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Sample records for recombinant corynebacterium glutamicum

  1. Transcriptome analysis of Corynebacterium glutamicum in the process of recombinant protein expression in bioreactors

    PubMed Central

    Sun, Yang; Guo, Wenwen; Wang, Fen; Zhan, Chunjun; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

    2017-01-01

    Corynebacterium glutamicum (C. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. Herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of C. glutamicum. Two C. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (EGFP), were cultured in parallel in 5-L bioreactors to study the change in metabolism in the process of EGFP expression. The results revealed that EGFP expression had great effects on the growth and metabolism of C. glutamicum and contributed to metabolism-like anaerobic conditions as follows: glycolysis was enhanced, the TCA cycle was shunted, and Glu, Val, Met, lactate and acetate were accumulated to produce sufficient ATP for EGFP production and transfer. Many differentially expressed genes related to ribosomal protein, transcriptional regulators, and energy metabolism were found to be expressed in the presence of EGFP, laying the foundation for identifying genomic loci to change the flow of the host cell metabolism to improve the ability of expressing foreign proteins in C. glutamicum. PMID:28369109

  2. The impact of PHB accumulation on L-glutamate production by recombinant Corynebacterium glutamicum.

    PubMed

    Liu, Qian; Ouyang, Shao-Ping; Kim, Jonghyok; Chen, Guo-Qiang

    2007-11-01

    Corynebacterium glutamicum, a gram-positive soil bacterium, has been used extensively for the industrial production of l-glutamate and other amino acids. In this study, an Escherichia coli-C. glutamicum shuttle expression plasmid harboring polyhydroxybutyrate (PHB) synthesis genes, phbCAB from Ralstonia eutropha, was constructed under the Ptrc promoter. C. glutamicum harboring this plasmid accumulated 3-13% PHB with a weight average molecular mass of 125,400 and a polydispersity of 11.3 when grown on glucose. PHB synthesis related enzyme activities including beta-ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB) and PHB synthase (PhbC) were found to be constitutively produced independent of IPTG. l-Glutamate production increased 39-68% in two C. glutamicum strains harboring PHB synthesis genes compared with their parent strains in shake flask experiments. In fermentor studies, the recombinant produced approximately 23% more l-glutamate compared with that of the wild type, and yielded less intermediate metabolites or by-products including alpha-ketoglutarate, l-glutamine and lactate. These results suggested that the expression of phbCAB genes in C. glutamicum could help regulate glutamate production metabolism. This demonstrated that the expression of PHB synthesis genes has a positive effect on l-glutamate production in C. glutamicum.

  3. Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

    PubMed

    Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

    2015-12-29

    In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C

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

    PubMed Central

    2014-01-01

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

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

    PubMed

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

    2016-03-01

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

  6. A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

    PubMed Central

    Yang, Peng; Liu, Wenjing; Cheng, Xuelian; Wang, Jing; Qi, Qingsheng

    2016-01-01

    ABSTRACT 5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. IMPORTANCE In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future. PMID:26921424

  7. A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield.

    PubMed

    Yang, Peng; Liu, Wenjing; Cheng, Xuelian; Wang, Jing; Wang, Qian; Qi, Qingsheng

    2016-05-01

    5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host,Corynebacterium glutamicum The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Productivity of cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB of Acinetobacter calcoaceticus.

    PubMed

    Doo, Eun-Hee; Lee, Won-Heong; Seo, Hyo-Seel; Seo, Jin-Ho; Park, Jin-Byung

    2009-06-15

    The biocatalytic efficiency of recombinant Corynebacterium glutamicum expressing the chnB gene encoding cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871 was investigated. Optimization of an expression system and induction conditions enabled the recombinant biocatalyst to produce CHMO to a specific activity of ca. 0.5 U mg(-1) protein. Tight control of feeding of an energy source (i.e., glucose) and dissolved oxygen tension during fed-batch culture-based biotransformation allowed the cells to produce epsilon-caprolactone to a concentration of 16.0 g l(-1). The specific and volumetric productivity for cyclohexanone oxidation were 0.12 g g drycells(-1)h(-1) (17.5 U g(-1) of dry cells) and 2.3 g l(-1)h(-1) (330 U l(-1)), respectively. These values correspond to over 5.4- and 2.7-fold of recombinant Escherichia coli expressing the same gene under similar reaction conditions. It could be concluded that the recombinant C. glutamicum is a promising biocatalyst for Baeyer-Villiger oxidations.

  9. 3-Amino-4-hydroxybenzoic acid production from sweet sorghum juice by recombinant Corynebacterium glutamicum.

    PubMed

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

    2015-12-01

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

  10. Ethambutol-mediated cell wall modification in recombinant Corynebacterium glutamicum increases the biotransformation rates of cyclohexanone derivatives.

