic network of C. glutamicum yielded 62 elementary modes by incorporating the accumulation of amino acids namely, lysine, alanine, va...

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

    OpenAIRE

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

    2013-01-01

    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 inhibitio...

  2. Analysis of the biotechnological production of 2-ketomethylvalerate with Corynebacterium glutamicum

    OpenAIRE

    Hagmann, Christian Thomas

    2012-01-01

    The current work deals with the biotechnological synthesis of alpha-ketomethylvalerate (KMV) with Corynebacterium glutamicum. KMV together with the two other branched-chain alpha-keto acids alpha-ketoisovalerate (KIV) and alpha-ketoisocaproate (KIC) is used as a pharmaceutical agent and also as ingredient of functional food. So far KMV has only been produced by chemical synthesis and the aim of this work was to initiate the development of a fermentative KMV production. When C. glutamicum was ...

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

    OpenAIRE

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

    2015-01-01

    Abstract 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; ac...

  4. Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

    OpenAIRE

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

    2014-01-01

    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 ...

  5. Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum▿

    OpenAIRE

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

    2008-01-01

    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 ...

  6. Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases▿

    OpenAIRE

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

    2011-01-01

    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 i...

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

    OpenAIRE

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

    2012-01-01

    Summary 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 h...

  8. Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis

    OpenAIRE

    Shioya Suteaki; Nagahisa Keisuke; Furusawa Chikara; Fujimura Koki; Shirai Tomokazu; Shimizu Hiroshi

    2007-01-01

    Abstract Background Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overprod...

  9. Improved L-lysine production in Corynebacterium glutamicum by rational strain engineering

    OpenAIRE

    Schiefelbein, Sarah

    2014-01-01

    The soil bacterium Corynebacterium glutamicum is the major organism for the production of the amino acid L-lysine, an important nutrient in animal feedstock. This study investigated new strategies for bioprocess and genetic engineering of C. glutamicum towards production of L-lysine. In aerobic cultivations, routinely performed in shake-flasks, the dissolved oxygen concentration is a critical often neglected parameter. Here, oxygen mass transfer was determined in disposable shake-flasks u...

  10. Production of Native-Type Streptoverticillium mobaraense Transglutaminase in Corynebacterium glutamicum

    OpenAIRE

    Date, Masayo; Yokoyama, Kei-ichi; Umezawa, Yukiko; Matsui, Hiroshi; Kikuchi, Yoshimi

    2003-01-01

    We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a...

  11. The antisense RNA involved in replication control of the plasmid pGAl from Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Venkova, Tatiana; Pátek, Miroslav; Espinosa, M.; Nešvera, Jan

    Berlin: European Commission DGXII, 2001. s. 451-53. [Symposium of the EU-concerted action on "Mobile genetic elements` contribution to bacterial adaptability and diversity" /3./. 21.09.2001-25.09.2001, Berlin] R&D Projects: GA ČR GA204/01/0998 Institutional research plan: CEZ:AV0Z5020903 Keywords : Corynebacterium glutamicum * RCR replicons Subject RIV: EE - Microbiology, Virology

  12. L-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Blombach, B.; Schreiner, M. E.; Holátko, Jiří; Bartek, T.; Oldiges, M.; Eikmanns, B. J.

    2007-01-01

    Roč. 73, č. 7 (2007), s. 2079-2084. ISSN 0099-2240 Grant ostatní: DE(DE) 04NR004/22000404 Institutional research plan: CEZ:AV0Z50200510 Source of funding: N - neverejné zdroje Keywords : corynebacterium glutamicum * plasmid-bound overexpression * fed-batch fermentations Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2007

  13. Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

    OpenAIRE

    2013-01-01

    In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contr...

  14. A Corynebacterium glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli.

    OpenAIRE

    Jäger, W; Kalinowski, J.; Pühler, A

    1997-01-01

    A chromosomal DNA fragment from the erythromycin-sensitive bacterium Corynebacterium glutamicum ATCC 13032 was shown to mediate resistance against erythromycin, tetracycline, puromycin, and bleomycin in Escherichia coli. Multicopy cloning of the fragment did not cause a resistance phenotype in C. glutamicum. The corresponding gene encodes a hydrophobic protein with 12 potential transmembrane-spanning ex-helical segments showing similarity to drug-H+ antiporters.

  15. Destabilized eYFP variants for dynamic gene expression studies in Corynebacterium glutamicum

    OpenAIRE

    Hentschel, Eva; Will, Cornelia; Mustafi, Nurije; Burkovski, Andreas; Rehm, Nadine; Frunzke, Julia

    2013-01-01

    Fluorescent reporter proteins are widely used for the non-invasive monitoring of gene expression patterns, but dynamic measurements are hampered by the extremely high stability of GFP and homologue proteins. In this study, we used SsrA-mediated peptide tagging for the construction of unstable variants of the GFP derivative eYFP (enhanced yellow fluorescent protein) and applied those for transient gene expression analysis in the industrial platform organism Corynebacterium glutamicum.

  16. Glutamate Dehydrogenase Is Not Essential for Glutamate Formation by Corynebacterium glutamicum

    OpenAIRE

    Kholy, Elke R. Börmann-El; Eikmanns, Bernhard J.; Gutmann, Marcella; Sahm, Hermann

    1993-01-01

    Two Corynebacterium glutamicum strains, one being glutamate dehydrogenase (GDH) negative and the other possessing 11-fold-higher specific GDH activity than the parental wild type, were constructed and used to analyze the role of GDH in C. glutamicum. The results indicate (i) that GDH is dispensable for glutamate synthesis required for growth and (ii) that although a high level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.

  17. Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

    OpenAIRE

    Billman-Jacobe, H; Hodgson, A L; Lightowlers, M; Wood, P. R.; Radford, A J

    1994-01-01

    Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small frac...

  18. Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.

    OpenAIRE

    Follettie, M T; Sinskey, A J

    1986-01-01

    The pheA gene of Corynebacterium glutamicum encoding prephenate dehydratase was isolated from a gene bank constructed in C. glutamicum. The specific activity of prephenate dehydratase was increased six-fold in strains harboring the cloned gene. Genetic and structural evidence is presented which indicates that prephenate dehydratase and chorismate mutase were catalyzed by separate enzymes in this species. The C. glutamicum pheA gene, subcloned in both orientations with respect to the Escherich...

  19. Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Elišáková, Veronika; Pátek, Miroslav; Holátko, Jiří; Nešvera, Jan; Leyval, D.; Goergen, J.-L.; Delaunay, S.

    2005-01-01

    Roč. 71, č. 1 (2005), s. 207-213. ISSN 0099-2240 R&D Projects: GA ČR GA525/01/0916; GA ČR GA525/04/0548 Grant ostatní: VALPAN(XE) QLK3-2000-00497 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * ahas Subject RIV: EE - Microbiology, Virology Impact factor: 3.818, year: 2005

  20. Mycothiol peroxidase MPx protects Corynebacterium glutamicum against acid stress by scavenging ROS

    OpenAIRE

    Tietao Wang; Fen Gao; Yiwen Kang; Chao Zhao; Muhang Li; Xihui Shen; Tao Su; Meiru Si

    2015-01-01

    Corynebacterium glutamicum mycothiol peroxidase (MPx) is a novel CysGPx family peroxidase that uses both the mycoredoxin and thioredoxin reducing systems as proton donors for peroxide detoxification. In this study, we revealed that MPx is also important for cellular survival under acid stress. A Δmpx mutant exhibited significantly decreased resistance to acid stress and markedly increased accumulation of reactive oxygen species (ROS) and protein carbonylation levels in vivo. Overexpression of...

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

    OpenAIRE

    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. Alr...

  2. MODELING THE FERMENTATIVE PRODUCTION OF L-GLUTAMIC ACID BY CORYNEBACTERIUM GLUTAMICUM IN A BATCH BIOREACTOR

    Directory of Open Access Journals (Sweden)

    N. S. Khan

    2013-01-01

    Full Text Available The fermentation kinetics of L-glutamic acid by Corynebacterium glutamicum was studied in a batch bioreactor. Mathematical model using the logistic equation for growth, Leudeking-Piret kinetic equation for product formation and Leudeking-Piret like equation for substrate consumption was proposed. Based on the analysis of experimental data followed by computer simulation, the model seemed to provide a reasonable description for L-glutamic acid fermentation.

  3. Flocculation of fine fluorite particles with Corynebacterium xerosis and commercial long chain polymers

    Directory of Open Access Journals (Sweden)

    Rigo Lisandra N.

    2002-01-01

    Full Text Available This work aimed to study, comparatively, the flocculation of fluorite particles with Corynebacterium xerosis cells and three commercial long chain polymers. Best flocculation results were obtained with cells of C. xerosis and with an anionic polyacrylamide. Both were effective in solids removal and water clarification, although flocculation with C. xerosis cells requires a higher dosage of reagent per mass unit of processed ore.

  4. Bioremediation of refinery wastewater using immobilised Burkholderia cepacia and Corynebacterium sp and their transconjugants

    OpenAIRE

    Abdullahi T. Ajao; Sabo E. Yakubu; Veronica J. Umoh; Joseph B. Ameh

    2013-01-01

    When oil spill occurs, it poses serious toxic hazards to all forms of life. Mixed culture of Burkholderia cepacia and Corynebacterium sp isolated from refinery sludge using selective enrichment technique was used for bioremediation of refinery wastewater in a laboratoryscale bioreactor. Physicochemical parameters of both raw and treated water were as determined and compared with Federal Environ - mental Protection Agency (FEPA-limit, Abuja, Nigeria) to asses the efficiency of the bioremediati...

  5. Selection and Characterization of a Lysine Yielding Mutant of Corynebacterium glutamicum - a Soil Isolate from Pakistan

    OpenAIRE

    Habib-ur-Rehman§٭, Abdul Hameed and Safia Ahmed

    2012-01-01

    L-lysine is the second limiting amino acid for poultry and supplemented in broiler feed for optimal performance. Lysine can be produced by inducing mutation in glutamate producing bacteria. The study was conducted to enhance lysine production from a local strain of Corynebacterium glutamicum. The bacterium was mutated by exposure to UV. Mutants resistant to s-2-aminoethyle L-cystein (AEC) and showing auxotrophy for L-homoserine were screened for lysine production qualitatively and quantitativ...

  6. REGULATION OF L-VALINE BIOSYNTHESIS IN CORYNEBACTERIUM GLUTAMICUM - ASPECTS OF PHYSIOLOGY AND BIOCHEMISTRY

    OpenAIRE

    Ilze Deniņa

    2010-01-01

    ANNOTATION This study investigates the optimum cellular physiology and intracellular biochemistry to achieve L-valine overproduction by Corynebacterium glutamicum recombinant strains. A transition in the bacterial growth rate to below maximum was found to be an optimum parameter of cellular physiology to increase L-valine synthesis rate by C. glutamicum during batch and fed-batch cultivations. The increase in acetohydroxyacid synthase activity was the determinant for the ...

  7. Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

    OpenAIRE

    Hasegawa, Satoshi; Suda, Masako; Uematsu, Kimio; Natsuma, Yumi; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2013-01-01

    We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gen...

  8. Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

    OpenAIRE

    Elišáková, Veronika; Pátek, Miroslav; Holátko, Jiří; Nešvera, Jan; Leyval, Damien; Goergen, Jean-Louis; Delaunay, Stéphane

    2005-01-01

    Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesi...

  9. Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection.

    Science.gov (United States)

    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

  10. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

    OpenAIRE

    Nesvera, J; Pátek, M; Hochmannová, J; Abrhámová, Z; Becvárová, V; Jelínkova, M; Vohradský, J

    1997-01-01

    The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheria...

  11. [Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum].

    Science.gov (United States)

    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

  12. Detection of Corynebacterium bovis infection in athymic nude mice from a research animal facility in Korea

    OpenAIRE

    Kim, Tae-Hyoun; Kim, Dong-Su; Han, Ju-Hee; Chang, Seo-Na; Kim, Kyung-Sul; Seok, Seung-Hyeok; Kim, Dong-Jae; Park, Jong-Hwan; Park, Jae-Hak

    2014-01-01

    Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, thi...

  13. Corynebacterium glutamicum as a Host for Synthesis and Export of d-Amino Acids▿

    OpenAIRE

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

    2011-01-01

    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-substr...

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

    Science.gov (United States)

    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

  15. Acetohydroxyacid Synthase, a Novel Target for Improvement of l-Lysine Production by Corynebacterium glutamicum▿ †

    OpenAIRE

    Blombach, Bastian; Hans, Stephan; Bathe, Brigitte; Eikmanns, Bernhard J.

    2008-01-01

    The influence of acetohydroxy acid synthase (AHAS) on l-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower Km for the substrate pyruvate and an about fourfold-lower Vmax; (ii) a slightly increased Km for the substrate α-ketobutyrate with...

  16. Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

    OpenAIRE

    Wittmann, Christoph; Kiefer, Patrick; Zelder, Oskar

    2004-01-01

    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 [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively hig...

  17. Reengineering of a Corynebacterium glutamicum l-Arginine and l-Citrulline Producer▿

    OpenAIRE

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

    2009-01-01

    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, argG92up, 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...

  18. Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems

    OpenAIRE

    Bröer, Stefan; Eggeling, Lothar; Krämer, Reinhard

    1993-01-01

    The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to...

  19. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.

    OpenAIRE

    Reinscheid, D J; Eikmanns, B J; Sahm, H

    1991-01-01

    From a Corynebacterium glutamicum mutant possessing a homoserine dehydrogenase resistant to feedback inhibition by L-threonine, the corresponding gene (homFBR) was analyzed and compared with the wild-type hom gene. DNA fragment exchange experiments between both genes showed that a 0.23-kb region close to the 3' terminus of homFBR was responsible for deregulation. Nucleotide sequence analysis revealed a single transition from G to A in homFBR leading to replacement of glycine-378 by glutamate ...

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

    OpenAIRE

    Komati Reddy, Gajendar; Wendisch, Volker F.

    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 en...

  1. Identification of channel-forming activity in the cell wall of Corynebacterium glutamicum.

    OpenAIRE

    Niederweis, M.; Maier, E. (Eva Maria); Lichtinger, T; Benz, R; Krämer, R

    1995-01-01

    The cell wall of the gram-positive Corynebacterium glutamicum was prepared. It contained an ion-permeable channel with a single-channel conductance of about 6 nS in 1 M KCl. The mobility sequence of the ions in the channel is similar to that in the aqueous phase, suggesting that it is a water-filled channel wide enough to allow unhindered diffusion of ions. The results indicate that we have identified the hydrophilic pathway through the mycolic acid layer of C. glutamicum.

  2. Epidemiological survey of Corynebacterium equi infections on five Ontario horse farms.

    OpenAIRE

    Prescott, J. F.; Travers, M.; Yager-Johnson, J A

    1984-01-01

    Corynebacterium equi was cultured from manure or soil on five horse-breeding farms in Ontario at monthly intervals on three occasions during the summer of 1982. The organism was widespread. Contamination by C. equi of the loafing paddock and pasture areas was significantly greater in a farm established 30 years than in two established for four and six years and there was a significant correlation between the C. equi burden in stables, paddocks and pastures and the length of use of the five fa...

  3. Recurrent Breast Abscesses due to Corynebacterium kroppenstedtii, a Human Pathogen Uncommon in Caucasian Women

    Directory of Open Access Journals (Sweden)

    Anne Le Flèche-Matéos

    2012-01-01

    Full Text Available Background. Corynebacterium kroppenstedtii (Ck was first described in 1998 from human sputum. Contrary to what is observed in ethnic groups such as Maori, Ck is rarely isolated from breast abscesses and granulomatous mastitis in Caucasian women. Case Presentation. We herein report a case of recurrent breast abscesses in a 46-year-old Caucasian woman. Conclusion. In the case of recurrent breast abscesses, even in Caucasian women, the possible involvement of Ck should be investigated. The current lack of such investigations, probably due to the difficulty to detect Ck, may cause the underestimation of such an aetiology.

  4. Linking Central Metabolism with Increased Pathway Flux: l-Valine Accumulation by Corynebacterium glutamicum

    OpenAIRE

    Radmacher, Eva; Vaitsikova, Adela; Burger, Udo; Krumbach, Karin; Sahm, Hermann; Eggeling, Lothar

    2002-01-01

    Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l-vali...

  5. l-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum▿

    OpenAIRE

    Blombach, Bastian; Schreiner, Mark E.; Holátko, Jiří; Bartek, Tobias; Oldiges, Marco; Eikmanns, Bernhard J.

    2007-01-01

    Corynebacterium glutamicum was engineered for the production of l-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ΔaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amount...

  6. Immobilazation of aerobic microorganisms on glassy sintered material, illustrated by the example of the production of L leucine using Corynebacterium glutamicum. Immobilisierung von aeroben Mikroorganismen an Glassintermaterial am Beispiel der L-Leucin-Produktion mit Corynebacterium glutamicum

    Energy Technology Data Exchange (ETDEWEB)

    Buechs, J.

    1988-12-01

    The aim of this study was to develop the carrier fixation of aerobic microorganisms on open-pore sintered glass material. The fermentative production of L-leucine from {alpha} cetonic isocaproic acid with Corynebacterium glutamicum was chosen as an example of a microbial process with a high demand of oxygen. (orig.).

  7. Complete Genome Sequence of Corynebacterium camporealensis DSM 44610, Isolated from the Milk of a Manchega Sheep with Subclinical Mastitis.

    Science.gov (United States)

    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

  8. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 04-7514, Isolated from a Dog in France

    Science.gov (United States)

    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

  9. Genome sequence of Corynebacterium pseudotuberculosis biovar equi strain 258 and prediction of antigenic targets to improve biotechnological vaccine production

    DEFF Research Database (Denmark)

    Soares, Siomar C; Trost, Eva; Ramos, Rommel T J;

    2013-01-01

    Corynebacterium pseudotuberculosis is the causative agent of several veterinary diseases in a broad range of economically important hosts, which can vary from caseous lymphadenitis in sheep and goats (biovar ovis) to ulcerative lymphangitis in cattle and horses (biovar equi). Existing vaccines ag...

  10. Draft Genome Sequence of Corynebacterium variabile Mu292, Isolated from Munster, a French Smear-Ripened Cheese

    Science.gov (United States)

    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

  11. Export of L-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family

    Czech Academy of Sciences Publication Activity Database

    Kennerknecht, N.; Sahm, H.; Yen, M. R.; Pátek, Miroslav; Saier, M. H.; Eggeling, L.

    2002-01-01

    Roč. 184, č. 14 (2002), s. 3947-3956. ISSN 0021-9193 R&D Projects: GA ČR GA525/01/0916 Keywords : l-isoleucine * corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology Impact factor: 3.959, year: 2002

  12. Modulating the metabolic flux through the biosynthesis pathways of branched-chain amino acids in corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Pátek, Miroslav; Elišáková, Veronika; Holátko, Jiří

    2004, s. 53. [Metabolic Engineering /5./. Squaw Creek (US), 19.09.2004-23.09.2004] R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * pathway Subject RIV: EE - Microbiology, Virology

  13. Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum: Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

    Czech Academy of Sciences Publication Activity Database

    Schreiner, M. E.; Riedel, Ch.; Holátko, Jiří; Pátek, Miroslav; Eikmanns, B. J.

    2006-01-01

    Roč. 188, č. 4 (2006), s. 1341-1350. ISSN 0021-9193 R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * pqo * molecular analysis Subject RIV: EE - Microbiology, Virology Impact factor: 3.993, year: 2006

  14. Identification and structural characterisation of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiAmutant

    OpenAIRE

    Tatituri, Raju Venkata Veera; Alderwick, Luke J.; Mishra, Arun K; Nigou, Jerome; Gilleron, Martine; Krumbach, Karin; Hitchen, Paul; Giordano, Assunta; Morris, Howard R.; Dell, Anne; Eggeling, Lothar; Besra, Gurdyal S.

    2007-01-01

    Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan, lipoarabinomannan (LAM). Herein, we show by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterised by single t-Araf residues linked to the

  15. Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Šilar, Radoslav; Rabatinová, Alžběta; Šanderová, Hana; Halada, Petr; Nešvera, Jan; Krásný, Libor; Pátek, Miroslav

    2012-01-01

    Roč. 96, č. 2 (2012), s. 521-529. ISSN 0175-7598 R&D Projects: GA ČR GC204/09/J015; GA ČR GPP302/12/P633 Institutional support: RVO:61388971 Keywords : Corynebacterium glutamicum * In vitro transcription * RNA polymerase Subject RIV: EE - Microbiology, Virology Impact factor: 3.689, year: 2012

  16. Genome Sequence of Corynebacterium glutamicum ATCC 14067, Which Provides Insight into Amino Acid Biosynthesis in Coryneform Bacteria

    OpenAIRE

    Lv, Yangyong; Liao, Juanjun; Wu, Zhanhong; Han, Shuangyan; Lin, Ying; Zheng, Suiping

    2012-01-01

    We report the genome sequence of Corynebacterium glutamicum ATCC 14067 (once named Brevibacterium flavum), which is useful for taxonomy research and further molecular breeding in amino acid production. Preliminary comparison with those of the reported coryneform strains revealed some notable differences that might be related to the difficulties in molecular manipulation.

  17. Osmolality, temperature, and membrane lipid composition modulate the activity of betaine transporter BetP in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Ozcan, Nuran; Ejsing, Christer S.; Shevchenko, Andrej; Lipski, Andrej; Morbach, Susanne; Krämer, Reinhard

    2007-01-01

    The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier...

  18. Complete Genome Sequence of Corynebacterium camporealensis DSM 44610, Isolated from the Milk of a Manchega Sheep with Subclinical Mastitis

    OpenAIRE

    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.

  19. Corynebacterium uropygiale sp. nov., isolated from the preen gland of Turkeys (Meleagris gallopavo).

    Science.gov (United States)

    Braun, Markus Santhosh; Zimmermann, Stefan; Danner, Maria; Rashid, Harun-Or; Wink, Michael

    2016-03-01

    A novel species of fastidious, lipophilic, club-shaped, Gram-positive bacteria was recovered from the preen glands of healthy Turkeys (Meleagris gallopavo) from two different locations. Phylogenetic analysis of the 16S rRNA gene showed highest similarity to Corynebacterium spheniscorum DSM 44757(T) (96.8%) with a 3.2kb stretch of rpoB sharing 82.4% sequence similarity to the same species. DNA fingerprinting by ERIC-PCR and polar lipid profiles clearly differentiated the Turkey isolates from the most closely related Corynebacteria, as did MALDI-TOF MS analysis. Chemotaxonomic tests revealed the presence of corynemycolic acids with C16:0, C18:0, C18:1ω9c and tuberculostearic acid as the major cellular fatty acids. The G+C content of the type strain was 60.7mol%. The species was susceptible to ampicillin, kanamycin A, streptomycin, amikacin, polymyxin B and vancomycin. From our results, it becomes evident that the isolated organisms represent a new species, for which the name Corynebacterium uropygiale sp. nov. is proposed. The type strain is Iso10(T) (=DSM 46817(T)=LMG 28616(T)). PMID:26776107

  20. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH2PO4 and K2HPO4 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 NaH2PO4 and K2HPO4 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 P6322, 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 Å3 Da−1. The structure was solved by molecular replacement. Structure refinement is in progress

  2. Investigation into the flux distribution of central carbon metabolism in Corynebacterium glutamicum using principal component analysis

    Directory of Open Access Journals (Sweden)

    Shang Chuanyu

    2015-01-01

    Full Text Available Central carbon metabolism is the main source of energy required by organisms and it provides precursors for other in vivo metabolic processes. The flux flowing through the pathways involved in central carbon metabolism characterizes its biological function and genetic readout between species or environments. In recent years, using a 13C tracer technique, researchers have measured the flux of central carbon metabolism in Corynebacterium glutamicum under a variety of nutritional and environmental changes or genetic modifications. However, there is no integrated and comparative analysis of these measured flux values. In this study, the flux values of central carbon metabolism in Corynebacterium glutamicum that were obtained in other recent studies were consolidated. A preliminary examination of the distribution characteristics of flux values in each metabolic pathway was conducted and the regression relationship between different fluxes was investigated. The principal components of the flux vector were further extracted and aggregated based on the components, and the general features of flux distribution of central carbon metabolism as well as the influence of environmental and genetic factors on the flux distribution were determined. This study provides a foundation for further investigation into the flux distribution and regulation characteristics of central carbon metabolism.

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

    International Nuclear Information System (INIS)

    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 Å3 Da−1. The structure was solved by molecular replacement. Structure refinement is in progress

  4. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production.

    Science.gov (United States)

    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

  5. Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

    Science.gov (United States)

    Katsukawa, Chihiro; Komiya, Takako; Umeda, Kaoru; Goto, Minami; Yanai, Tokuma; Takahashi, Motohide; Yamamoto, Akihiko; Iwaki, Masaaki

    2016-03-01

    Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans. PMID:26853714

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung-Jin, E-mail: kkj@postech.ac.kr [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Sujin [21C Frontier Microbial Genomics and Applications Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Lee, Sujin [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of); Kang, Beom Sik [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Lee, Heung-Soo [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of); Oh, Tae-Kwang; Kim, Myung Hee, E-mail: kkj@postech.ac.kr [21C Frontier Microbial Genomics and Applications Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Soo-Young [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of); Kang, Beom Sik [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Ghyung-Hwa; Kim, Kyung-Jin, E-mail: kkj@postech.ac.kr [Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784 (Korea, Republic of)

    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.

  8. The pan-genome of the animal pathogen Corynebacterium pseudotuberculosis reveals differences in genome plasticity between the biovar ovis and equi strains

    DEFF Research Database (Denmark)

    Soares, Siomar C; Silva, Artur; Trost, Eva;

    2013-01-01

    Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Cory...

  9. Toxigenic Corynebacterium ulcerans isolated from a wild bird (ural owl) and its feed (shrew-moles): comparison of molecular types with human isolates

    OpenAIRE

    Katsukawa, Chihiro; Umeda, Kaoru; Inamori, Ikuko; Kosono, Yuka; Tanigawa, Tomokazu; Komiya, Takako; Iwaki, Masaaki; Yamamoto, Akihiko; Nakatsu, Susumu

    2016-01-01

    Background Corynebacterium ulcerans is a pathogen causing diphtheria-like illness to humans. In contrast to diphtheria by Corynebacterium diphtheriae circulating mostly among humans, C. ulcerans infection is zoonotic. The present study aimed to clarify how a zoonotic pathogen C. ulcerans circulates among wild birds and animals. Results By screening 380 birds, a single strain of toxigenic C. ulcerans was isolated from a carnivorous bird, ural owl (Strix uralensis). The bacterium was also isola...

  10. Bioconversion of sugar cane molasses into glutamic acid by gamma irradiated corynebacterium glutamicum

    International Nuclear Information System (INIS)

    Corynebacterium glutamicum (ATCC 13058) was used for glutamic acid production from sugar cane molasses which contain sufficient. The addition of 5 units ml4 of penicillin G was superior in glutamic acid production (11.5 g L 4). Tweens and their saturated fatty acids were effective on the accumulation of glutamic acid in the culture medium and the maximum yield (16.6 g L4) was the addition of 5 mg ml4 Tween 40. Gamma irradiation prior to Tween-40 treatment of bacterial cells resulted in an obvious increase in glutamic acid production and it was maximum (23.72 g L4) at 0.1 k Gy exposure dose of inocula. 5 tabs

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

    International Nuclear Information System (INIS)

    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 λpSL5. 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+ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA+ 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+ and lysA+. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

  12. Toxigenic Corynebacterium ulcerans isolated from a free-roaming red fox (Vulpes vulpes).

    Science.gov (United States)

    Sting, Reinhard; Ketterer-Pintur, Sandra; Contzen, Matthias; Mauder, Norman; Süss-Dombrowski, Christine

    2015-01-01

    Corynebacterium (C.) ulcerans could be isolated from the spleen of a red fox (Vulpes vulpes) that had been found dead in the state of Baden-Württemberg, Germany. Pathohistological examination suggested that the fox had died of distemper, as was confirmed by PCR. The isolate was identified biochemically, by MALDI-TOF MS, FT-IR and by partial 16S rRNA, rpoB and tox gene sequencing. Using the Elek test the C. ulcerans isolate demonstrated diphtheria toxin production. FT-IR and sequencing data obtained from the C. ulcerans isolate from the red fox showed higher similarity to isolates from humans than to those from wild game. PMID:26054226

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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 Å3 Da−1 and a solvent content of about 41%

  20. BIOCHEMICAL AND PHYLOGENETIC STUDIES OF CreD OF Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Muhammad Tausif Chaudhry

    2015-06-01

    Full Text Available CreD characterized as Mg2+-dependent phosphohydrolase with conserved HD domain was involved in 4-cresol metabolism in Corynebacterium glutamicum. Native molecular mass of 54 kDa suggested that the biological unit is a dimer. No deoxynucleotide triphosphate triphosphohydrolase (dNTPase activity was detected for CreD. The apparent Km and Vmax values for 4-nitrophenyl phosphate were 0.35 mM and 16.23 M min-1 mg-1, respectively, while calculated values for kcat and kcat/Km were 0.4 s-1 and 1.14103 M-1 s-1, respectively. Among thiol group inhibitors, iodoacetic acid significantly inhibited phosphohydrolase activity. Sequence identity and phylogenetic analysis suggested universal existence of CreD homologues. Involvement of HD-domain hydrolase in aromatic degradation has not been reported before.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Schoepe, Jan, E-mail: jschoepe@smail.uni-koeln.de; Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar [Institute for Biochemistry, University of Köln, Zülpicher Strasse 47, Köln, NRW 50974 (Germany)

    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%.

  3. Influence of Corynebacterium parvum on the phagocytosis of 198Au colloids in rats

    International Nuclear Information System (INIS)

    The kinetics of the phagocytosis of gelatin-protected 198Au colloids in Wistar rats treated with Corynebacterium Parvum (CBP), was studied in order to explain its mechanism of immunomodulation. A previously developed extracorporeal blood circulation technique was used. The changes in the rate of phagocytosis, v, after the administration of CBP, for a dose of the 198Au colloid smaller or higher than the substratum constant, were studied. In the first case, no significant changes of v were observed; in the second case, significant increases of v were determined, which reached a maximum 6 days after the CBP administration. The kinetic analysis of the obtained data indicates that the action of CBP is exerted on the stage of the entrance of the colloidal particle into the reticuloendothelial cell. (author)

  4. Brain and lung cryptococcoma and concurrent corynebacterium pseudotuberculosis infection in a goat: a case report

    Directory of Open Access Journals (Sweden)

    MCR Luvizotto

    2009-01-01

    Full Text Available A four-year-old male goat with a history of neurological disorder was euthanized. It presented uncommon nodules in the brain and lungs associated with multiple abscesses, predominantly in the spleen and liver. Histological examination of brain and lung sections revealed yeast forms confirmed to be Cryptococcus gattii after a combination of isolation and polymerase chain reaction (PCR procedures. Moreover, Corynebacterium pseudotuberculosis infection was diagnosed by PCR of samples from the lung, spleen and liver. The present report highlights the rare concurrent infection of C. gatti and C. pseudotuberculosis in an adult goat from São Paulo state, Brazil, and indicates the necessity of surveillance in the treatment of goats with atypical pulmonary infections associated with neurological disorders.

  5. Corynebacterium striatum infecting a malignant cutaneous lesion: the emergence of an opportunistic pathogen Corynebacterium striatum infectando lesão cutânea maligna: a emergência de um patógeno oportunista

    Directory of Open Access Journals (Sweden)

    Silvana Vargas Superti

    2009-04-01

    Full Text Available We described a case of a 27-year old male patient with skin and soft tissue infection of a neoplastic lesion caused by Corynebacterium striatum, an organism which has been rarely described as a human pathogen. Identification was confirmed by DNA sequencing. Successful treatment with penicillin was achieved. The role of the C. striatum as an emerging opportunistic pathogen is discussed.Descrevemos infecção de lesão neoplásica em paciente masculino de 27 anos, envolvendo pele e partes moles, causada por Corynebacterium striatum, um microrganismo raramente descrito como patógeno humano. A identificação foi confirmada por seqüenciamento de DNA. O paciente foi tratado com penicilina, com sucesso. O papel do C. striatum como patógeno oportunista é discutido.

  6. Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

    Directory of Open Access Journals (Sweden)

    Jerônimo C Ruiz

    Full Text Available BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL. CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep. Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/ under accession numbers CP001809 and CP001829.

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

    Science.gov (United States)

    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

  8. Phenotypic, molecular characterization, antimicrobial susceptibility and draft genome sequence of Corynebacterium argentoratense strains isolated from clinical samples

    OpenAIRE

    Fernández-Natal, I.; J.A. Sáez-Nieto; Rodríguez-Lázaro, D.; Valdezate-Ramos, S.; Parras-Padilla, T.; Medina, M.J.; R.H. Rodríguez-Pollán; Van Der Blom, J; Tauch, A.; Soriano, F

    2016-01-01

    During a 12-year period we isolated five Corynebacterium argentoratense strains identified by phenotypic methods, including the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and 16S rRNA gene sequencing. In addition, antimicrobial susceptibility was determined, and genome sequencing for the detection of antibiotic resistance genes was performed. The organisms were isolated from blood and throat cultures and could be identified by all methods u...

  9. Impact of trehalose and mycolate biosynthesis on the cell envelope of a Corynebacterium glutamicum L-lysine production strain

    OpenAIRE

    Gebhardt, Henrike

    2005-01-01

    In contrast to other Gram-positive bacteria all members of the suborder of Corynebacterineae, including Corynebacterium glutamicum, contain a cell envelope that comprises an outer lipid bilayer, the mycolate layer, which is considered as permeability barrier. Trehalose is an important component of the mycolate layer and involved in the biosynthesis of mycolate. The first step of mycolate biosynthesis, the condensation of trehalose monomycolate was proven to be located in the cell envelope. Th...

  10. In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

    OpenAIRE

    Krömer, Jens Olaf; Sorgenfrei, Oliver; Klopprogge, Kai; Heinzle, Elmar; Wittmann, Christoph

    2004-01-01

    An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isot...