    PubMed

    Yun, Ji-Yeong; Lee, Jung-Eun; Yang, Kyung-Mi; Cho, Suekyung; Kim, Arim; Kwon, Yong-Uk; Kwon, Yong-Euk; Park, Jin-Byung

    2012-01-01

    The effects of structural modification of cell wall on the biotransformation capability by recombinant Corynebacterium glutamicum cells, expressing the chnB gene encoding cyclohexanone monooxygenase of Acinetobacter calcoaceticus NCIMB 9871, were investigated. Baeyer-Villiger oxygenation of 2-(2'-acetoxyethyl) cyclohexanone (MW 170 Da) into R-7-(2'-acetoxyethyl)-2-oxepanone was used as a model reaction. The whole-cell biotransformation followed Michaelis-Menten kinetics. The V (max) and K (S) values were estimated as 96.8 U g(-1) of dry cells and 0.98 mM, respectively. The V (max) was comparable with that of cyclohexanone oxygenation, whereas the K (S) was almost eightfold higher. The K (S) value of 2-(2'-acetoxyethyl) cyclohexanone oxygenation was reduced by ca. 30% via altering the cell envelop structure of C. glutamicum with ethambutol, which inhibits arabinosyl transferases involved in the biosynthesis of cell wall arabinogalactan and mycolate layers. The higher whole-cell biotransformation rate was also observed in the oxygenation of ethyl 2-cyclohexanone acetate upon ethambutol treatment of the recombinant C. glutamicum. Therefore, it was assumed that the biotransformation efficiency of C. glutamicum-based biocatalysts, with respect to medium- to large-sized lipophilic organic substrates (MW > ca. 170), can be enhanced by engineering their cell wall outer layers, which are known to function as a formidable barrier to lipophilic molecules.

  11. Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum.

    PubMed

    Lubitz, Dorit; Wendisch, Volker F

    2016-10-07

    Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope. Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin. Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.

  12. Putrescine production by engineered Corynebacterium glutamicum.

    PubMed

    Schneider, Jens; Wendisch, Volker F

    2010-10-01

    Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).

  13. Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2012-03-01

    Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

  14. Specific γ-aminobutyric acid decomposition by gabP and gabT under neutral pH in recombinant Corynebacterium glutamicum.

    PubMed

    Ni, Yalan; Shi, Feng; Wang, Nannan

    2015-11-01

    Corynebacterium glutamicum that expresses the exogenous L-glutamate decarboxylase (GAD) gene can synthesize γ-aminobutyric acid (GABA). To prevent GABA decomposition in the recombinant C. glutamicum GAD strain, GABA uptake and the GABA shunt pathway were blocked. GABA uptake is catalyzed by GABA permease encoded by gabP. The first reaction of the GABA shunt pathway is catalyzed by the GABA transaminase encoded by gabT. Initially, the effects of pH on GABA decomposition in recombinant C. glutamicum co-expressing two GAD genes (gadB1 and gadB2) were analyzed, demonstrating that GABA could be decomposed under neutral pH. Next, the gabP and gabT were individually deleted, and the GABA production of the related GAD strains was investigated by controlling the pH of the final fermentation stage at a neutral state. During this stage, the GABA concentration of the gabT-deleted GAD strain decreased from 23.9 ± 1.8 to 17.7 ± 0.7 g/l. However, the GABA concentration of the gabP-deleted GAD strain remained at 18.6-19.4 g/l. This study demonstrated that GABA was decomposed under neutral pH and that the deletion of gabP could effectively alleviate GABA decomposition in C. glutamicum.

  15. The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse.

    PubMed

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

    2016-05-28

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

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

    PubMed

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

    2016-05-20

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

  17. Deletion of odhA or pyc improves production of γ-aminobutyric acid and its precursor L-glutamate in recombinant Corynebacterium glutamicum.

    PubMed

    Wang, Nannan; Ni, Yalan; Shi, Feng

    2015-07-01

    To enhance γ-aminobutyric acid (GABA) production in recombinant Corynebacterium glutamicum, metabolic engineering strategies were used to improve the supply of the GABA precursor, L-glutamate. C. glutamicum ATCC13032 co-expressing two glutamate decarboxylase genes (gadB1 and gadB2) was constructed in a previous study Shi et al. (J Ind Microbiol Biotechnol 40:1285-1296, 2013) to synthesize GABA from endogenous L-glutamate. To improve its L-glutamate supply, new strains were constructed here. First, the odhA and pyc genes were deleted separately. Then, a gadB1-gadB2 co-expression plasmid was transferred into ΔodhA, Δpyc, and ATCC13032, resulting in recombinant strains SNW201, SNW202, and SNW200, respectively. After fermenting for 72 h, GABA production increased to 29.5 ± 1.1 and 24.9 ± 0.7 g/l in SNW201 and SNW202, respectively, which was significantly higher than that in SNW200 (19.4 ± 2.6 g/l). The GABA conversion ratios of SNW201 and SNW202 reached 0.98 and 0.96 mol/mol, respectively. The recombinant strains SNW201 and SNW202 can be used as candidates for GABA production.

  18. Glycerol-3-phosphatase of Corynebacterium glutamicum.

    PubMed

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

    2012-06-15

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

  19. Engineering Corynebacterium glutamicum for violacein hyper production.

    PubMed

    Sun, Hongnian; Zhao, Dongdong; Xiong, Bin; Zhang, Chunzhi; Bi, Changhao

    2016-08-24

    Corynebacterium glutamicum was used as a metabolic engineering chassis for production of crude violacein (mixture of violacein and deoxyviolacein) due to Corynebacterium's GRAS status and advantages in tryptophan fermentation. The violacein is a commercially potential compound with various applications derived from L-tryptophan. Corynebacterium glutamicum ATCC 21850 that could produce 162.98 mg L(-1) tryptophan was employed as a novel host for metabolic engineering chassis. Heterologous vio operon from Chromobacterium violaceum was over-expressed in ATCC 21850 strain with constitutive promoter to have obtained 532 mg L(-1) violacein. Considering toxicity of violacein, vio operon was expressed with inducible promoter and 629 mg L(-1) violacein was obtained in batch culture. Due to the economical coding nature of vio operon, the compressed RBS of vio genes were replaced with complete strong C. glutamicum ones. And extended expression units were assembled to form a synthetic operon. With this strategy, 1116 mg L(-1) violacein in batch culture was achieved. Fermentation process was then optimized by studying induction time, induction concentration, culture composition and fermentation temperature. as a result, a titer of 5436 mg L(-1) and a productivity of 47 mg L(-1) h(-1) were achieved in 3 L bioreactor. With metabolic engineering and fermentation optimization practice, C. glutamicum 21850 (pEC-C-vio1) was able to produce violacein with both titer and productivity at the highest level ever reported. Due to advantages of mature C. glutamicum fermentation industry, this work has built basis for commercial production of violacein.

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

    PubMed Central

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

    2016-01-01

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

  1. Engineering Corynebacterium glutamicum for isobutanol production.

    PubMed

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

    2010-07-01

    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 alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by approximately 25% to 4.9 g/L isobutanol in a pycldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.

  2. Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

    PubMed Central

    2012-01-01

    Background Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ɛ-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained. Conclusion C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions

  3. Engineering Corynebacterium glutamicum for isobutanol production

    PubMed Central

    Smith, Kevin Michael; Cho, Kwang-Myung

    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 alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2522-6) contains supplementary material, which is available to authorized users. PMID:20376637

  4. Synthetic promoter libraries for Corynebacterium glutamicum.

    PubMed

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

    2014-03-01

    The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.

  5. Functional Characterization of Corynebacterium glutamicum Mycothiol S-Conjugate Amidase

    PubMed Central

    Si, Meiru; Long, Mingxiu; Chaudhry, Muhammad Tausif; Xu, Yixiang; Zhang, Pan; Zhang, Lei; Shen, Xihui

    2014-01-01

    The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. PMID:25514023

  6. 3-Methyl-1-butanol Biosynthesis in an Engineered Corynebacterium glutamicum.

    PubMed

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

    2016-05-01

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

  7. Functional analysis of arabinofuranosidases and a xylanase of Corynebacterium alkanolyticum for arabinoxylan utilization in Corynebacterium glutamicum.

    PubMed

    Kuge, Takayuki; Watanabe, Akira; Hasegawa, Satoshi; Teramoto, Haruhiko; Inui, Masayuki

    2017-06-01

    Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-L-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-L-arabinofuranoside with k cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.

  8. Corynebacterium glutamicum possesses β-N-acetylglucosaminidase.

    PubMed

    Matano, Christian; Kolkenbrock, Stephan; Hamer, Stefanie N; Sgobba, Elvira; Moerschbacher, Bruno M; Wendisch, Volker F

    2016-08-05

    In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes. Chitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA 2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and β-N-acetylglucosaminidase NagA2. C. glutamicum possesses β-N-acetylglucosaminidase. In the wild type, β-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active β-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme

  9. Corynebacterium glutamicum promoters: a practical approach

    PubMed Central

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

    2013-01-01

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

  10. Corynebacterium glutamicum tailored for efficient isobutanol production.

    PubMed

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

    2011-05-01

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

  11. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

    PubMed

    Shi, Feng; Jiang, Junjun; Li, Yongfu; Li, Youxin; Xie, Yilong

    2013-11-01

    γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

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

    NASA Astrophysics Data System (ADS)

    Wittmann, Christoph

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

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

    PubMed Central

    2016-01-01

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

  14. Silencing of cryptic prophages in Corynebacterium glutamicum.

    PubMed

    Pfeifer, Eugen; Hünnefeld, Max; Popa, Ovidiu; Polen, Tino; Kohlheyer, Dietrich; Baumgart, Meike; Frunzke, Julia