  11. Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes

    OpenAIRE

    Cremer, Josef; Eggeling, Lothar; Sahm, Hermann

    1991-01-01

    The gene cluster that codes for feedback-resistant aspartate kinase (lysCα and lysCβ) and aspartate semialdehyde dehydrogenase (asd) was cloned from a mutant strain of Corynebacterium glutamicum. Its functional analysis by subcloning, enzyme assays, and type of aspartate kinase regulation enabled the isolation of a fragment for separate expression of the feedback-resistant kinase without aspartate semialdehyde dehydrogenase expression. This was used together with other clones constructed (J. ...

  12. Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l-Lysine Production ▿

    OpenAIRE

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

    2010-01-01

    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 glycolyt...

  13. Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

    OpenAIRE

    Guillouet, S.; Rodal, A. A.; An, G.-H.; Lessard, P. A.; Sinskey, A J

    1999-01-01

    The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same stra...

  14. Mechanism of Concerted Inhibition of α2β2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum*

    OpenAIRE

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

    2010-01-01

    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 comple...

  15. Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

    OpenAIRE

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

    2004-01-01

    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 env...

  16. Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

    OpenAIRE

    Menkel, E; Thierbach, G; Eggeling, L; Sahm, H

    1989-01-01

    Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion o...

  17. Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

    OpenAIRE

    Morbach, S; Sahm, H; Eggeling, L

    1995-01-01

    The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were construc...

  18. Reductive Cleavage of Demeton-S-Methyl by Corynebacterium glutamicum in Cometabolism on More Readily Metabolizable Substrates

    OpenAIRE

    Girbal, Laurence; Hilaire, Didier; Leduc, Sébastien; Delery, Laure; Rols, Jean-Luc; Lindley, Nicholas D.

    2000-01-01

    Corynebacterium glutamicum is able to biotransform demeton-S-methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. Among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton-S-methyl biotransformation. The reaction mechanism of demeton-S-methyl biotransformation involves reductive cleavage of an S-C bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.

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

    OpenAIRE

    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 m...

  20. Izolace a charakterizace promotorů Corynebacterium glutamicum regulovaných změnami teploty

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Martina; Pátek, Miroslav; Nešvera, Jan

    2004. s. 313-314. [Mezioborové setkání mladých biologů, biochemiků a chemiků /4./. 09.06.2004-12.06.2004, Žďárské vrchy] R&D Projects: GA ČR GA525/04/0548 Institutional research plan: CEZ:AV0Z5020903 Keywords : corynebacterium glutamicum * promoters Subject RIV: EE - Microbiology, Virology

  1. A Gain-of-Function Mutation in Gating of Corynebacterium glutamicum NCgl1221 Causes Constitutive Glutamate Secretion

    OpenAIRE

    Nakayama, Yoshitaka; Yoshimura, Kenjiro; Iida, Hidetoshi

    2012-01-01

    The A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion in Corynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

  2. Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum.

    OpenAIRE

    Oguiza, J A; Tao, X; Marcos, A T; Martín, J F; Murphy, J R

    1995-01-01

    A homolog of the Corynebacterium diphtheriae dtxR gene was isolated from Brevibacterium lactofermentum. The product of the B. lactofermentum dtxR gene was immunoreactive with polyclonal anti-DtxR antibodies and functioned as an iron-activated repressor capable of regulating the expression of beta-galactosidase from a diphtheria tox promoter/operator transcriptional fusion in recombinant Escherichia coli. The extents of induction by increasing concentrations of the chelator 2,2'-dipyridyl were...

  3. Effects of bovine subclinical mastitis caused by Corynebacterium spp. on somatic cell count, milk yield and composition by comparing contralateral quarters.

    Science.gov (United States)

    Gonçalves, Juliano Leonel; Tomazi, Tiago; Barreiro, Juliana Regina; Beuron, Daniele Cristine; Arcari, Marcos André; Lee, Sarah Hwa In; Martins, Cristian Marlon de Magalhães Rodrigues; Araújo Junior, João Pessoa; dos Santos, Marcos Veiga

    2016-03-01

    Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content. PMID:26831159

  4. A single method to stain Malassezia furfur and Corynebacterium minutissimum in scales Um método simples para corar Malassezia furfur e Corynebacterium minutissimum nas escamas

    Directory of Open Access Journals (Sweden)

    Antar Padilha-Gonçalves

    1996-08-01

    Full Text Available A single and practical method to slain Malassezia furfur and Corynebacterium minutissimum in lesions' scales is described. The scales are collected by pressing small pieces of scotch tape (about 4 cm lenght and 2 cm width onto the lesions and following withdrawl the furfuraceous scales will remain on the glue side. These pieces are then immersed for some minutes in lactophenol-cotton blue stain. Following absorption of the stain the scales are washed in current water to remove the excess of blue stain, dried with filter paper, dehydrated via passage in two bottles containing absolute alcohol and then placed in xylene in a centrifugation tube. The xylene dissolves the scotch tape glue and the scales fall free in the tube. After centrifugation and decantation the scales concentrated on the bottom of the tube are collected with a platinum-loop, placed in Canada balsam on a microscopy slide and closed with a cover slip. The preparations are then ready to be submitted to microscopic examination. Other stains may also be used instead of lactophenol-cotton blue. This method is simple, easily performed, and offers good conditions to study these fungi as well as being useful for the diagnosis of the diseases that they cause.É descrito um método simples e prático para corar Malassezia furfur e Corynebacterium minutissimum nas escamas das lesões. O material é colhido com o auxílio de fita durex que será usada na maior parte das etapas do método para ajudar a fácil execução do processo de coloração. Para colher as escamas, pequenos pedaços de fita durex com cerca de 4 cm de comprimento por 2 cm de largura são colocados e pressionados sobre as lesões, e quando retirados trazem aderidas as escamas furfuráceas na face com goma. Esses pedaços de fita durex são imersos por alguns minutos no corante lactofenol-azul cotton e logo que as escamas estiverem coradas em azul são lavadas em água corrente para remover o excesso de corante azul, secos

  5. Biochemical and molecular characterization of the gentisate transporter GenK in Corynebacterium glutamicum.

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    Ying Xu

    Full Text Available BACKGROUND: Gentisate (2,5-dihydroxybenzoate is a key ring-cleavage substrate involved in various aromatic compounds degradation. Corynebacterium glutamicum ATCC13032 is capable of growing on gentisate and genK was proposed to encode a transporter involved in this utilization by its disruption in the restriction-deficient mutant RES167. Its biochemical characterization by uptake assay using [(14C]-labeled gentisate has not been previously reported. METHODOLOGY/PRINCIPAL FINDINGS: In this study, biochemical characterization of GenK by uptake assays with [(14C]-labeled substrates demonstrated that it specifically transported gentisate into the cells with V(max and K(m of 3.06 ± 0.16 nmol/min/mg of dry weight and 10.71 ± 0.11 µM respectively, and no activity was detected for either benzoate or 3-hydoxybenzoate. When GenK was absent in strain RES167 ΔgenK, it retained 85% of its original transport activity at pH 6.5 compared to that of strain RES167. However, it lost 79% and 88% activity at pH 7.5 and 8.0, respectively. A number of competing substrates, including 3-hydroxybenzoate, benzoate, protocatechuate and catechol, significantly inhibited gentisate uptake by more than 40%. Through site-directed mutagenesis, eight amino acid residues of GenK, Asp-54, Asp-57 and Arg-386 in the hydrophobic transmembrane regions and Arg-103, Trp-309, Asp-312, Arg-313 and Ile-317 in the hydrophilic cytoplasmic loops were shown to be important for gentisate transport. When conserved residues Asp-54 and Asp-57 respectively were changed to glutamate, both mutants retained approximately 50% activity and were able to partially complement the ability of strain RES167 ΔgenK to grow on gentisate. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that GenK is an active gentisate transporter in Corynebacterium glutamicum ATCC13032. The GenK-mediated gentisate transport was also shown to be a limiting step for the gentisate utilization by this strain. This enhances our

  6. Reproductive Pathological Changes Associated with Experimental Subchronic Corynebacterium pseudotuberculosis Infection in Nonpregnant Boer Does

    Directory of Open Access Journals (Sweden)

    A. M. Othman

    2016-01-01

    Full Text Available Corynebacterium pseudotuberculosis causes caseous lymphadenitis (CLA, which is a contagious and chronic disease in sheep and goats. In order to assess the histopathological changes observed in the reproductive organs of nonpregnant does infected with the bacteria, 20 apparently healthy adult Boer does were divided into four inoculation groups, intradermal, intranasal, oral, and control, consisting of five goats each. Excluding the control group, which was unexposed, other does were inoculated with 107 CFU/1 mL of live C. pseudotuberculosis through the various routes stated above. Thirty days after infection, the ovaries, uterus, and iliac lymph nodes were collected for bacterial recovery and molecular detection, as well as histopathological examination. The mean changes in necrosis, congestion, inflammatory cell infiltration, and oedema varied in severity among the ovaries, uterus, and iliac lymph nodes following different inoculation routes. Overall, the intranasal route of inoculation showed more severe (p<0.05 lesions in all the organs examined. The findings of this study have shown that C. pseudotuberculosis could predispose to infertility resulting from pathological lesions in the uterus and ovaries of does.

  7. In silico identification of Corynebacterium pseudotuberculosis antigenic targets and application in immunodiagnosis.

    Science.gov (United States)

    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

  8. Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum

    Institute of Scientific and Technical Information of China (English)

    CHEN Xue Lan; ZHANG Bin; TANG Li; JIAO Hai Tao; XU Heng Yi; XU Feng; XU Hong; WEI Hua; XIONG Yong Hua

    2014-01-01

    Objective Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. Methods Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer. Results Arginine production assays showed a 69.9%reduction in arginine from 9.01±0.22 mg/mL in C. crenatum MT to 2.71±0.13 mg/mL (P Conclusion The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.

  9. The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

    Institute of Scientific and Technical Information of China (English)

    RUAN Hong; R. Gerstmeir; S. Schnicke; B.J. Eikmanns

    2005-01-01

    During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the pta-ack operon.

  10. Three-stage fermentation and kinetic modeling of bioflocculant by Corynebacterium glutamicum

    Institute of Scientific and Technical Information of China (English)

    Liang Shen; Zhongtao An; Qingbiao Li; Chuanyi Yao; Yajuan Peng; Yuanpeng Wang; Ruihua Lai; Xu Deng; Ning He

    2015-01-01

    Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling. Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were pro-posed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose con-centrations from 10.0 to 17.5 g·L−1. The initial biomass concentration could shorten the lag time of cel growth, while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of 16.22 g·L−1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of 2.23 g·L−1 was obtained and bioflocculant concentration was enhanced to 176.32 mg·L−1, 18.62% and 403.63%higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.

  11. Evolution, epidemiology and diversity of Corynebacterium diphtheriae: New perspectives on an old foe.

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. Crystallization and preliminary crystallographic analysis of cgHle, a homoserine acetyltransferase homologue, from Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    The enzyme cgHle from C. glutamicum, which has acetyl ester hydrolase activity, was crystallized in four different crystal forms. X-ray diffraction data have been collected to a resolution of 1.2 Å. CgHle is an enzyme that is encoded by gene cg0961 from Corynebacterium glutamicum. The physiological function of cgHle is so far unclear. Bioinformatic annotations based on sequence homology indicated that cgHle may be an acetyl-CoA:homoserine acetyl transferase and as such may be involved in methionine biosynthesis, but recent evidence has shown that it is an esterase that catalyzes the hydrolysis of acetyl esters. Here, the crystallization of cgHle in two orthorhombic crystal forms, a trigonal crystal form and a monoclinic crystal form is described. The trigonal crystals have a solvent content of 83.7%, which is one of the highest solvent contents ever found for protein crystals. One of the orthorhombic crystals diffracted X-rays to at least 1.2 Å resolution

  16. Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge.

    Science.gov (United States)

    Ribeiro, Dayana; Rocha, Flávia de Souza; Leite, Kátia Morais Costa; Soares, Siomar de Castro; Silva, Artur; Portela, Ricardo Wagner Dias; Meyer, Roberto; Miyoshi, Anderson; Oliveira, Sérgio Costa; Azevedo, Vasco; Dorella, Fernanda Alves

    2014-01-01

    Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA. PMID:24597857

  18. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    International Nuclear Information System (INIS)

    Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals. FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively

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

    Science.gov (United States)

    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

  20. Quantitative proteomic overview on the Corynebacterium glutamicuml-lysine producing strain DM1730.

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Volker Fritz Wendisch

    2012-10-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  4. Mycothiol peroxidase MPx protects Corynebacterium glutamicum against acid stress by scavenging ROS

    Directory of Open Access Journals (Sweden)

    Tietao Wang

    2015-09-01

    Full Text Available Corynebacterium glutamicum mycothiol peroxidase (MPx is a novel CysGPx family peroxidase that uses both the mycoredoxin and thioredoxin reducing systems as proton donors for peroxide detoxification. In this study, we revealed that MPx is also important for cellular survival under acid stress. A Δmpx mutant exhibited significantly decreased resistance to acid stress and markedly increased accumulation of reactive oxygen species (ROS and protein carbonylation levels in vivo. Overexpression of mpx increased the resistance of C. glutamicum to acid stress by reducing ROS accumulation. Elevated expression of the mpx gene was consistently observed when the C. glutamicum wild-type strain was exposed to acid stress conditions, which in turn directly contributed to tolerance to acid stress. The acid-induced expression of mpx was mediated by the stress-responsive extracytoplasmic function-sigma (ECF-σ factor, SigH. The results unequivocally show that MPx is essential for combating acid stress by reducing intracellular ROS levels induced by acid stress in C. glutamicum, which adds a new dimension to the general physiological functions of CysGPx

  5. 3-Amino-4-hydroxybenzoic acid production from sweet sorghum juice by recombinant Corynebacterium glutamicum.

    Science.gov (United States)

    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

  6. Crystallization and preliminary crystallographic analysis of the global nitrogen regulator AmtR from Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    AmtR is a rare example of a member of the TetR family of bacterial transcription regulators that is not regulated by a small-molecule effector but by interaction with a protein named GlnK. Wild-type and SeMet-substituted AmtR have been produced and crystallized and preliminary electron-density maps have been obtained to 3.0 Å resolution. AmtR, a member of the TetR family of transcription regulators, is a global regulator of nitrogen control in Corynebacterium glutamicum. Unlike other TetR-family members, which are regulated by small-molecule effectors, AmtR is regulated by a protein called GlnK. It has been shown that a GlnK trimer has to become adenylylated prior to formation of a complex with AmtR. The physiological function of AmtR has been very well studied, but structural characterization of the mechanistic aspects of AmtR-regulated transcription has yet to be accomplished. AmtR has successfully been crystallized in space group P21212, with six molecules in the asymmetric unit and unit-cell parameters a = 153.34, b = 163.10, c = 51.93 Å. Preliminary phases were obtained using Se-SAD

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

    Science.gov (United States)

    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

  8. Expression, crystallization and preliminary crystallographic study of GluB from Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    GluB, a substrate-binding protein from C. glutamicum, was expressed, purified and crystallized, followed by X-ray diffraction data collection and preliminary crystallographic analysis. GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 82.50, c = 72.69 Å

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Sticht Heinrich

    2009-07-01

    Full Text Available Abstract 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.

  11. Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dharmendra; Subramanian, Kartik; Bisaria, Virendra S.; Sreekrishnan, T.R.; Gomes, James [Indian Inst. of Technology, Dept. of Biochemical Engineering and Biotechnology, New Delhi (India)

    2005-02-01

    The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l{sup -1} of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l{sup -1} h{sup -1}) every 2 h to the production medium increased the production of methionine to 3.39 g l{sup -1}. A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers. (Author)

  12. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis in Camels (Camelus dromedarius in Jordan

    Directory of Open Access Journals (Sweden)

    Azmi D. Hawari

    2008-01-01

    Full Text Available Problem statement: This study was conducted to describe & report for the first time outbreaks of natural C.pseudotuberculosis infection in adult camel herds (Camelus dromedarius in Jordan. An infectious disease syndrome was reported in three camel herds (Camelus dromedarius intensively raised at south province in Jordan. Approach: The herds included over 160 adult camels out of which about 8% were affected with multiple muscle and subcutaneous abscesses at various sites of the body. The camels were also heavily infested with ticks. Results: The infected camels did not respond favorably to several broad spectrum antibiotics. Post-mortem examination of 5 carcasses revealed emaciation and presence of external and internal multiple abscesses particularly in the lungs. The abscesses were encapsulated by fibrous tissue and contained creamy yellowish white pus. The lymph nodes were slightly congested and swollen. Conclusion: Corynebacterium pseudotuberculosis type I strain or biovar ovis (the known cause of caseous lymphadenitis in sheep was isolated from pus, lymph nodes, ticks, milk, blood and liver samples. The clinical symptoms, nature and distribution of lesions of caseous lymphadenitis in camels are not as typical as in sheep. Recommendations for pseudotuberculosis control were given.

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

    Energy Technology Data Exchange (ETDEWEB)

    Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J. (Saskatchewan)

    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.