    2016-12-01

    DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Silencing of cryptic prophages in Corynebacterium glutamicum

    PubMed Central

    Pfeifer, Eugen; Hünnefeld, Max; Popa, Ovidiu; Polen, Tino; Kohlheyer, Dietrich; Baumgart, Meike; Frunzke, Julia

    2016-01-01

    DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum. CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction. PMID:27492287

  16. Metabolic engineering of Corynebacterium glutamicum for methanol metabolism.

    PubMed

    Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan; Bott, Michael; Marienhagen, Jan

    2015-03-01

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

  17. Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

    PubMed Central

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

    2015-01-01

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

  18. Mapping the membrane proteome of Corynebacterium glutamicum.

    PubMed

    Schluesener, Daniela; Fischer, Frank; Kruip, Jochen; Rögner, Matthias; Poetsch, Ansgar

    2005-04-01

    In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.

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

    PubMed

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

    2013-11-01

    Gene deletion techniques are important for modifying Corynebacterium glutamicum, the bacterium for industrial production of amino acids. In this study, a novel multiple-gene-deletion system for C. glutamicum was developed. The system is composed of three plasmids pDTW109, pDTW201 and pDTW202. pDTW109 is a temperature-sensitive vector which harbors a cat gene under the tacM promoter, a cre gene under the tac promoter, an origin oriE for replicating in Escherichia coli, and another origin rep(TS) for replicating in C. glutamicum only at low temperatures; it has high transformation efficiency in C. glutamicum and can be easily eliminated by growing at 37°C. pDTW201 and pDTW202 carry loxp-flanked or mutant lox-flanked kan, respectively. This deletion system combines homologous recombination and Cre/lox site-specific recombination, could efficiently delete the aceE gene from the chromosome of C. glutamicum ATCC13032, ATCC13869 or ATCC14067, respectively, and could also delete both genes of aceE and ilvA from the chromosome of C. glutamicum ATCC14067. The system is simple and efficient, and can be easily implemented for multiple-gene-deletion in C. glutamicum. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-02-01

    The yellow-pigmented soil bacterium Corynebacterium glutamicum ATCC13032 is accumulating the cyclic C50 carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C50 carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C50 carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C50 carotenoids were glucosylated. We here also demonstrate production of the C40 carotenoids β-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C40 and C50 carotenoids.

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

    PubMed

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

    2016-06-30

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. Hyaluronic acid production with Corynebacterium glutamicum: effect of media composition on yield and molecular weight.

    PubMed

    Hoffmann, J; Altenbuchner, J

    2014-09-01

    Corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (HA). A set of expression vectors containing hasA, encoding HA synthase from Streptococcus equi subsp. zooepidemicus, alone or in combination with genes encoding enzymes for HA precursor production (hasB, hasC, glmU from Pseudomonas putida KT2440) or bacterial haemoglobin (vgb from Vitreoscilla sp.) was constructed. Recombinant Coryne. glutamicum strains were cultivated in two different minimal media, CGXII and MEK700. HA was isolated from the culture broth by ethanol precipitation or ultrafiltration. Analyses of the isolated HA revealed that overall production was higher in CGXII medium (1241 mg l(-1)) than in MEK700 medium (363 mg l(-1)), but molecular weight of the product was higher in MEK700 (>1·4 MDa) than in CGXII (<270 kDa). Coexpression of hasB, hasC or glmU had no effect on HA yield and did not improve molecular weight of the product. Coexpression of vgb lowered HA yield about 1·5-fold and did not affect molecular weight of the product. Microscopy of negative-stained cultures revealed that Coryne. glutamicum produces no distinct HA capsule. Regulation of cell growth and gene expression level of hasA are reasonable starting points for controlling the molecular weight of HA produced by Coryne. glutamicum. Corynebacterium glutamicum has a great potential as an alternative production host for HA. The fact that Coryne. glutamicum produces no distinct HA capsule facilitates HA isolation and improves overall yield. © 2014 The Society for Applied Microbiology.

  6. High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production.

    PubMed

    Kang, Mi-Suk; Han, Sang-Soo; Kim, Mi-Young; Kim, Bu-Youn; Huh, Jong-Pil; Kim, Hak-Sung; Lee, Jin-Ho

    2014-05-01

    The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

  7. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

    PubMed

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

    2015-01-27

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

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

    PubMed

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

    2014-12-20

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-08-28

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

  11. Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum.

    PubMed

    Siebert, Daniel; Wendisch, Volker F

    2015-01-01

    Production of the versatile bulk chemical 1,2-propanediol and the potential biofuel 1-propanol is still dependent on petroleum, but some approaches to establish bio-based production from renewable feed stocks and to avoid toxic intermediates have been described. The biotechnological workhorse Corynebacterium glutamicum has also been shown to be able to overproduce 1,2-propanediol by metabolic engineering. Additionally, C. glutamicum has previously been engineered for production of the biofuels ethanol and isobutanol but not for 1-propanol. In this study, the improved production of 1,2-propanediol by C. glutamicum is presented. The product yield of a C. glutamicum strain expressing the heterologous genes gldA and mgsA from Escherichia coli that encode methylglyoxal synthase gene and glycerol dehydrogenase, respectively, was improved by additional expression of alcohol dehydrogenase gene yqhD from E. coli leading to a yield of 0.131 mol/mol glucose. Deletion of the endogenous genes hdpA and ldh encoding dihydroxyacetone phosphate phosphatase and lactate dehydrogenase, respectively, prevented formation of glycerol and lactate as by-products and improved the yield to 0.343 mol/mol glucose. To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol. Thus, B12-dependent diol dehydratase activity may be limiting 1-propanol production. Production of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes. Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

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

    PubMed

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

    2015-10-01

    Thermochemical processing provides continuous production of bio-oils from lignocellulosic biomass. Levoglucosan, a pyrolytic sugar substrate C6H10O5 in a bio-oil, has been used for ethanol production using engineered Escherichia coli. Here we provide the first example for succinate production from levoglucosan with Corynebacterium glutamicum, a well-known industrial amino acid producer. Heterologous expression of a gene encoding a sugar kinase from Lipomyces starkeyi, Gibberella zeae or Pseudomonas aeruginosa was employed for levoglucosan conversion in C. glutamicum because the wild type was unable to utilize levoglucosan as sole carbon source. As result, expression of a levoglucosan kinase (LGK) of L. starkeyi only enabled growth with levoglucosan as sole carbon source in CgXII minimal medium by catalyzing conversion of levoglucosan to glucose-6-phosphate. Subsequently, the lgk gene was expressed in an aerobic succinate producer of C. glutamicum, strain BL-1. The recombinant strain showed a higher succinate yield (0.25 g g(-1)) from 2% (w/v) levoglucosan than the reference strain BL-1 from 2% (w/v) glucose (0.19 g g(-1)), confirming that levoglucosan is an attractive carbon substrate for C. glutamicum producer strains. In summary, we demonstrated that a pyrolytic sugar could be a potential carbon source for microbial cell factories. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032†

    PubMed Central

    Ordóñez, Efrén; Letek, Michal; Valbuena, Noelia; Gil, José A.; Mateos, Luis M.

    2005-01-01

    Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite. PMID:16204540

  14. Corynebacterium glutamicum Tailored for Efficient Isobutanol Production ▿ †

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-02-20

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

  16. Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations.

    PubMed

    Harst, Andreas; Albaum, Stefan P; Bojarzyn, Tanja; Trötschel, Christian; Poetsch, Ansgar

    2017-05-08

    The metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ΔaceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ΔaceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE. Metabolic pathways in C. glutamicum WT and ΔaceE, devoid of functional pyruvate dehydrogenase, were compared to understand proteome changes that contribute to the high production of branched chain amino acids (BCAA) in the ΔaceE strain. The data complements previous metabolome studies and corroborates the role of malate provided by the glyoxylate cycle and increased activity of glycolysis and pyruvate carboxylase reaction to replenish the TCA cycle. A slight increase in acetohydroxyacid synthase (ILV subunit B) substantiates the previously reported increased pyruvate pool in C. glutamicumΔaceE, and the benefit of additional ilv gene cluster overexpression for BCAA production. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background The soil bacterium Corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. Glutamate is the precursor of the amino acid proline. Proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. Alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. The direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. An ornithine overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production. Results Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium. Conclusions The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach. PMID:23806148

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

    PubMed

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

    2015-11-01

    Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

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

    2015-01-01

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

  20. Metabolic engineering of Corynebacterium glutamicum for L-arginine production.

    PubMed

    Park, Seok Hyun; Kim, Hyun Uk; Kim, Tae Yong; Park, Jun Seok; Kim, Suok-Su; Lee, Sang Yup

    2014-08-05

    L-arginine is an important amino acid for diverse industrial and health product applications. Here we report the development of metabolically engineered Corynebacterium glutamicum ATCC 21831 for the production of L-arginine. Random mutagenesis is first performed to increase the tolerance of C. glutamicum to L-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of NADPH level, disruption of L-glutamate exporter to increase L-arginine precursor and flux optimization of rate-limiting L-arginine biosynthetic reactions. Fed-batch fermentation of the final strain in 5 l and large-scale 1,500 l bioreactors allows production of 92.5 and 81.2 g l(-1) of L-arginine with the yields of 0.40 and 0.35 g L-arginine per gram carbon source (glucose plus sucrose), respectively. The systems metabolic engineering strategy described here will be useful for engineering Corynebacteria strains for the industrial production of L-arginine and related products.