  14. Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

    Directory of Open Access Journals (Sweden)

    Monica Cristina de Souza

    2012-06-01

    Full Text Available Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2 cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

  15. L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains

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    Nagai Kazuo

    2001-10-01

    Full Text Available Abstract Background A non-pathogenic species of coryneform bacteria, Corynebacterium glutamicum, was originally isolated as an L-glutamate producing bacterium and is now used for fermentative production of various amino acids. A mutation in the C. glutamicum ltsA gene caused susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production. Results The characteristics of eight lysozyme-sensitive mutants which had been isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis were examined. Complementation analysis with the cloned wild-type ltsA gene and DNA sequencing of the ItsA region revealed that four mutants had a mutation in the ltsA gene. Among them, two mutants showed temperature-sensitive growth and overproduced L-glutamate at higher temperatures, as well as the previously reported ltsA mutant. Other two showed temperature-resistant growth: one missense mutant produced L-glutamate to some extent but the other nonsense mutant did not. These two mutants remained temperature-resistant in spite of introduction of ltsA::kan mutation that causes temperature-sensitive growth in the wild-type background. Conclusions These results indicate that a defect caused by the ltsA mutations is responsible for temperature-sensitive growth and L-glutamate overproduction by C. glutamicum. The two temperature-resistant mutants seem to carry suppressor mutations that rendered cells temperature-resistance and abolished L-glutamate overproduction.

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

    Science.gov (United States)

    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

  17. Epidemiological survey of Corynebacterium equi infections on five Ontario horse farms.

    Science.gov (United States)

    Prescott, J F; Travers, M; Yager-Johnson, J A

    1984-01-01

    Corynebacterium equi was cultured from manure or soil on five horse-breeding farms in Ontario at monthly intervals on three occasions during the summer of 1982. The organism was widespread. Contamination by C. equi of the loafing paddock and pasture areas was significantly greater in a farm established 30 years than in two established for four and six years and there was a significant correlation between the C. equi burden in stables, paddocks and pastures and the length of use of the five farms for horses. In all farms, numbers of C. equi in pasture soil exceeded numbers in fresh manure, suggesting that environmental multiplication of the organism might occur. A farm with an endemic C. equi pneumonia problem differed significantly from the other four farms, where disease was not endemic, in the larger number of C. equi isolated in the stable area. By contrast the farm with a C. equi pasture soil burden significantly heavier than on all other farms had no deaths due to C. equi pneumonia. There was a correlation (r = 0.78, p = 0.061) between the number of cases of C. equi pneumonia on the farms and numbers of C. equi in the area of the stables, but not on the paddocks or pastures. About two-thirds of randomly chosen isolates from the farms belonged to the three capsular serotypes most commonly found in pneumonic foals. PMID:6713248

  18. Physico-chemical parameter for production of lactic acid or ethanol of (corynebacterium glutamicum) bacteria

    International Nuclear Information System (INIS)

    The interest to obtain products for the bio-fuel industry from renewable resources has directed research to find resistant and costs-effective biotechnological systems. Corynebacterium glutamicum, is a microorganism used to produce amino acids, that grows in wide variety of substrates and its resistance during fermentation to pH, temperature, osmotic pressure variations and alcohol aggregate, renders this organism a suitable candidate to improve by genetic modifications lactic acid and ethanol synthesis. However, some aspects of its physiology remain unknown, such us increase lactic acid and ethanol production from C5 and C6 sugars. For this reason, the main aim in our work was to identify the most important variables with impact on culture and the best culture conditions to produce lactic acid or ethanol in batch culture. To achieve this objective, eight variables were tested in culture using a statistical model. The best culture conditions were obtained and tested in a bacth bioreactor system. Temperature, biotin and glucose concentration were the variables with most impact (p-1, 16 g/l of lactic acid was obtained after 15 h of culture with an efficiency of 32%. High glucose consumption was observed during bacterial growth, which leads to low concentration of substrate for the production process; this suggests a culture feeding at the end of exponential growth phase, which can increase the production yield.

  19. Bioremediation of refinery wastewater using immobilised Burkholderia cepacia and Corynebacterium sp and their transconjugants

    Directory of Open Access Journals (Sweden)

    Abdullahi T. Ajao

    2013-07-01

    Full Text Available When oil spill occurs, it poses serious toxic hazards to all forms of life. Mixed culture of Burkholderia cepacia and Corynebacterium sp isolated from refinery sludge using selective enrichment technique was used for bioremediation of refinery wastewater in a laboratoryscale bioreactor. Physicochemical parameters of both raw and treated water were as determined and compared with Federal Environ - mental Protection Agency (FEPA-limit, Abuja, Nigeria to asses the efficiency of the bioremediation process. Each of the bacterium was screened for the presence of plasmid DNA and for the involvement or otherwise of plasmid in the bioremediation of wastewater. The immobilised cells showed percentage decrease in chemical oxygen demand (97%, biochemical oxygen demand (94%, phenol (98%, total petroleum hydrocarbon (79%, oil and grease (90% of the refinery waste water after 20 days of treatment while their transconjugants showed the multiplicative effect by achieving the same percentage after 10 days of treatment. Therefore, the findings revealed that bioaugmentation of wastewater using transmissible catabolic plasmid will enhance efficiency of the bioremediation by spreading the plasmid among indigenous microbial community either through horizontal gene transfer or transformation.

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

    Science.gov (United States)

    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

  1. Teste de pele em caprinos vacinados e infectados com Corynebacterium pseudotuberculosis

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    Francisco Selmo Fernandes Alves

    1999-07-01

    Full Text Available Dez caprinos foram vacinados com toxóide a 3%, outros dez com uma bacterina e mais dois grupos-controle de cinco animais cada, submetidos à inoculação de infusão de cérebro e coração e solução salina, respectivamente. Todos os animais foram examinados e avaliados com um teste de pele. Tanto o toxóide quanto a bacterina foram produzidos a partir de amostra de Corynebacterium pseudotuberculosis. Todos os caprinos foram desafiados com C. pseudotuberculosis, trinta dias após as vacinações. Nenhuma das vacinas induziu reação de hipersensibilidade na pele dos caprinos antes do desafio. Após o desafio, todos os animais desenvolveram reações mensuráveis na primeira, quinta e décima semana em resposta ao teste de pele. Os diâmetros da reação dérmica aumentaram do décimo dia à quinta semana após o desafio. As medidas alcançaram tamanho maior na décima semana. O resultado deste estudo indica que antígeno específico do C. pseudotuberculosis pode ser utilizado em caprinos no diagnóstico da linfadenite caseosa como teste de pele ou como instrumento experimental para monitorar o desenvolvimento da doença.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  6. PARÁMETROS FISICOQUÍMICOS PARA LA SÍNTESIS DE ÁCIDO LÁCTICO O ETANOL DE LA BACTERIA (Corynebacterium glutamicum) Physico-Chemical Parameter for Production of Lactic Acid or Ethanol of (Corynebacterium glutamicum) Bacteria

    OpenAIRE

    ANGÉLICA CASTELLANOS; LINA MARCELA GARCIA; MYRIAM ASTUDILLO; JORGE ENRIQUE LÓPEZ GALÁN; LUZ MARINA FLOREZ PARDO

    2011-01-01

    El interés por obtener productos para la industria de biocombustibles a partir de desechos agrícolas, conduce a la búsqueda de nuevos sistemas biotecnológicos resistentes y costo-efectivos. Corynebacterium glutamicum, es un microorganismo usado para producir amino-ácidos que crece en gran variedad de sustratos y es resistente durante la fermentación, a variaciones de pH, temperatura, presión osmótica y acumulación de alcohol, características que lo hacen candidato a ser mejorado para la sínte...

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

    Science.gov (United States)

    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

  8. Cystic Neutrophilic Granulomatous Mastitis: Further Characterization of a Distinctive Histopathologic Entity Not Always Demonstrably Attributable to Corynebacterium Infection.

    Science.gov (United States)

    D'Alfonso, Timothy M; Moo, Tracy-Ann; Arleo, Elizabeth K; Cheng, Esther; Antonio, Lilian B; Hoda, Syed A

    2015-10-01

    Granulomatous lobular mastitis (GLM) is an uncommon condition that typically occurs in parous, reproductive-aged women and can simulate malignancy on the basis of clinical and imaging features. A distinctive histologic pattern termed cystic neutrophilic granulomatous mastitis (CNGM) is seen in some cases of GLM and has been associated with Corynebacterium infection. We sought to further characterize the clinical, imaging, and histopathologic features of CNGM by studying 12 cases and attempted to establish the relationship of this disease with Corynebacterium infection. Patients were women ranging in age from 25 to 49 years (median: 34 y), and all presented with a palpable mass that was painful in half of the cases. In 2 of 9 cases, imaging was highly suspicious for malignancy (BI-RADS 5). CNGM was characterized by lobulocentric granulomas with mixed inflammation and clear vacuoles lined by neutrophils within granulomas. Gram-positive bacilli were identified in 5/12 cases. In 4 patients, the disease process worsened after the diagnostic core biopsy, with the development of a draining sinus in 2 cases. No growth of bacteria was seen in any microbial cultures. No bacterial DNA was identified by 16S rDNA polymerase chain reaction for 1 case that showed gram-positive bacilli on histology. Patients were treated with variable combinations of surgery, antibiotics, and steroids. The time to significant resolution of symptoms ranged from 2 weeks to 6 months. Similar to other forms of GLM, CNGM can mimic malignancy clinically and on imaging. When encountered in a needle core biopsy sample, recognition of the characteristic histologic pattern and its possible association with Corynebacterium infection can help guide treatment. PMID:26200100

  9. Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum▿ †

    OpenAIRE

    Kawaguchi, Hideo; Sasaki, Miho; Vertès, Alain A.; Inui, Masayuki; Yukawa, Hideaki

    2009-01-01

    Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (L...

  10. Brote de mastitis clínica por Corynebacterium spp. y Streptococcus dysgalactiae en cabras en Salta, Argentina

    Directory of Open Access Journals (Sweden)

    Micheloud, J.F.

    2014-04-01

    Full Text Available Intramammary infections are a serious problem for goat’s milk production worldwide. Staphylococcus spp. are the most prevalent pathogens responsible for intramammary infection in small ruminants; however, there is only little information about goat mastitis in Argentina. The objective of this communication was to describe an outbreak of clinical mastitis affecting 12 of 24 lactating goats. Corynebacterium spp. and Streptococcus dysgalactiae were isolated in pure culture from all milk samples. All the clinical isolates were identified by biochemical tests and subjected to antibiotic susceptibility tests.

  11. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

    OpenAIRE

    Schneider Jens; Peters-Wendisch Petra; Stansen K Corinna; Götker Susanne; Maximow Stanislav; Krämer Reinhard; Wendisch Volker F

    2012-01-01

    Abstract Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 m...

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

    OpenAIRE

    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 toward...

  13. Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

    OpenAIRE

    Lange, C.; Rittmann, D.; Wendisch, V. F.; Bott, M.; Sahm, H

    2003-01-01

    Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild ...

  14. Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

    OpenAIRE

    Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-01-01

    Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenz...

  15. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine

    OpenAIRE

    Lange, C.; Rittmann, D.; Wendisch, V. F.; Bott, M.; Sahm, H

    2003-01-01

    Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJCilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in th...

  16. Utilization of fermentation waste (Corynebacterium glutamicum) for biosorption of Reactive Black 5 from aqueous solution

    International Nuclear Information System (INIS)

    A fermentation waste, Corynebacterium glutamicum, was successfully employed as a biosorbent for Reactive Black 5 (RB5) from aqueous solution. This paper initially studied the effect of pretreatment on the biosorption capacity of C. glutamicum toward RB5, using several chemical agents, such as HCl, H2SO4, HNO3, NaOH, Na2CO3, CaCl2 and NaCl. Among these reagents, 0.1 M HNO3 gave the maximum enhancement of the RB5 uptake, exhibiting 195 mg/g at pH 1 with an initial RB5 concentration of 500 mg/l. The solution pH and temperature were found to affect the biosorption capacity, and the biosorption isotherms derived at different pHs and temperatures revealed that a low pH (pH 1) and high temperature (35 deg. C) favored biosorption. The biosorption isotherm was well represented using three-parameter models (Redlich-Peterson and Sips) compared to two-parameter models (Langmuir and Freundlich models). As a result, high correlation coefficients and low average percentage error values were observed for three-parameter models. A maximum RB5 uptake of 419 mg/g was obtained at pH 1 and a temperature of 35 deg. C, according to the Langmuir model. The kinetics of the biosorption process with different initial concentrations (500-2000 mg/l) was also monitored, and the data were analyzed using pseudo-first and pseudo-second order models, with the latter describing the data well. Various thermodynamic parameters, such as ΔGo, ΔHo and ΔSo, were calculated, indicating that the present system was a spontaneous and endothermic process. The use of a 0.1 M NaOH solution successfully desorbed almost all the dye molecules from dye-loaded C. glutamicum biomass at different solid-to-liquid ratios examined

  17. Transcription of malP is subject to phosphotransferase system-dependent regulation in Corynebacterium glutamicum.

    Science.gov (United States)

    Kuhlmann, Nora; Petrov, Dimitar P; Henrich, Alexander W; Lindner, Steffen N; Wendisch, Volker F; Seibold, Gerd M

    2015-09-01

    The Gram-positive Corynebacterium glutamicum co-metabolizes most carbon sources such as the phosphotransferase system (PTS) sugar glucose and the non-PTS sugar maltose. Maltose is taken up via the ABC-transporter MusEFGK2I, and is further metabolized to glucose phosphate by amylomaltase MalQ, maltodextrin phosphorylase MalP, glucokinase Glk and phosophoglucomutase Pgm. Surprisingly, growth of C. glutamicum strains lacking the general PTS components EI or HPr was strongly impaired on the non-PTS sugar maltose. Complementation experiments showed that a functional PTS phosphorelay is required for optimal growth of C. glutamicum on maltose, implying its involvement in the control of maltose metabolism and/or uptake. To identify the target of this PTS-dependent control, transport measurements with 14C-labelled maltose, Northern blot analyses and enzyme assays were performed. The activities of the maltose transporter and enzymes MalQ, Pgm and GlK were not decreased in PTS-deficient C. glutamicum strains, which was corroborated by comparable transcript amounts of musE, musK and musG, as well as of malQ, in C. glutamicum ΔptsH and WT. By contrast, MalP activity was significantly reduced and only residual amounts of malP transcripts were detected in C. glutamicum ΔptsH when compared to WT. Promoter activity assays with the malP promoter in C. glutamicum ΔptsH and WT confirmed that malP transcription is reduced in the PTS-deficient strain. Taken together, we show here for what is to the best of our knowledge the first time a regulatory function of the PTS in C. glutamicum and identify malP transcription as its target. PMID:26296766

  18. Proteome scale comparative modeling for conserved drug and vaccine targets identification in Corynebacterium pseudotuberculosis.

    Science.gov (United States)

    Hassan, Syed Shah; Tiwari, Sandeep; Guimarães, Luís Carlos; Jamal, Syed Babar; Folador, Edson; Sharma, Neha Barve; de Castro Soares, Siomar; Almeida, Síntia; Ali, Amjad; Islam, Arshad; Póvoa, Fabiana Dias; de Abreu, Vinicius Augusto Carvalho; Jain, Neha; Bhattacharya, Antaripa; Juneja, Lucky; Miyoshi, Anderson; Silva, Artur; Barh, Debmalya; Turjanski, Adrian Gustavo; Azevedo, Vasco; Ferreira, Rafaela Salgado

    2014-01-01

    Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens. PMID:25573232

  19. Bayesian Geostatistical Analysis and Ecoclimatic Determinants of Corynebacterium pseudotuberculosis Infection among Horses

    Science.gov (United States)

    Boysen, Courtney; Davis, Elizabeth G.; Beard, Laurie A.; Lubbers, Brian V.; Raghavan, Ram K.

    2015-01-01

    Kansas witnessed an unprecedented outbreak in Corynebacterium pseudotuberculosis infection among horses, a disease commonly referred to as pigeon fever during fall 2012. Bayesian geostatistical models were developed to identify key environmental and climatic risk factors associated with C. pseudotuberculosis infection in horses. Positive infection status among horses (cases) was determined by positive test results for characteristic abscess formation, positive bacterial culture on purulent material obtained from a lanced abscess (n = 82), or positive serologic evidence of exposure to organism (≥1:512)(n = 11). Horses negative for these tests (n = 172)(controls) were considered free of infection. Information pertaining to horse demographics and stabled location were obtained through review of medical records and/or contact with horse owners via telephone. Covariate information for environmental and climatic determinants were obtained from USDA (soil attributes), USGS (land use/land cover), and NASA MODIS and NASA Prediction of Worldwide Renewable Resources (climate). Candidate covariates were screened using univariate regression models followed by Bayesian geostatistical models with and without covariates. The best performing model indicated a protective effect for higher soil moisture content (OR = 0.53, 95% CrI = 0.25, 0.71), and detrimental effects for higher land surface temperature (≥35°C) (OR = 2.81, 95% CrI = 2.21, 3.85) and habitat fragmentation (OR = 1.31, 95% CrI = 1.27, 2.22) for C. pseudotuberculosis infection status in horses, while age, gender and breed had no effect. Preventative and ecoclimatic significance of these findings are discussed. PMID:26473728

  20. Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds.