  1. Purification and characterization of fumarase from Corynebacterium glutamicum.

    PubMed

    Genda, Tomoko; Watabe, Shoji; Ozaki, Hachiro

    2006-05-01

    Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.

  2. Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

    PubMed Central

    Farwick, M; Siewe, R M; Krämer, R

    1995-01-01

    Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system. PMID:7642496

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

    PubMed

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

    2013-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-10

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

    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, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally

  9. Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum.

    PubMed

    Nguyen, Anh Q D; Schneider, Jens; Wendisch, Volker F

    2015-05-10

    Corynebacterium glutamicum has been engineered for production of the polyamide monomer putrescine or 1,4-diaminobutane. Here, N-acetylputrescine was shown to be a significant by-product of putrescine production by recombinant putrescine producing C. glutamicum strains. A systematic gene deletion approach of 18 (putative) N-acetyltransferase genes revealed that the cg1722 gene product was responsible for putrescine acetylation. The encoded enzyme was purified and characterized as polyamine N-acetyltransferase. The enzyme accepted acetyl-CoA and propionyl-CoA as donors for acetylation of putrescine and other diamines as acceptors, but showed highest catalytic efficiency with the triamine spermidine and the tetraamine spermine and, hence, was named SnaA. Upon deletion of snaA in the putrescine producing strain PUT21, no acteylputrescine accumulated, but about 41% more putrescine as compared to the parent strain. Moreover, a transcriptome approach identified increased expression of the cgmAR operon encoding a putative permease and a transcriptional TetR-family repressor upon induction of putrescine production in C. glutamicum PUT21. CgmR is known to bind to cgmO upstream of cgmAR and gel mobility shift experiments with purified CgmR revealed that putrescine and other diamines perturbed CgmR-cgmO complex formation, but not migration of free cgmO DNA. Deletion of the repressor gene cgmR resulted in expression changes of a number of genes and increased putrescine production of C. glutamicum PUT21 by 19% as compared to the parent strain. Overexpression of the putative transport gene cgmA increased putrescine production by 24% as compared to the control strain. However, cgmA overexpression in PUT21ΔsnaA did not further improve putrescine production, hence, the beneficial effects of both targets were not synergistic at the highest described yield of 0.21 g g(-1).

  10. Glutamate Fermentation-2: Mechanism of L-Glutamate Overproduction in Corynebacterium glutamicum.

    PubMed

    Hirasawa, Takashi; Wachi, Masaaki

    The nonpathogenic coryneform bacterium, Corynebacterium glutamicum, was isolated as an L-glutamate-overproducing microorganism by Japanese researchers and is currently utilized in various amino acid fermentation processes. L-Glutamate production by C. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and β-lactam antibiotics. These treatments affect the cell surface structures of C. glutamicum. After the discovery of C. glutamicum, many researchers have investigated the underlying mechanism of L-glutamate overproduction with respect to the cell surface structures of this organism. Furthermore, metabolic regulation during L-glutamate overproduction by C. glutamicum, particularly, the relationship between central carbon metabolism and L-glutamate biosynthesis, has been investigated. Recently, the role of a mechanosensitive channel protein in L-glutamate overproduction has been reported. In this chapter, mechanisms of L-glutamate overproduction by C. glutamicum have been reviewed.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2016-06-01

    Amylomaltase (α-1,4-glucanotransferase, AM; EC 2.4.1.25) from Corynebacterium glutamicum expressed in Escherichia coli was used to prepare the enzyme-modified cassava starch for food application. About 5% to 15% (w/v) of cassava starch slurries were incubated with 1, 3, or 5 units of amylomaltase/g starch. Apparent amylose, amylopectin chain length distribution, thermal properties, freeze-thaw stability, thermo-reversibility, and gel strength of the obtained modified starches were measured. The apparent amylose content and retrogradation enthalpy were lower, whereas the retrogradation temperatures, freeze-thaw stability, and thermo-reversibility were higher than those of the native cassava starch. However, when amylomaltase content was increased to 20 units of amylomaltase/g starch and for 24 h, the modified starch showed an improvement in the thermo-reversibility property. When used in panna cotta, the gel strength of the sample using the 20 units/24 h modified cassava starch was similar to that of using gelatin. © 2016 Institute of Food Technologists®

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

    PubMed

    Woo, Han Min; Park, Jin-Byung

    2014-06-20

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

  15. Biosynthesis of pinene from glucose using metabolically-engineered Corynebacterium glutamicum.

    PubMed

    Kang, Min-Kyoung; Eom, Jin-Hee; Kim, Yunje; Um, Youngsoon; Woo, Han Min

    2014-10-01

    Pinene is a monoterpenes (C10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. Herein, we have metabolically-engineered Corynebacterium glutamicum, to produce pinene and studied its toxicity in C. glutamicum. Geranyl diphosphate synthases (GPPS) and pinene synthases (PS), obtained from Pinus taeda and Abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and isopentenyl diphosphate isomerase (Idi) from C. glutamicum using CoryneBrick vector. Most strains expressing PS-GPPSs produced detectable amounts of pinene, but co-expression of DXS and IDI with PS (P. taeda) and GPPS (A. grandis) resulted in 27 μg ± 7 α-pinene g(-1) cell dry weight, which is the first report in C. glutamicum. Further engineering of PS and GPPS in the C. glutamicum strain may increase pinene production.