    Science.gov (United States)

    Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K

    2016-01-01

    Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection. PMID:26817981

  1. Selection and Characterization of a Lysine Yielding Mutant of Corynebacterium glutamicum - a Soil Isolate from Pakistan

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    Habib-ur-Rehman§٭, Abdul Hameed and Safia Ahmed

    2012-01-01

    Full Text Available L-lysine is the second limiting amino acid for poultry and supplemented in broiler feed for optimal performance. Lysine can be produced by inducing mutation in glutamate producing bacteria. The study was conducted to enhance lysine production from a local strain of Corynebacterium glutamicum. The bacterium was mutated by exposure to UV. Mutants resistant to s-2-aminoethyle L-cystein (AEC and showing auxotrophy for L-homoserine were screened for lysine production qualitatively and quantitatively. A mutant showing highest production of lysine (8.2 mg/mL was selected for optimization of physical and nutritional parameters for maximum production of lysine in shake flask. An initial pH 7.6, 30˚C temperature, 300 rpm and 60 h incubation time were the optimized values of physical requirements. Cane molasses and corn starch hydrolysate were required at 15% (w/v in the fermentation media which provided around 9% total sugars to produce maximum lysine (17 to 18 mg/mL. When amonium sulphate was used at 3.5% (w/v level in molasses or corn starch hydrolysate based fermentation media, production of lysine slightly increased above 18 mg/mL. It is concluded that industrial by products like cane molasses, corn steep liquor, and corn starch hydrolysate can be used as carbon and organic nitrogen sources in fermentation medium for scale up process of lysine production and this lysine enriched broth may be used in broiler feed later. However, more potent lysine producing mutant and additional in vivo trials would be required to commercialize this product.

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

    Science.gov (United States)

    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

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    Anh Q. D. Nguyen

    2015-04-01

    Full Text Available 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(dine 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.

  4. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Shioya Suteaki

    2007-06-01

    Full Text Available Abstract Background Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis, which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition. Results We performed a metabolic flux analysis (MFA with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS. The flux from phosphoenolpyruvate (PEP to oxaloacetate (Oxa catalyzed by phosphoenolpyruvate carboxylase (PEPc was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc. Conclusion It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Schluesener Daniela

    2007-01-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

  20. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Hirasawa Takashi

    2009-08-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  5. Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine

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    S Abulmagd

    2013-01-01

    Full Text Available Background: Diphtheria is a highly communicable disease caused by toxin-producing strains of Corynebacterium diphtheriae. Objectives: To evaluate the efficacy of A and B subunits of diphtheria toxin (DT-A, DT-B as potential vaccines against C. diphtheriae. A culture of C. diphtheriae (strain PW 8 was grown on Loeffler plates while Lingood medium was used for production of diphtheria toxin (DT. Materials and Methods: DT was purified and digested to obtain pure DT-A and DT-B and detoxified to obtain diphtheria toxin. Four groups of mice were immunised with different antigens (Ag of C. diphtheriae. Results: The antibody (Ab titres were significantly increased with immunised groups subsequent to three injections. On the other hand, Ab titres were estimated after the three immunisations and the levels of different Ab isotypes were comparatively measured. The levels of various isotypes immune responses showed variation between immunised groups where the IgG subclasses were significantly increased mainly with DPT immunised group. The IgM and IgA were significantly increased with DT-A more than others. Additionally, the evaluation of the cellular immune responses demonstrated that spleen cells from DPT and DT-A groups gave highly significant proliferative response with production of high levels of IL-2 and IFN-γ (Th1/Th2. Separation and purification of DT gene were performed using polymerase chain reaction (PCR and sub-cloned in pGEM-T vector, for further studying of recombinant vaccine. Conclusion: Our results showed the possibility to prepare a potent recombinant vaccine containing whole DT gene or DT-A against C. diphtheriae or could be used in treatment of cancer as it give high levels of IL-2 and IFN-γ.

  6. Implantation of Corynebacterium pseudodiphtheriticum for elimination of Staphylococcus aureus from the nasal cavity in volunteers

    Science.gov (United States)

    Viacheslav, Ilyin; Kiryukhina, Nataliya

    Nasal carriage of Staphylococcus aureus is a well-documented risk factor of infection and inflammation of the skin, soft tissues and bacteremia. It is also known that most often etiology of these disorders is associated with autoinfection. The present-day methods of opportunistic pathogens eradication from the nasal cavity are based principally on the use of antiseptic and antibacterial agents. For instance, a local antibiotic mupirocin in the form of nasal ointment is considered to be the gold standard for the treatment of S. aureus carriage. The literature describes investigations showing how mupirocin can strengthen antibiotic resistance in S. aureus strains, including those with methicillin resistance (MRSA). It is also common knowledge that recolonization of the nasal mucous membrane takes place within several months after mupirocin treatment. This circumstance dictates the necessity to look for alternative ways of preventing the S. aureus carriage and methods of elimination. One of the methods of nasal S. aureus elimination is implantation of nonpathogenic microorganisms which will extrude opportunistic pathogens without impinging the symbiotic microbiota. Effectiveness of saline suspension of Corynebacterium pseudodiphtheriticum containing spray was assessed in a several chamber experiments with simulation of some spaceflight factors (dry immersion, isolation). Various schemes of application of preparations were applied. In all cases of corynebacteria application the strong inhibiting effect against S. aureus was detected. This fact opens a prospect of using nonpathogenic corynebacteria as a nasal probiotic. Administration of the nasal corynebacteria spray possibly prevented cross-infection by MRSA and appearance of staphylococcal infection. Further pre-clinical and clinical study of this bacterial therapy method is under development.

  7. Technetium-99m labeling and fibronectin binding ability of Corynebacterium diphtheriae

    International Nuclear Information System (INIS)

    The use of radionuclides has permitted advances in areas of clinical and scientific knowledge. Several molecules and cells have been labelled with Technetium-99m (99mTc). The stannous chloride (SnCl2) has a significant influence on the labeling and stability of 99mTc radiotracers. The frequent risk of diphtheria epidemics has intensified interest in the virulence factors of Corynebacterium diphtheriae. Although studies have looked at potential adhesins including haemagglutinins and exposed sugar residues, the molecular basis of mechanisms of adherence remains unclear. Adherence of pathogens to mammalian tissues may be mediated by fibronectin (FN) found in body fluids, matrix of connective tissues, and cell surfaces. In the present study we evaluated the binding ability to human plasma FN by 99mTc labeled-C.diphtheriae. Due to adverse effects of stannous ions, microorganisms were submitted to survival and filamentation induction assays. Data showed a dose dependent susceptibility to SnCl2 bactericidal effects. Cell filamentation was observed for concentrations of SnCl2 > 110 μg/ml. Adherence levels of 99mTc labelled 241strain to coverslips coated with 20 μg/ml FN were higher (P = 0.0037) than coated with bovine serum albumin. FN binding by the sucrose fermenting 241 C. diphtheriae strain (8.9% + 2.6) was significantly lower (P=0.0139) than Staphylococcus aureus Cowan I strain (34.1% ± 1.2). Therefore, bacterial 99mTc labeling represents an additional tool that may contribute to the comprehension of C. diphtheriae interactions with host receptors such as FN that act as biological organizers by holding bacterial cells in position and guiding their migration. (author)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  10. Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

    Directory of Open Access Journals (Sweden)

    Luciene de Fátima Costa Torres

    2013-05-01

    Full Text Available Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp, 16S rRNA (C. ulcerans and C. pseudotuberculosis, pld (C. pseudotuberculosis, dtxR (C. diphtheriae and tox [diphtheria toxin (DT ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

  11. Functional analysis of sequences adjacent to dapE of Corynebacterium glutamicum reveals the presence of aroP, which encodes the aromatic amino acid transporter.

    OpenAIRE

    Wehrmann, A; Morakkabati, S; Krämer, R; Sahm, H; Eggeling, L

    1995-01-01

    An initially nonclonable DNA locus close to a gene of L-lysine biosynthesis in Corynebacterium glutamicum was analyzed in detail. Its stepwise cloning and its functional identification by monitoring the amino acid uptakes of defined mutants, together with mechanistic studies, identified the corresponding structure as aroP, the general aromatic amino acid uptake system.

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

    Science.gov (United States)

    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

  13. Increased Glucose Utilization in Corynebacterium glutamicum by Use of Maltose, and Its Application for the Improvement of l-Valine Productivity▿

    OpenAIRE

    Krause, Felix S.; Henrich, Alexander; Blombach, Bastian; Krämer, Reinhard; Eikmanns, Bernhard J.; Seibold, Gerd M.

    2009-01-01

    Corynebacterium glutamicum efficiently utilizes maltose as a substrate. We show here that the presence of maltose increases glucose utilization by raising the expression of ptsG, which encodes the glucose-specific EII permease of the phosphotransferase system. Consequently, the l-valine productivity of a pyruvate dehydrogenase complex-deficient C. glutamicum strain was improved by the presence of maltose.

  14. Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Ins Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

    Czech Academy of Sciences Publication Activity Database

    Hüser, A.T.; Chassagnole, Ch.; Lindley, N.D.; Merkamm, M.; Guyonvarch, A.; Elišáková, Veronika; Pátek, Miroslav; Kalinowski, J.; Brune, I.; Pühler, A.; Tauch, A.

    2005-01-01

    Roč. 71, č. 6 (2005), s. 3255-3268. ISSN 0099-2240 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * metabolic flux Subject RIV: EE - Microbiology, Virology Impact factor: 3.818, year: 2005

  15. Dissection of Ammonium Uptake Systems in Corynebacterium glutamicum: Mechanism of Action and Energetics of AmtA and AmtB▿

    OpenAIRE

    Walter, Britta; Küspert, Melanie; Ansorge, Daniel; Krämer, Reinhard; Burkovski, Andreas

    2008-01-01

    Corynebacterium glutamicum has two different Amt-type proteins. While AmtB has a low substrate affinity and is not saturable up to 3 mM methylammonium, AmtA has a high substrate affinity and mediates saturable, membrane potential-dependent transport, resulting in a high steady-state accumulation of methylammonium, even in the absence of metabolic trapping.

  16. The effect of isoleucine limitation on valine precursor levels and acetohydroxyacid synthase activity in stringent (rel+) and relaxed (rel-) Corynebacterium glutamicum mutant

    Czech Academy of Sciences Publication Activity Database

    Denina, I.; Paegle, L.; Ruklisha, M.; Prouza, Marek; Pátek, Miroslav

    Praha: Verlag, 2006, s. 156. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Institutional research plan: CEZ:AV0Z50200510 Keywords : isoleucine * corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology

  17. RamB, the Transcriptional Regulator of Acetate Metabolism in Corynebacterium glutamicum, Is Subject to Regulation by RamA and RamB▿

    OpenAIRE

    Cramer, Annette; Auchter, Marc; Frunzke, Julia; Bott, Michael; Eikmanns, Bernhard J.

    2006-01-01

    In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that RamB represses its own expression by direct interaction with a 13-bp motif in the ramB promoter region. Additionally, ramB expression is subject to carbon source-dependent positive control by RamA.

  18. Atypical Location of Double-Strand Origin of Replication (nic site) on the Plasmid pGA1 from Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Abrhámová, Zdeňka; Pátek, Miroslav; Nešvera, Jan

    2002-01-01

    Roč. 47, č. 4 (2002), s. 307-310. ISSN 0015-5632 R&D Projects: GA ČR GA204/97/0528; GA ČR GA204/01/0998 Institutional research plan: CEZ:AV0Z5020903 Keywords : plasmid * pga1 * corynebacterium glutamicum Subject RIV: EE - Microbiology, Virology Impact factor: 0.979, year: 2002

  19. l-Valine Production during Growth of Pyruvate Dehydrogenase Complex- Deficient Corynebacterium glutamicum in the Presence of Ethanol or by Inactivation of the Transcriptional Regulator SugR▿

    OpenAIRE

    Blombach, Bastian; Arndt, Annette; Auchter, Marc; Eikmanns, Bernhard J.

    2008-01-01

    Pyruvate dehydrogenase complex-deficient strains of Corynebacterium glutamicum produce l-valine from glucose only after depletion of the acetate required for growth. Here we show that inactivation of the DeoR-type transcriptional regulator SugR or replacement of acetate by ethanol already in course of the growth phase results in efficient l-valine production.

  20. Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    The cysteine free derivative of the arsenic repressor ArsR from Corynebacterium glutamicum was expressed, purified, crystallized and X-ray diffraction data up to 1.86 Å resolution have been collected. The crystals belonged to the space group P4 with the unit-cell parameters a = b = 41.84, c = 99.47 Å. ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å

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

    Science.gov (United States)

    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

  2. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

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

    Science.gov (United States)

    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

  4. Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Venkatesh KV

    2006-10-01

    Full Text Available Abstract Background Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. Results Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. Conclusion We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at

  5. Utilization of fermentation waste (Corynebacterium glutamicum) for biosorption of Reactive Black 5 from aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Vijayaraghavan, K. [Division of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Chonju 561-756 (Korea, Republic of)]. E-mail: drkvijy@chonbuk.ac.kr; Yun, Yeoung-Sang [Division of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Chonju 561-756 (Korea, Republic of)]. E-mail: ysyun@chonbuk.ac.kr

    2007-03-06

    A fermentation waste, Corynebacterium glutamicum, was successfully employed as a biosorbent for Reactive Black 5 (RB5) from aqueous solution. This paper initially studied the effect of pretreatment on the biosorption capacity of C. glutamicum toward RB5, using several chemical agents, such as HCl, H{sub 2}SO{sub 4}, HNO{sub 3}, NaOH, Na{sub 2}CO{sub 3}, CaCl{sub 2} and NaCl. Among these reagents, 0.1 M HNO{sub 3} gave the maximum enhancement of the RB5 uptake, exhibiting 195 mg/g at pH 1 with an initial RB5 concentration of 500 mg/l. The solution pH and temperature were found to affect the biosorption capacity, and the biosorption isotherms derived at different pHs and temperatures revealed that a low pH (pH 1) and high temperature (35 deg. C) favored biosorption. The biosorption isotherm was well represented using three-parameter models (Redlich-Peterson and Sips) compared to two-parameter models (Langmuir and Freundlich models). As a result, high correlation coefficients and low average percentage error values were observed for three-parameter models. A maximum RB5 uptake of 419 mg/g was obtained at pH 1 and a temperature of 35 deg. C, according to the Langmuir model. The kinetics of the biosorption process with different initial concentrations (500-2000 mg/l) was also monitored, and the data were analyzed using pseudo-first and pseudo-second order models, with the latter describing the data well. Various thermodynamic parameters, such as {delta}G{sup o}, {delta}H{sup o} and {delta}S{sup o}, were calculated, indicating that the present system was a spontaneous and endothermic process. The use of a 0.1 M NaOH solution successfully desorbed almost all the dye molecules from dye-loaded C. glutamicum biomass at different solid-to-liquid ratios examined.

  6. Prediction of DtxR regulon: Identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae

    Directory of Open Access Journals (Sweden)

    Hasnain Seyed

    2004-09-01

    Full Text Available Abstract Background The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae. Result Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within C. diphtheriae genome. In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation. Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay. The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG, an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps which is involved in iron storage and oxidative stress defense. In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin. Conclusions We have used an in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae. Our analysis shows that DtxR could provide a molecular link between Fe+2-induced Fenton's reaction and protection of DNA from oxidative damage. DtxR-regulated Dps prevents lethal combination of Fe+2 and H2O2 and also protects DNA by nonspecific DNA-binding. In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase

  7. Antigenic Studies of Oral and Nonoral Black-Pigmented Bacteroides Strains

    OpenAIRE

    Reed, Michael J; Slots, Jørgen; Mouton, Christian; Genco, Robert J.

    1980-01-01

    Antigens of several oral and nonoral strains of Bacteroides asaccharolyticus (proposed classification of oral B. asaccharolyticus, Bacteroides gingivalis), Bacteroides melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus, and B. melaninogenicus subsp. levii were identified in soluble preparations obtained by sonication, autoclaving, and NaOH treatment of whole bacterial cells. The sonicate preparations contained the most complete representation of soluble antigens usi...

  8. L-lysin export in Corynebacterium glutamicum: Physiological and molecular-biological characterisation of the carrier-mediated export of a primary metabolite; L-Lysinexport bei Corynebacterium glutamicum: Physiologische und molekularbiologische Charakterisierung des Carrier-vermittelten Exportes eines Primaermetaboliten

    Energy Technology Data Exchange (ETDEWEB)

    Vrljic, M.M.

    1997-02-01

    The aim of the present study was to isolate the gene encoding the lysin export carrier of Corynebacterium glutamicum by way of identifying the molecular make-up of the lysin exporter. Neither mutants for heterologous complementation nor sequence information for hybridisation experiments were available for this purpose. The first step therefore was to isolate a mutant of C. glutamicum specifically deficient in lysin export. In contrast to strains deficient in amino acid uptake, which can be selected via their resistance to toxic amino acid analogues, mutants deficient in amino acid export can only be isolated by testing strains singly for loss of the export function in question. Furthermore, as the wildtype of C. glutamicum shows no lysin excretion at all unless manipulated to increase its cellular lysin concentration, it was first necessary to establish a system for induction of lysin excretion in the wildtype. This would then permit isolation of lysin export deficient mutants of C. glutamicum for subsequent homologous complementation experiments.

  9. Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan.

    Science.gov (United States)

    Seto, Yukiji; Komiya, Takako; Iwaki, Masaaki; Kohda, Tomoko; Mukamoto, Masafumi; Takahashi, Motohide; Kozaki, Shunji

    2008-03-01

    Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae. PMID:18362399

  10. Conjugative plasmid in Corynebacterium flaccumfaciens subsp. oortii that confers resistance to arsenite, arsenate, and antimony(III)

    Energy Technology Data Exchange (ETDEWEB)

    Hendrick, C.A.; Haskins, W.P.; Vidaver, A.K.

    1984-07-01

    Gene transfer systems for phytopathogenic corynebacteria have not been reported previously. In this paper a conjugative 46-megadalton plasmid (pDG101) found in Corynebacterium flaccumfaciens subsp. oorii CO101 is described that mediates resistance to arsenite, arsenate, and antimony(III). Transfer of the plasmid from CO101 to four other strains from the C. flaccumfaciens group occurred between cells immobilized on nitrocellulose filters or on agar surfaces. Transconjugant strains expressed the same levels of metal resistance as the donor strain and were able to act as donor strains in subsequent matings. The physical presence of the plasmid was detected by agarose gel electrophoresis. Arsenite-sensitive derivatives of the donor and transconjugant strains were obtained after heat treatment; these were cured of pDG101.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and biochar.

    Science.gov (United States)

    Zhang, Hairong; Tang, Jingchun; Wang, Lin; Liu, Juncheng; Gurav, Ranjit Gajanan; Sun, Kejing

    2016-09-01

    The present work aimed to develop a novel strategy to bioremediate the petroleum hydrocarbon contaminants in the environment. Salt tolerant bacterium was isolated from Dagang oilfield, China and identified as Corynebacterium variabile HRJ4 based on 16S rRNA gene sequence analysis. The bacterium had a high salt tolerant capability and biochar was developed as carrier for the bacterium. The bacteria with biochar were most effective in degradation of n-alkanes (C16, C18, C19, C26, C28) and polycyclic aromatic hydrocarbons (NAP, PYR) mixture. The result demonstrated that immobilization of C. variabile HRJ4 with biochar showed higher degradation of total petroleum hydrocarbons (THPs) up to 78.9% after 7-day of incubation as compared to the free leaving bacteria. The approach of this study will be helpful in clean-up of petroleum-contamination in the environments through bioremediation process using eco-friendly and cost effective materials like biochar. PMID:27593267

  15. Whole-Genome Sequence of Corynebacterium auriscanis Strain CIP 106629 Isolated from a Dog with Bilateral Otitis from the United Kingdom

    Science.gov (United States)

    Tiwari, Sandeep; Jamal, Syed Babar; Oliveira, Leticia Castro; Clermont, Dominique; Bizet, Chantal; Mariano, Diego; de Carvalho, Paulo Vinicius Sanches Daltro; Souza, Flavia; Pereira, Felipe Luiz; de Castro Soares, Siomar; Dorella, Fernanda; Carvalho, Alex; Leal, Carlos; Barh, Debmalya; Figueiredo, Henrique; Hassan, Syed Shah; Azevedo, Vasco

    2016-01-01

    In this work, we describe a set of features of Corynebacterium auriscanis CIP 106629 and details of the draft genome sequence and annotation. The genome comprises a 2.5-Mbp-long single circular genome with 1,797 protein-coding genes, 5 rRNA, 50 tRNA, and 403 pseudogenes, with a G+C content of 58.50%. PMID:27516502

  16. Draft Genome Sequence for the Type Strain of Corynebacterium afermentans LCDC 88-0199T, Isolated from a Human Blood Culture

    Science.gov (United States)

    Bernier, Anne-Marie

    2016-01-01

    A draft genome for Corynebacterium afermentans LCDC 88-0199T was investigated. The size of the genome was 2,345,615 bp with an observed G+C content of 64.85%. Annotation revealed 2 rRNA sequences, 54 tRNA genes, and 2,164 coding sequences. Genome coverage was 85× and consisted of 24 contigs with an N50 of 187,988 bp. PMID:27389276

  17. Regulation of Corynebacterium glutamicum Heat Shock Response by the Extracytoplasmic-Function Sigma Factor SigH and Transcriptional Regulators HspR and HrcA▿ §

    OpenAIRE

    Ehira, Shigeki; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2009-01-01

    Heat shock response in Corynebacterium glutamicum was characterized by whole-genome expression analysis using a DNA microarray. It was indicated that heat shock response of C. glutamicum included not only upregulation of heat shock protein (HSP) genes encoding molecular chaperones and ATP-dependent proteases, but it also increased and decreased expression of more than 300 genes related to disparate physiological functions. An extracytoplasmic-function sigma factor, SigH, was upregulated by he...

  18. In Vitro Activities of the New Semisynthetic Glycopeptide Telavancin (TD-6424), Vancomycin, Daptomycin, Linezolid, and Four Comparator Agents against Anaerobic Gram-Positive Species and Corynebacterium spp.

    OpenAIRE

    Goldstein, Ellie J. C.; Citron, Diane M.; Merriam, C. Vreni; Warren, Yumi A.; Tyrrell, Kerin L.; Fernandez, Helen T.

    2004-01-01

    Telavancin is a new semisynthetic glycopeptide anti-infective with multiple mechanisms of action, including inhibition of bacterial membrane phospholipid synthesis and inhibition of bacterial cell wall synthesis. We determined the in vitro activities of telavancin, vancomycin, daptomycin, linezolid, quinupristin-dalfopristin, imipenem, piperacillin-tazobactam, and ampicillin against 268 clinical isolates of anaerobic gram-positive organisms and 31 Corynebacterium strains using agar dilution m...

  19. Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis.

    OpenAIRE

    Pátek, M; Krumbach, K.; Eggeling, L; Sahm, H

    1994-01-01

    Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes...

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

    OpenAIRE

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

    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 Bl...

  1. The three-dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase-NADPH-L-2-amino-6-methylene-pimelate.

    OpenAIRE

    Cirilli, M.; Scapin, G.; Sutherland, A; Vederas, J. C.; Blanchard, J. S.

    2000-01-01

    The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate D-dehydrogenase in a ternary complex with NADPH and L-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 A. L-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 microM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+ -dependen...

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

    OpenAIRE

    Kabus, A.; Georgi, T; Wendisch, V. F.; Bott, M

    2007-01-01

    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-pr...

  3. Determination of Immobilization Process Parameters of Corynebacterium glutamicum on Kappa carrageenan, Its Application in L-lysine Fermentation and The Investigation Into Its Storage Conditions

    OpenAIRE

    Suong Thi Hong Nguyen

    2014-01-01

    The parameters of the immobilized process of Corynebacterium glutamicum on kappa carrageenan were identified by Plackett-Burman matrix, and the experiments were designed by response surface methodology having the central composite designs (RSM-CCD). The maximum yield of cell immobilization on kappa carrageenan carrier reached at 78% ± 2%. Optimal parameters were 3 grams kappa carrageenan per 100 militters sterile water and 58.58 million cfu/mL, forming gels at 100C for 25 minutes ...

  4. 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

    OpenAIRE

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

    2005-01-01

    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 significa...

  5. Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

    OpenAIRE

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

    2004-01-01

    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 β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were ab...

  6. Comparative studies for the biotechnological production of l-Lysine by immobilized cells of wild-type Corynebacterium glutamicum ATCC 13032 and mutant MH 20-22 B

    OpenAIRE

    Razak, Meerza Abdul; Viswanath, Buddolla

    2015-01-01

    Establishing a cost and time efficient approach for bioprocess optimization is desired but is challenging. In the present work, we have addressed the effectiveness of using immobilized cells for aerobic processes, behaviour of immobilized cells, optimization and upstream bioprocess analysis for the production of lysine by immobilized cells of Corynebacterium glutamicum ATCC 13032 and MH 20-22 B in stirred tank bioreactor. Optimized operational conditions for maximal yield and productivity wer...

  7. Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae.

    OpenAIRE

    Boyd, J.; Oza, M N; Murphy, J. R.

    1990-01-01

    Although the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of tox expression is controlled, to a large extent, by its bacterial host Corynebacterium diphtheriae. Optimal yields of tox gene products are obtained only when iron becomes the growth-rate-limiting substrate. Previous studies suggest that regulation of tox expression is mediated through an iron-binding aporepressor. To facilitate molecular cloning of the tox...

  8. 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

    OpenAIRE

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

    2012-01-01

    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 demon...

  9. Changes in growth and in uptake, distribution and translocation of phosphorus in susceptible and resistant alfalfa plants induced by Corynebacterium insidiosum

    International Nuclear Information System (INIS)

    The weight of alfalfa plants, especially roots of susceptible strain decreased when inoculated with Corynebacterium insidiosum. At the 6th week after inoculation the 32P uptake per plant and its translocation into the above-ground organs considerably decreased in susceptible plants. On the other hand, the 32P uptake increased and radiophosphorus was accumulated in above-ground organs in resistant plants. (author)

  10. Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

    Science.gov (United States)

    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

  11. Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

    Directory of Open Access Journals (Sweden)

    Ju-Sim Kim

    Full Text Available 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(2O(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(2O(2. In contrast, exposure of C. diphtheriae C7(β to H(2O(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(2O(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(2O(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(2O(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(2O(2.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. A role for sigma factor SigE in Corynebacterium pseudotuberculosis resistance to nitric oxide/ peroxide stress

    Directory of Open Access Journals (Sweden)

    Luis G. C. Pacheco

    2012-04-01

    Full Text Available Pathogenic intracellular bacteria can respond to antimicrobial mechanisms of the host cell through transient activation of stress-responsive genes by alternative sigma (σ factors of the RNA polymerase. We evaluated the contribution of the extracytoplasmic function sigma factor σE for Corynebacterium pseudotuberculosis resistance to stress conditions resembling those found intracellularly during infection. A sigE null mutant strain (delta-sigE of this bacterium was more susceptible in vitro to acidic pH, cell surface stressors, and biologically relevant concentrations of nitric oxide (NO. The same mutant strain was unable to persist in C57BL/6 mice but remained infective in mice lacking inducible nitric oxide synthase (iNOS, confirming the significance of σE for resistance to nitric oxide/peroxide stress in vivo. High-throughput proteomic analysis identified NO-responsive extracellular proteins of C. pseudotuberculosis and demonstrated the participation of σE in composition of this bacterium´s exoproteome.

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

    Science.gov (United States)

    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

  17. Phenotypic, molecular characterization, antimicrobial susceptibility and draft genome sequence of Corynebacterium argentoratense strains isolated from clinical samples

    Directory of Open Access Journals (Sweden)

    I. Fernández-Natal

    2016-03-01

    Full Text Available During a 12-year period we isolated five Corynebacterium argentoratense strains identified by phenotypic methods, including the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF and 16S rRNA gene sequencing. In addition, antimicrobial susceptibility was determined, and genome sequencing for the detection of antibiotic resistance genes was performed. The organisms were isolated from blood and throat cultures and could be identified by all methods used. All strains were resistant to cotrimoxazole, and resistance to β-lactams was partly present. Two strains were resistant to erythromycin and clindamycin. The draft genome sequences of theses isolates revealed the presence of the erm(X resistance gene that is embedded in the genetic structure of the transposable element Tn5423. Although rarely reported as a human pathogen, C. argentoratense can be involved in bacteraemia and probably in other infections. Our results also show that horizontal transfer of genes responsible for antibiotic resistance is occurring in this species.

  18. Effects of the removal of the primary tumor and immunotherapy with Corynebacterium parvum on metastatic tumor proliferation.

    Directory of Open Access Journals (Sweden)

    Hashimoto,Osamu

    1983-08-01

    Full Text Available The effects of surgical intervention by removal of the primary focus, and the effectiveness of an immunomodulator, Corynebacterium parvum (Cp, on the proliferation of metastatic tumor tissue were investigated by following the postoperative changes in the 3H-thymidine labelling rate of metastatic tissue in an experimental model of metastasis in mice. In addition, the delayed type hypersensitivity reaction (DTH was studied to investigate the immune capacity of the host. The labelling rate of mice that had the primary focus removed remained high with little variation, while that of the mice not operated on decreased gradually. On the other hand, in mice undergoing a sham operation, the rate was the same as that of the mice with the primary focus removed for a short while, but then gradually decreased. When Cp was administered, especially before removal of the primary focus, the rate was lower than that of the tumor bearing control group and decreased steadily. The number of pulmonary metastatic nodules was increased by removal of the primary focus, but this increase was inhibited by the administration of Cp which prolonged life. The depression in the DTH was less in the group given Cp preoperativeLy than in either the group of mice having the primary focus removed or those not having it removed.

  19. Phenotypic, molecular characterization, antimicrobial susceptibility and draft genome sequence of Corynebacterium argentoratense strains isolated from clinical samples.

    Science.gov (United States)

    Fernández-Natal, I; Sáez-Nieto, J A; Rodríguez-Lázaro, D; Valdezate-Ramos, S; Parras-Padilla, T; Medina, M J; Rodríguez-Pollán, R H; Blom, J; Tauch, A; Soriano, F

    2016-03-01

    During a 12-year period we isolated five Corynebacterium argentoratense strains identified by phenotypic methods, including the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and 16S rRNA gene sequencing. In addition, antimicrobial susceptibility was determined, and genome sequencing for the detection of antibiotic resistance genes was performed. The organisms were isolated from blood and throat cultures and could be identified by all methods used. All strains were resistant to cotrimoxazole, and resistance to β-lactams was partly present. Two strains were resistant to erythromycin and clindamycin. The draft genome sequences of theses isolates revealed the presence of the erm(X) resistance gene that is embedded in the genetic structure of the transposable element Tn5423. Although rarely reported as a human pathogen, C. argentoratense can be involved in bacteraemia and probably in other infections. Our results also show that horizontal transfer of genes responsible for antibiotic resistance is occurring in this species. PMID:26933505

  20. Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074.

    Science.gov (United States)

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi

    2016-09-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue. PMID:27215832

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9

    Directory of Open Access Journals (Sweden)

    Dalong Zhang

    2014-12-01

    Full Text Available L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

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

    Science.gov (United States)

    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

  13. Expression, purification, crystallization and preliminary crystallographic analysis of Cg1458: a novel oxaloacetate decarboxylase from Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    To elucidate the mechanism of oxaloacetate decarboxylation by Cg1458, recombinant Cg1458 has been purified and crystallized. Oxaloacetate decarboxylase catalyses the decarboxylation of oxaloacetate to pyruvate and CO2. Recently, the Corynebacterium glutamicum gene product Cg1458 was determined to be a soluble oxaloacetate decarboxylase. To elucidate the mechanism of oxaloacetate decarboxylation by Cg1458, recombinant Cg1458 was purified and crystallized. The best crystal was grown from 0.2 M MgCl2, 0.1 M Bis-Tris pH 6.0, 25%(w/v) polyethylene glycol 3350 using the hanging-drop method. The crystals belonged to space group P43212, with unit-cell parameters a = b = 124.1, c = 73.6 Å. The crystals are most likely to contain a dimer in the asymmetric unit, with a VM value of 2.27 Å3 Da−1. A full data set was collected at 1.9 Å resolution using synchrotron radiation on beamline BL17U of SSRF, Shanghai, China. Structure-solution attempts by molecular replacement were successful with PDB entries 3qdf or 2dfu as the template

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Peifer Susanne

    2012-10-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  4. Expression, purification, crystallization and preliminary crystallographic analysis of SpaA, a major pilin from Corynebacterium diphtheriae

    International Nuclear Information System (INIS)

    SpaA, one of the major pilins of C. diphtheriae, has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.6 Å resolution. Bacterial pili are cell-surface organelles that are critically involved in adhesion to host cells, leading to the colonization of host tissues and the establishment of infections. Whereas the pili of Gram-negative bacteria have been extensively studied, those of Gram-positive bacteria came to light only recently after the discovery and characterization of Corynebacterium diphtheriae pili. These newly discovered pili are formed by the covalent polymerization of pilin subunits catalyzed by sortase enzymes, making them fundamentally different from the noncovalent pilin assemblies of Gram-negative bacteria. Here, the expression, crystallization and preliminary crystallographic analysis of SpaA, which forms the shaft of one of the three types of pili expressed by C. diphtheriae, are reported. SpaA53–486 crystals diffracted to 1.6 Å resolution and belonged to space group P212121, with unit-cell parameters a = 34.9, b = 64.1, c = 198.7 Å, α = β = γ = 90°

  5. Key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes.

    Science.gov (United States)

    Serrano, Ana; Frago, Susana; Herguedas, Beatriz; Martínez-Júlvez, Marta; Velázquez-Campoy, Adrián; Medina, Milagros

    2013-01-01

    Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis. PMID:22892871

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

    Science.gov (United States)

    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

  7. 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

    Science.gov (United States)

    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

  8. Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum

    OpenAIRE

    Radoš, Dušica; David L Turner; Fonseca, Luís L.; Carvalho, Ana Lúcia; Blombach, Bastian; Eikmanns, Bernhard J.; Neves, Ana Rute; Santos, Helena

    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 i...