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

    PubMed Central

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

    2014-01-01

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

  17. Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Krämer, Reinhard; Stephanopoulos, Gregory; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum. PMID:14711665

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

    PubMed Central

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

    2016-01-01

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

  19. A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose.

    PubMed

    Jorge, João M P; Leggewie, Christian; Wendisch, Volker F

    2016-11-01

    Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L(-1) of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L(-1), and the volumetric productivity was increased to 0.31 g L(-1) h(-1); the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.

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

    PubMed

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

    2008-10-01

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

  1. Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?

    PubMed

    Shen, Xi-Hui; Zhou, Ning-Yi; Liu, Shuang-Jiang

    2012-07-01

    With the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by Corynebacterium glutamicum has been emerging. This review summarizes recent findings on degradation of aromatic compounds by C. glutamicum. Among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in C. glutamicum. Other important knowledge derived from C. glutamicum would be the discovery of linkages among aromatic degradation and primary metabolisms such as gluconeogenesis and central carbon metabolism. Various transporters in C. glutamicum have also been identified, and they play an essential role in microbial assimilation of aromatic compounds. Regulation on aromatic degradation occurs mainly at transcription level via pathway-specific regulators, but global regulator(s) is presumably involved in the regulation. It is concluded that C. glutamicum is a very useful model organism to disclose new knowledge of biochemistry, physiology, and genetics of the catabolism of aromatic compounds in high GC content Gram-positive bacteria, and that the new physiological properties of aromatic degradation and assimilation are potentially important for industrial applications of C. glutamicum.

  2. Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

    PubMed Central

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

    2013-01-01

    To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63up, and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the β-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63up mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63up fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic

  3. Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869.

    PubMed

    Kikuchi, Yoshimi; Date, Masayo; Itaya, Hiroshi; Matsui, Kazuhiko; Wu, Long-Fei

    2006-11-01

    Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.

  4. Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum.

    PubMed

    Li, Pan-Pan; Liu, Ya-Jun; Liu, Shuang-Jiang

    2009-10-01

    Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as 'conserved hypothetical protein' in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.

  5. Global transcriptomic analysis of the response of Corynebacterium glutamicum to ferulic acid.

    PubMed

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Xiao, He; Zhang, Yaoling; Si, Meiru; Shen, Xihui; Wang, Yao

    2017-03-01

    Corynebacterium glutamicum can survive by using ferulic acid as the sole carbon source. In this study, we assessed the response of C. glutamicum to ferulic acid stress by means of a global transcriptional response analysis. The transcriptional data showed that several genes involved in degradation of ferulic acid were affected. Moreover, several genes related to the stress response; protein protection or degradation and DNA repair; replication, transcription and translation; and the cell envelope were differentially expressed. Deletion of the katA or sigE gene in C. glutamicum resulted in a decrease in cell viability under ferulic acid stress. These insights will facilitate further engineering of model industrial strains, with enhanced tolerance to ferulic acid to enable easy production of biofuels from lignocellulose.

  6. Bio-based production of chemicals, materials and fuels -Corynebacterium glutamicum as versatile cell factory.

    PubMed

    Becker, Judith; Wittmann, Christoph

    2012-08-01

    Since their discovery almost 60 years ago, Corynebacterium glutamicum and related subspecies are writing a remarkable success story in industrial biotechnology. Today, these gram-positive soil bacteria, traditionally well-known as excellent producers of L-amino acids are becoming flexible, efficient production platforms for various chemicals, materials and fuels. This development is intensively driven by systems metabolic engineering concepts integrating systems biology and synthetic biology into strain engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Growth response of Avena sativa in amino-acids-rich soils converted from phenol-contaminated soils by Corynebacterium glutamicum.