  9. Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

    OpenAIRE

    Morabbi Heravi, Kambiz; 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 reveal...

  10. Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids

    OpenAIRE

    Reinscheid, Dieter J.; Kronemeyer, Wolfgang; Eggeling, Lothar; Eikmanns, Bernhard J.; Sahm, Hermann

    1994-01-01

    The hom-1-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies o...

  11. Characterization of myo-Inositol Utilization by Corynebacterium glutamicum: the Stimulon, Identification of Transporters, and Influence on l-Lysine Formation▿

    OpenAIRE

    Krings, Eva; Krumbach, Karin; Bathe, Brigitte; Kelle, Ralf; Wendisch, Volker F.; Sahm, Hermann; Eggeling, Lothar

    2006-01-01

    Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h−1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >1...

  12. Characterization of myo-Inositol Utilization by Corynebacterium glutamicum: the Stimulon, Identification of Transporters, and Influence on L-Lysine Formation

    OpenAIRE

    Krings, E.; Krumbach, K.; Bahte, B.; Kelle, R.; Wendisch, V.; Sahm, H; Eggeling, L

    2006-01-01

    Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >1...

  13. Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

    OpenAIRE

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

    2013-01-01

    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 wa...

  14. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans.

    OpenAIRE

    Jäger, W; Schäfer, A.; Pühler, A; Labes, G; Wohlleben, W.

    1992-01-01

    The expression of the structural gene (sacB) encoding Bacillus subtilis levansucrase in two gram-positive soil bacteria, Corynebacterium glutamicum ATCC 13032 and Streptomyces lividans 1326, was investigated. sacB expression in the presence of sucrose is lethal to C. glutamicum but not to S. lividans. While S. lividans secretes levansucrase into the medium, we could show that the enzyme is retained by C. glutamicum cells. Our results imply that the sacB gene can be used as a positive selectio...

  15. Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

    OpenAIRE

    Oikawa Tadao; Matsui Daisuke; Stansen K Corinna; Youn Jung-Won; Kato Osamu; Wendisch Volker F

    2010-01-01

    Abstract Background Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The abso...

  16. The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Tsai, Ching-Ju; Ejsing, Christer S.; Shevchenko, Andrej;

    2007-01-01

    The osmoregulated and chill-sensitive glycine-betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence...... crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for...

  17. PARÁMETROS FISICOQUÍMICOS PARA LA SÍNTESIS DE ÁCIDO LÁCTICO O ETANOL DE LA BACTERIA (Corynebacterium glutamicum Physico-Chemical Parameter for Production of Lactic Acid or Ethanol of (Corynebacterium glutamicum Bacteria

    Directory of Open Access Journals (Sweden)

    ANGÉLICA CASTELLANOS

    2011-08-01

    Full Text Available El interés por obtener productos para la industria de biocombustibles a partir de desechos agrícolas, conduce a la búsqueda de nuevos sistemas biotecnológicos resistentes y costo-efectivos. Corynebacterium glutamicum, es un microorganismo usado para producir amino-ácidos que crece en gran variedad de sustratos y es resistente durante la fermentación, a variaciones de pH, temperatura, presión osmótica y acumulación de alcohol, características que lo hacen candidato a ser mejorado para la síntesis de ácido láctico y etanol. Aún se desconocen aspectos de su fisiología que aumenten su eficiencia en convertir azúcares (C5 y C6 en estos dos metabolitos. Por tanto, este trabajo buscó identificar los parámetros fisicoquímicos que tuvieron un mayor efecto sobre crecimiento bacteriano y síntesis de ácido láctico o etanol en un sistema por lotes. Para lograr este objetivo, ocho variables fueron evaluadas en un modelo estadístico producido en erlenmeyer; con los resultados obtenidos, se hallaron las mejores condiciones que fueron puestas a prueba en un cultivo en biorreactor. La temperatura, concentración de biotina y azúcar fueron las variables de mayor impacto (pThe interest to obtain products for the bio-fuel industry from renewable resources has directed research to find resistant and costs-effective biotechnological systems. Corynebacterium glutamicum, is a microorganism used to produce amino acids, that grows in wide variety of substrates and its resistance during fermentation to pH, temperature, osmotic pressure variations and alcohol aggregate, renders this organism a suitable candidate to improve by genetic modifications lactic acid and ethanol synthesis. However, some aspects of its physiology remain unknown, such us increase lactic acid and ethanol production from C5 and C6 sugars. For this reason, the main aim in our work was to identify the most important variables with impact on culture and the best culture conditions

  18. Technetium-99m labeling and fibronectin binding ability of Corynebacterium diphtheriae; Marcacao de Corynebacterium diphtheriae com Tecnecio-99m e avaliacao da capacidade de ligacao a fibronectina de plasma humano

    Energy Technology Data Exchange (ETDEWEB)

    Souza, S.M.S.; Nagao, P.E.; Bernardo-Filho, M. [Universidade do Estado do Rio de Janeiro, RJ (Brazil). Inst. de Biologia Roberto Alcantara Gomes; Pereira, G.A.; Napoleao, F.; Andrade, A.F.B.; Hirata Junior, R.; Mattos-Guaraldi, A.L. [Universidade do Estado do Rio de Janeiro, RJ (Brazil). Faculdade de Ciencias Medicas

    2004-04-15

    The use of radionuclides has permitted advances in areas of clinical and scientific knowledge. Several molecules and cells have been labelled with Technetium-99m ({sup 99m}Tc). The stannous chloride (SnCl{sub 2}) has a significant influence on the labeling and stability of {sup 99m}Tc radiotracers. The frequent risk of diphtheria epidemics has intensified interest in the virulence factors of Corynebacterium diphtheriae. Although studies have looked at potential adhesins including haemagglutinins and exposed sugar residues, the molecular basis of mechanisms of adherence remains unclear. Adherence of pathogens to mammalian tissues may be mediated by fibronectin (FN) found in body fluids, matrix of connective tissues, and cell surfaces. In the present study we evaluated the binding ability to human plasma FN by {sup 99m}Tc labeled-C.diphtheriae. Due to adverse effects of stannous ions, microorganisms were submitted to survival and filamentation induction assays. Data showed a dose dependent susceptibility to SnCl{sub 2} bactericidal effects. Cell filamentation was observed for concentrations of SnCl{sub 2} > 110 {mu}g/ml. Adherence levels of {sup 99m}Tc labelled 241strain to coverslips coated with 20 {mu}g/ml FN were higher (P = 0.0037) than coated with bovine serum albumin. FN binding by the sucrose fermenting 241 C. diphtheriae strain (8.9% + 2.6) was significantly lower (P=0.0139) than Staphylococcus aureus Cowan I strain (34.1% {+-} 1.2). Therefore, bacterial {sup 99m}Tc labeling represents an additional tool that may contribute to the comprehension of C. diphtheriae interactions with host receptors such as FN that act as biological organizers by holding bacterial cells in position and guiding their migration. (author)

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

    Science.gov (United States)

    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

  20. Assessment of blood changes post-challenge with Corynebacterium pseudotuberculosis and its exotoxin (phospholipase D: A comprehensive study in goat

    Directory of Open Access Journals (Sweden)

    Z. K. H. Mahmood

    2015-09-01

    Full Text Available Aim: There is very little information regarding blood changes during the challenge of phospholipase D (PLD in goats. Therefore, this experiment was conducted to study the changes in blood after the challenge with Corynebacterium pseudotuberculosis and its exotoxin, PLD to fill in the gap of caseous lymphadenitis (CLA research. Materials and Methods: Twenty-six crossbred Boer goats aged 12-14 months were divided into 3 groups; the first group n=6 was inoculated with 1 ml phosphate buffered solution s.c. as the control. The second group n=10 was inoculated with C. pseudotuberculosis 1 × 109 cfu s.c. The third group n=10 was intravenous injected with PLD 1 ml/20 kg body weight. Serial blood collections were done at 1 h, 3 h, 5 h, 8 h, and 12 h then every 24 h post-inoculation for the first 30 days of the experiment. Subsequently, the blood collection continued twice a week till the end of the experiment (90 days post-challenge. Results: Both C. pseudotuberculosis and PLD treated groups showed significant changes (p<0.05 in red blood cell count, hemoglobin (Hb, packed cell volume, mean corpuscular volume, mean corpuscular Hb concentration, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, globulin, and total plasma proteins. Similarly, both treated groups showed significant changes (p<0.05 in alanine transaminase, alkaline phosphatase, aspartate transaminase, total bilirubin, calcium concentration, creatine phosphokinase, creatinine, gamma-glutamyl transpeptidase, urea concentration, lactate dehydrogenase, prothrombin time, and activated partial thromboplastin time. Conclusion: It concluded that C. pseudotuberculosis and PLD have a negative impact on the goat’s health in general reflected by all those changes recorded in the hemogram, leukogram, and the blood chemistry.

  1. Chemoimmunotherapy of small cell bronchogenic carcinoma with VP-16-213, ifosfamide, vincristine, adriamycin, and Corynebacterium parvum

    International Nuclear Information System (INIS)

    Thirty-five consecutive patients with small cell bronchogenic carcinoma (SCBC) received chemoimmunotherapy with VP-16-213, Ifosfamide, vincristine, Adriamycin, and Corynebacterium parvum. Of 33 evaluable patients, 26 (79%) responded with complete (55%) or partial (24%) remissions. Complete remissions were more common among patients with limited disease (11/14 patients, 79%) compared with those with extensive disease (7/19 patients, 37%) and among patients (11/14 patients, 79%) compared with those with extensive disease (7/19 patients, 37%) and among patients who were ambulatory prior to therapy (16/25 patients, 64%) compared with those who were nonambulatory (2/8 patients, 25%). Myelosuppression consisted primarily of neutropenia. Eight percent of the treatment courses in 29% of the patients were associated with hematuria and/or documented episodes of infection during neutropenia. There were three deaths possibly related to treatment, in two of which there was no evidence of disease at post-mortem examination. Six patients relapsed in the central nervous system (CNS). In four instances, CNS relapse was the only site of tumor progression. Central nervous system relapse was more common among evaluable patients who did not receive prophylactic brain irradiation (5/17 patients, 29%, vs. 1/15 patients, 7%; P . 0.23). The median survival duration for all patients was 63 weeks, being slightly longer for patients with limited disease than for those with extensive disease (70.9 weeks vs. 56 weeks; P . 0.18). This was also true for patients who achieved complete rather than partial remissions (71 weeks vs. 50 weeks; P . 0.09). Patients receiving prophylactic brain irradiation experienced longer survival

  2. Assessment of heavy metal tolerance and hexavalent chromium reducing potential of Corynebacterium paurometabolum SKPD 1204 isolated from chromite mine seepage

    Directory of Open Access Journals (Sweden)

    Satarupa Dey

    2016-07-01

    Full Text Available Corynebacterium paurometabolum SKPD 1204 (MTCC 8730, a heavy metal tolerant and chromate reducing bacterium isolated from chromite mine seepage of Odisha, India has been evaluated for chromate reduction under batch culture. The isolate was found to tolerate metals like Co(II, Cu(II, Ni(II, Mn(II, Zn(II, Fe(III and Hg(II along with Cr(VI and was resistant to different antibiotics as evaluated by disc-diffusion method. The isolate, SKPD 1204 was found to reduce 62.5% of 2 mM Cr(VI in Vogel Bonner broth within 8 days of incubation. Chromate reduction capability of SKPD 1204 decreased with increase in Cr(VI concentration, but increased with increase in cell density and attained its maximum at 1010 cells/mL. Chromate reducing efficiency of SKPD 1204 was promoted in the presence of glycerol and glucose, while the highest reduction was recorded at pH 7.0 and 35 °C. The reduction process was inhibited by divalent cations Zn(II, Cd(II, Cu(II, and Ni(II, but not by Mn(II. Anions like nitrate, phosphate, sulphate and sulphite was found to be inhibitory to the process of Cr(VI reduction. Similarly, sodium fluoride, carbonyl cyanide m-chlorophenylhydrazone, sodium azide and N, N,-Di cyclohexyl carboiimide were inhibitory to chromate reduction, while 2,4-dinitrophenol appeared to be neither promotive nor inhibitory to the process.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Adnan Shah

    Full Text Available 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.

  8. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Schneider Jens

    2012-01-01

    Full Text Available Abstract Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation.

  9. Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

    Institute of Scientific and Technical Information of China (English)

    SHEN Xihui; LIU Shuangjiang

    2005-01-01

    Although the protocatechuate branch of the β-ketoadipate pathway in Gram bacteria has been well studied, this branch is less understood in Gram+ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C.glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  13. Transcriptional Analysis of the groES-groEL1, groEL2, and dnaK genes in Corynebacterium glutamicum: Characterization of Heat Shock-Induced Promoters

    Czech Academy of Sciences Publication Activity Database

    Barreiro, C.; González-Lavado, E.; Pátek, Miroslav; Martin, J. F.

    2004-01-01

    Roč. 186, č. 14 (2004), s. 4813-4817. ISSN 0021-9193 R&D Projects: GA AV ČR KSK5052113 Keywords : corynebacterium glutamicum * mrna Subject RIV: EE - Microbiology, Virology Impact factor: 4.146, year: 2004

  14. Factors enhancing L-valine production by the growth-limited L-isoleucine auxotrophic strain Corynebacterium glutamicum D-eltailvA D-panB ilvNM13 (pECKAilvBNC)

    Czech Academy of Sciences Publication Activity Database

    Denina, I.; Paegle, L.; Prouza, Marek; Holátko, Jiří; Pátek, Miroslav; Nešvera, Jan; Ruklisha, M.

    2010-01-01

    Roč. 37, č. 7 (2010), s. 689-699. ISSN 1367-5435 R&D Projects: GA ČR GC204/07/J012 Institutional research plan: CEZ:AV0Z50200510 Keywords : Corynebacterium glutamicum * L-valine synthesis * Limited growth Subject RIV: EE - Microbiology, Virology Impact factor: 2.416, year: 2010

  15. 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

    Czech Academy of Sciences Publication Activity Database

    Busche, T.; Šilar, Radoslav; Pičmanová, Martina; Pátek, Miroslav; Kalinowski, J.

    2012-01-01

    Roč. 13, č. 445 (2012), s. 445-464. ISSN 1471-2164 R&D Projects: GA ČR GC204/09/J015 Institutional research plan: CEZ:AV0Z50200510 Keywords : Corynebacterium glutamicum * ECF sigma factor * Anti-sigma factor Subject RIV: EE - Microbiology, Virology Impact factor: 4.397, year: 2012

  16. Radiation-induced osteogenic sarcoma of C3H mouse: effects of Corynebacterium parvum and WBI on its natural history and response to irradiation

    International Nuclear Information System (INIS)

    An osteogenic sarcoma, which appeared at 316 days following single dose 5000 rad to the leg of C3H mouse, has been studied as early generation F2 and F3 transplants in syngeneic hosts with respect to local growth, pattern of spread, and response to local irradiation in normal, C. parvum treated, and whole body irradiated hosts. Mean survival of untreated mice was 126 days after transplantation. Grossly evident metastatic tumor appeared in 85% of these mice; in 47 of 48 animals metastases were seen in the lung. Corynebacterium parvum given intravenously as a single dose of 350 μg at 96 hr after tumor transplant retarded tumor growth: regression was observed in 10 of 19 tumors, 3 of 19 mice were cured of their osteosarcomas and mean survival was prolonged from 126 to 173 days, in one study where i.v. C. parvum was given when tumor was 5 mm, 1 of 13 mice was cured by C. parvum alone. Although 5000 rad resulted in 100% of tumor destruction in normal mice; 49% died of metastatic tumor to the lung. In C. parvum treatment mice only 16% died of metastatic tumor. The radiation doses which achieved control of half of the irradiated 8mm diameter tumors were 4350 and 3600 rad for normal and C. parvum treated hosts, respectively. While body irradiation 600 rad given 24 hr prior to tumor transplant had an opposite effect to Corynebacterium parvum. (author)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  19. A TatABC-type Tat translocase is required for unimpaired aerobic growth of Corynebacterium glutamicum ATCC13032.

    Directory of Open Access Journals (Sweden)

    Dan Oertel

    Full Text Available 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.

  20. Isolation of Corynebacterium Xerosis from Jordanian Soil and a Study on its Antimicrobial Activity against a Range of Bacteria and Fungi

    International Nuclear Information System (INIS)

    A bacterial strain which has been identified as Corneybacterium Xerosis NB-2 was isolated from a soil sample from Jerash Private University, Jerash, Jordan. This isolate was found to produce an antimicrobial substance active only against filamentous fungi and yeasts (Aspergillus niger SQ 40, Fusarium oxysporium SQ11, Verticillium dahliae SQ 42, Saccharomyces SQ 46 and Candida albicans SQ 47). However, all tested gram-positive bacteria and gram negative bacteria (Bacillus megaterium SQ5, Bacillus cereus SQ6, Staphylococcus aureus SQ9, Streptococcus pyogens SQ10, Eschericshia coli SQ 22, Klepsiella spp SQ33 and SQ33 and Pseudonomas mallei SQ 34) were found to be resistant. In batch culture, the isolated NB-2 produced the antimicrobial substance late in the growth phase and antimicrobial activity of Corynebacterium Xerosis against filamentous fungi and yeasts which was not previously described. (author)

  1. The uptake, distribution and translocation of 86Rb in alfalfa plants susceptible and resistant to the bacterial wilt and the effect of Corynebacterium insidiosum upon these processes

    International Nuclear Information System (INIS)

    Alfalfa (Medicago sativa L.) plants susceptible (S) and resistant (R) to bacterial wilt were fed via roots with a nutrient solution labelled with 86Rb+, at different times after inoculation with Corynebacterium insidiosum (McCull.) H.L. Jens. The infection did not affect 86Rb+ uptake per plant in the course of a 14-day-period following inoculation; however, it affected its distribution differently in the S- and the R-plants. 86Rb+ uptake significantly decreased due to the infection in the S-plants on the day 49 after inoculation (a 4-h-exposure to 86Rb+), with the ions more slowly translocated to the shoots in diseased S-plants than in diseased R-plants. Likely factors causing these effects and their relationship to alfalfa resistance to bacterial wilt are discussed. (author)

  2. Quinone-dependent D-lactate dehydrogenase Dld (Cg1027 is essential for growth of Corynebacterium glutamicum on D-lactate

    Directory of Open Access Journals (Sweden)

    Oikawa Tadao

    2010-12-01

    Full Text Available Abstract Background Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer. Conclusions Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

  3. Study on the fermentation process conditions of L-valine produced by Corynebacterium glutamicum%L-缬氨酸的发酵工艺条件研究

    Institute of Scientific and Technical Information of China (English)

    曾青兰; 孙连连; 王志勇

    2012-01-01

    以谷氨酸棒杆菌(Corynebacterium glutamicum)CICC20887为生产菌株,采用单因素实验研究了发酵工艺条件对L-缬氨酸产量的影响.结果表明,该菌发酵生产L-缬氨酸的适宜初糖浓度、生物素添加量、VB1添加量、玉米浆添加量分别为90 g/L、80 μg/L、0.20 mg/L、30 g/L,发酵期间,24 h前pH值应控制在6.5~6.7、后48 h应控制在7.0~7.2,温度30~31℃,发酵周期应控制在66~72 h.%Using Corynebacterium glutamicum CICC20887 as producing strain,the effect of fermentation process condition on the yield of L-valine were studied with single factor design; The results showed that the optimum concentration of initial glucose, D-biotin, VB1 and corn syrup were 90g/L,80μg/L,0. 20 mg /L, 30g/L respectively;The optimal fermentation conditions of pH, temperature and fermentation period were 6. 5~6. 7 (for the initial 24 hours) ,7. 0~7. 2 (during 24 to 72 hours), 30~31℃ , 66~72 h respectively. This study could offe a basis for industrial production of L-valine.

  4. Response of the cytoplasmic and membrane proteome of Corynebacterium glutamicum ATCC 13032 to pH changes

    Directory of Open Access Journals (Sweden)

    Poetsch Ansgar

    2008-12-01

    Full Text Available Abstract Background C. glutamicum has traditionally been grown in neutral-pH media for amino acid production, but in a previous article we reported that this microorganism is a moderate alkaliphile since it grows optimally at pH 7.0–9.0, as shown in fermentor studies under tightly controlled pH conditions. We determined the best pH values to study differential expression of several genes after acidic or basic pH conditions (pH 6.0 for acidic expression and pH 9.0 for alkaline expression. Thus, it was interesting to perform a detailed analysis of the pH-adaptation response of the proteome of C. glutamicum ATCC 13032 to clarify the circuits involved in stress responses in this bacterium. In this paper we used the above indicated pH conditions, based on transcriptional studies, to confirm that pH adaptation results in significant changes in cytoplasmatic and membrane proteins. Results The cytoplasmatic and membrane proteome of Corynebacterium glutamicum ATCC 13032 at different pH conditions (6.0, 7.0 and 9.0 was analyzed by classical 2D-electrophoresis, and by anion exchange chromatography followed by SDS-PAGE (AIEC/SDS-PAGE. A few cytoplasmatic proteins showed differential expression at the three pH values with the classical 2D-technique including a hypothetical protein cg2797, L-2.3-butanediol dehydrogenase (ButA, and catalase (KatA. The AIEC/SDS-PAGE technique revealed several membrane proteins that respond to pH changes, including the succinate dehydrogenase complex (SdhABCD, F0F1-ATP synthase complex subunits b, α and δ (AtpF, AtpH and AtpA, the nitrate reductase II α subunit (NarG, and a hypothetical secreted/membrane protein cg0752. Induction of the F0F1-ATP synthase complex β subunit (AtpD at pH 9.0 was evidenced by Western analysis. By contrast, L-2.3-butanediol dehydrogenase (ButA, an ATPase with chaperone activity, the ATP-binding subunit (ClpC of an ATP-dependent protease complex, a 7 TMHs hypothetical protein cg0896, a conserved

  5. Scientific Opinion on the safety and efficacy of L-valine (ValAMINO®) produced by Corynebacterium glutamicum (DSM 25202) for all animal species, based on a dossier submitted by Evonik Industries AG

    OpenAIRE

    EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)

    2014-01-01

    The product L-valine, feed grade (ValAMINO®), is a feed additive produced by fermentation with a genetically modified strain of Corynebacterium glutamicum (DSM 25202). Neither the production strain nor its recombinant DNA was detected in the final product. Therefore, the final product does not give rise to any safety concerns with regard to the genetic modification. The additive L-valine, feed grade, produced by C. glutamicum (DSM 25202) is safe for all target animals when supplemented in app...

  6. Complete genome sequence, lifestyle, and multi-drug resistance of the human pathogen Corynebacterium resistens DSM 45100 isolated from blood samples of a leukemia patient

    Directory of Open Access Journals (Sweden)

    Schröder Jasmin

    2012-04-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

    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

  8. A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence

    Science.gov (United States)

    Reardon-Robinson, Melissa E.; Osipiuk, Jerzy; Jooya, Neda; Chang, Chungyu; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2016-01-01

    Summary The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae. PMID:26294390

  9. Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

    Directory of Open Access Journals (Sweden)

    Miriam F. Rebouças

    2013-11-01

    Full Text Available Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA, a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  12. [Comparative electron-microscopic study of 8 representatives of the genus Corynebacterium grown on solid nutrient medium during the stationary phase of development].

    Science.gov (United States)

    Vysotskiĭ, V V; Mazurova, I K; Shmeleva, E A

    1976-09-01

    After 18 hours of growth on selective serum-agar medium C diphtheriae cultures with different toxicogenic activity, and also diphtheroid and Hoffmann's baccillus cultures were removed, washed of the remnants of the nutrient medium and fixed under cold conditions by two combined methods (with glutaric aldehyde-osmic acid--uranyl acetate, and potassium permeanganate--uranyl acetate). The preparations were studied in ultrathin sections. It appeared that corynebacteria had during the stationary phase of development a general structural plan characteristic of Gram positive microorganisms and for all the corynebacterium genus. Cells of diphtheria toxicognic strains had signs of the accelerated (in comparison with other strains) rate of development; the principal mass of toxicogenic cells after 18 hours of growth had morphological signs of the stage of rest. The majority of cells whose toxicogenicity was inconstant had an extensive microcapsule which was also a characteristic element of the diphtheroid and Hoffmann's bacillus ultrastructure. The total thickness of the walls in the cells of toxicogenic strains and of the strains whose toxicogenicity was inconstant constituted 190-200 A; in nontoxicogenic strains, diphtheroid and Hoffmann's bacillus it was from 230 to 320 A. Surface structures of corynebacteria were differentiated better in the cells with toxicogenic activity. In the majority of cells of nontoxicogenic strains and also diphtheroid and Hoffmann's bacillus individual wall layers were differentiated with difficulty. PMID:827882

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. 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.

    Science.gov (United States)

    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

  16. CO₂ /HCO₃⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum.

    Science.gov (United States)

    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

  17. Determination of Immobilization Process Parameters of Corynebacterium glutamicum on Kappa carrageenan, Its Application in L-lysine Fermentation and The Investigation Into Its Storage Conditions

    Directory of Open Access Journals (Sweden)

    Suong Thi Hong Nguyen

    2014-10-01

    Full Text Available The parameters of the immobilized process of Corynebacterium glutamicum on kappa carrageenan were identified by Plackett-Burman matrix, and the experiments were designed by response surface methodology having the central composite designs (RSM-CCD. The maximum yield of cell immobilization on kappa carrageenan carrier reached at 78% ± 2%. Optimal parameters were 3 grams kappa carrageenan per 100 militters sterile water and 58.58 million cfu/mL, forming gels at 100C for 25 minutes and the speed when soaking particles of 150 rpm for 120 minutes in 0.58 M potassium chlorua solvent. The immobile finished products are applied in L-lysine production, their reusing ability is 3 times and the total yield of L-lysine was accumulated 93 g/L in medium during 96 fermented hours. The L-lysine productivity of the batch fermentation was 0.969 g.L-1 .h-1 . And the set-up storage conditions are the mixed solvent of CaCl2 0.5% (w/v and KCl 0.5% (w/v; pH is 7.0 in 40C. After 60 storage days, the survival cell rate was remained 51%.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  20. Development of a potential stationary-phase specific gene expression system by engineering of SigB-dependent cg3141 promoter in Corynebacterium glutamicum.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  4. Conservación por congelación de Bordetella pertussis y Corynebacterium diphtheriae, empleados en la producción de vacunas para uso humano

    Directory of Open Access Journals (Sweden)

    Yilian Plasencia,

    2000-11-01

    Full Text Available En el presente estudio se evaluó el método de congelación a –70ºC para la preservación de Bordetella pertussis y Corynebacterium diphtheriae. Para verificar el sustento de los cultivos se realizó un adecuado control de calidad, que incluyó comprobación de pureza, viabilidad y estabilidad de las propiedades de interés. En este trabajo se probaron diferentes formulaciones. Se seleccionó la que arrojó los mejores resultados y se realizó un estudio de mantenimiento de las características evaluadas durante el tiempo. Para medir determinados parámetros se realizaron procesos a escala industrial, empleándose para esto un biorreactor Chemap de 35 L. Se tomaron como referencia los valores obtenidos por las cepas conservadas por liofilización. De esta forma se buscaron alternativas y soluciones a problemas presentados en su conservación. Los resultados obtenidos sugieren la posible inclusión en el Programa de Mantenimiento establecido.

  5. Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity.

    Science.gov (United States)

    Dalili, Dina; Amini, Mohsen; Faramarzi, Mohammad Ali; Fazeli, Mohammad Reza; Khoshayand, Mohammad Reza; Samadi, Nasrin

    2015-11-01

    Herein we reported the structure and several properties of a new biosurfactants produced by Corynebacterium xerosis strain NS5. This strain was capable of producing a novel lipopeptide biosurfactant that we have named coryxin. The biosurfactant structure was characterized by using Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (NMR), and Liquid chromatography-mass spectrometry (LC-MS). It contained a hydrophobic moiety of 3-hydroxydecanoic acid and a peptide part predicted as a sequence of seven amino acids including Asn-Arg-Asn-Gln-Pro-Asn-Ser. Coryxin lowered the surface tension of water to 31.4 mN/m, with a critical micelle concentration of 25mg/l. It was a strong emulsifier with an emulsification index of 61% against n-hexane. Coryxin showed antibacterial activity against test organisms belonging to Gram-positive and Gram-negative bacteria and disrupted preformed biofilms of Staphylococcus aureus (82.5%), Streptococcus mutans (80%), Escherichia coli (66%) and Pseudomonas aeruginosa (30%). In conclusion, microbial surfactant from C. xerosis exhibited inhibitory and disruptive activities against biofilm formation that could be of use in biofilm-related menace. PMID:26280817

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  8. Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

    Directory of Open Access Journals (Sweden)

    Maiara G. Blagitz

    2015-01-01

    Full Text Available This study evaluated the expression of CD14, toll-like receptor (TLR 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

  9. Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

    Science.gov (United States)

    Abbouni, Bouziane; Elhariry, Hesham M; Auling, Georg

    2003-01-01

    Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain. PMID:12882290

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

    Science.gov (United States)

    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

  11. Transcriptional Cross-Regulation between Gram-Negative and Gram-Positive Bacteria, Demonstrated Using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum

    OpenAIRE

    Marbaniang, Carmelita N.; J.Gowrishankar

    2012-01-01

    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...

  12. Parámetros fisicoquímicos para la síntesis de ácido láctico ó etanol de la bacteria (corynebacterium glutamicum)

    OpenAIRE

    Garcia Lina Marcela; Castellanos Sánchez Angélica María; Lopez Galan Jorge Enrique; Astudillo Myriam; Florez Pardo Luz Marina

    2011-01-01

    El interés por obtener productos para la industria de biocombustibles a partir de desechos agrícolas, conduce a las investigaciones en la búsqueda de sistemas microbianos resistentes y costo-efectivos. La Corynebacterium glutamicum, es un microorganismo usado para producir amino-ácidos, crece en gran variedad de sustratos y es resistente durante la fermentación, a variaciones en el pH, temperatura, presión osmótica y acumulación de alcohol, características que lo hacen candidato a ser mejora...

  13. Competition of Reactive red 4, Reactive orange 16 and Basic blue 3 during biosorption of Reactive blue 4 by polysulfone-immobilized Corynebacterium glutamicum

    International Nuclear Information System (INIS)

    Competition of Reactive red 4 (RR4), Reactive orange 16 (RO16) and Basic blue 3 (BB3) during biosorption of Reactive blue 4 (RB4) by polysulfone-immobilized protonated Corynebacterium glutamicum (PIPC) was investigated in batch and column mode of operations. Through potentiometric titrations, and with the aid of proton-binding model, carboxyl, phosphonate and amine were identified as functional groups of PIPC, with apparent pKa values of 3.47 ± 0.05, 7.08 ± 0.07 and 9.90 ± 0.05 mmol/g, respectively. Since reactive dyes release dye anions (ROSO3-) in solutions, the positively charged amine groups were responsible for biosorption. PIPC favored biosorption at pH 3 when RB4 was studied/used as single-solute; while the presence of RR4 and RO16 severely affected the RB4 biosorption. When present as a single-solute, PIPC recorded 184.5 mg RB4/g; while PIPC exhibited 126.9, 120.9 and 169.6 mg RB4/g in the presence of RR4, RO16 and BB3, respectively. In general, the accessibility of amine group depends on the molecular size, number of sulfonate groups and reactivity of each reactive dye. Single and multicomponent Freundlich equations successfully described the biosorption isotherms. With 0.1 M NaOH, it is possible to reuse PIPC for RB4 biosorption in 10 repeated cycles. Column experiments in an up-flow packed column coincided with batch results, that is PIPC showed strong preference towards highly reactive and relatively small RB4 anions; however, the presence of competing dyes hinder the RB4 column biosorption performance

  14. Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

    Science.gov (United States)

    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

  15. Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

    Science.gov (United States)

    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

  16. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production.

    Science.gov (United States)

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

    2012-07-01

    N-acetyl-L-glutamate kinase (EC 2.7.2.8) is first committed in the specific L-arginine pathway of Corynebacterium sp. A limited increase of L-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that L-arginine feedback inhibition plays an influence on the L-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I0.5R efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I0.5R of L-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust L-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The L-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway. PMID:21901472

  17. Immunoregulation of antitumor response; differential secretion of arachidonic acid metabolites by macrophages during stimulation ''in vitro'' with BCG and ''Corynebacterium parvum''

    International Nuclear Information System (INIS)

    The level of arachidonic acid (AA) metabolites in the supernatants of cultures peritoneal exudate cells (PEC) were studied under various conditions using BCG and ''Corynebacterium parvum'' as stimulators. The metabolite levels were analyzed by thin layer chromatography (TLC). The degree of macrophage cytotoxic/cytostatic activity was dependent on the dose and character of stimulators used and the source of macrophages. The application of micro cytotoxicity assay for the evaluation of tumor cell lysis (lung sarcoma SaL-1) ''in vitro'' revealed that peritoneal macrophages from healthy and tumor bearing BALB/c mice may affect the degree of antitumor response. In the supernatants of cultured PEC from tumor bearing mice AA level increased (by 10-fold) in comparison with PEC from healthy mice. Stimulation with BCG induced over a double level of AA in PEC isolated from tumor bearing mice non-stimulated or stimulated with ''C.parvum''. A lower level of prostaglandins (PGs) was found in the supernatants of cultured PEC isolated from healthy mice (stimulated and non-stimulated), but the highest level of PGs was observed in the supernatants of cultured PEC isolated from tumor bearing mice stimulated with BCG. The unique metabolite of AA was found only in the supernatants form non-stimulated PEC from tumor bearing mice. PEC from tumor bearing mice produced metabolites of AA which were not detected in control group. These results suggest that macrophages also play a regulatory role by secretion of AA. This process can be modified by bacterial antigens. (author). 21 refs, 7 figs

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Hong-Il Kim

    2016-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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