    PubMed

    Lee, Soo Youn; Kim, Bit-Na; Choi, Yong Woo; Yoo, Kye Sang; Kim, Yang-Hoon; Min, Jiho

    2012-04-01

    The biodegradation of phenol in laboratory-contaminated soil was investigated using the Gram-positive soil bacterium Corynebacterium glutamicum. This study showed that the phenol degradation caused by C. glutamicum was greatly enhanced by the addition of 1% yeast extract. From the toxicity test using Daphnia magna, the soil did not exhibit any hazardous effects after the phenol was removed using C. glutamicum. Additionally, the treatment of the phenolcontaminated soils with C. glutamicum increased various soil amino acid compositions, such as glycine, threonine, isoleucine, alanine, valine, leucine, tyrosine, and phenylalanine. This phenomenon induced an increase in the seed germination rate and the root elongation of Avena sativa (oat). This probably reflects that increased soil amino acid composition due to C. glutamicum treatment strengthens the plant roots. Therefore, the phenol-contaminated soil was effectively converted through increased soil amino acid composition, and additionally, the phenol in the soil environment was biodegraded by C. glutamicum.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2016-02-01

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

  11. Transcriptome and Multivariable Data Analysis of Corynebacterium glutamicum under Different Dissolved Oxygen Conditions in Bioreactors

    PubMed Central

    Sun, Yang; Guo, Wenwen; Wang, Fen; Peng, Feng; Yang, Yankun; Dai, Xiaofeng; Liu, Xiuxia; Bai, Zhonghu

    2016-01-01

    Dissolved oxygen (DO) is an important factor in the fermentation process of Corynebacterium glutamicum, which is a widely used aerobic microbe in bio-industry. Herein, we described RNA-seq for C. glutamicum under different DO levels (50%, 30% and 0%) in 5 L bioreactors. Multivariate data analysis (MVDA) models were used to analyze the RNA-seq and metabolism data to investigate the global effect of DO on the transcriptional distinction of the substance and energy metabolism of C. glutamicum. The results showed that there were 39 and 236 differentially expressed genes (DEGs) under the 50% and 0% DO conditions, respectively, compared to the 30% DO condition. Key genes and pathways affected by DO were analyzed, and the result of the MVDA and RNA-seq revealed that different DO levels in the fermenter had large effects on the substance and energy metabolism and cellular redox balance of C. glutamicum. At low DO, the glycolysis pathway was up-regulated, and TCA was shunted by the up-regulation of the glyoxylate pathway and over-production of amino acids, including valine, cysteine and arginine. Due to the lack of electron-acceptor oxygen, 7 genes related to the electron transfer chain were changed, causing changes in the intracellular ATP content at 0% and 30% DO. The metabolic flux was changed to rebalance the cellular redox. This study applied deep sequencing to identify a wealth of genes and pathways that changed under different DO conditions and provided an overall comprehensive view of the metabolism of C. glutamicum. The results provide potential ways to improve the oxygen tolerance of C. glutamicum and to modify the metabolic flux for amino acid production and heterologous protein expression. PMID:27907077

  12. Transcriptome and Multivariable Data Analysis of Corynebacterium glutamicum under Different Dissolved Oxygen Conditions in Bioreactors.

    PubMed

    Sun, Yang; Guo, Wenwen; Wang, Fen; Peng, Feng; Yang, Yankun; Dai, Xiaofeng; Liu, Xiuxia; Bai, Zhonghu

    2016-01-01

    Dissolved oxygen (DO) is an important factor in the fermentation process of Corynebacterium glutamicum, which is a widely used aerobic microbe in bio-industry. Herein, we described RNA-seq for C. glutamicum under different DO levels (50%, 30% and 0%) in 5 L bioreactors. Multivariate data analysis (MVDA) models were used to analyze the RNA-seq and metabolism data to investigate the global effect of DO on the transcriptional distinction of the substance and energy metabolism of C. glutamicum. The results showed that there were 39 and 236 differentially expressed genes (DEGs) under the 50% and 0% DO conditions, respectively, compared to the 30% DO condition. Key genes and pathways affected by DO were analyzed, and the result of the MVDA and RNA-seq revealed that different DO levels in the fermenter had large effects on the substance and energy metabolism and cellular redox balance of C. glutamicum. At low DO, the glycolysis pathway was up-regulated, and TCA was shunted by the up-regulation of the glyoxylate pathway and over-production of amino acids, including valine, cysteine and arginine. Due to the lack of electron-acceptor oxygen, 7 genes related to the electron transfer chain were changed, causing changes in the intracellular ATP content at 0% and 30% DO. The metabolic flux was changed to rebalance the cellular redox. This study applied deep sequencing to identify a wealth of genes and pathways that changed under different DO conditions and provided an overall comprehensive view of the metabolism of C. glutamicum. The results provide potential ways to improve the oxygen tolerance of C. glutamicum and to modify the metabolic flux for amino acid production and heterologous protein expression.

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

    PubMed

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

    2016-02-01

    Corynebacterium glutamicum, typically used as industrial workhorse for amino acid production, is a moderately salt-alkali-tolerant microorganism with optimal growth at pH 7-9. However, little is known about the mechanisms of salt-alkali tolerance in C. glutamicum. Here, the catalytic capacity of three putative Na(+)/H(+) antiporters from C. glutamicum (designated as Cg-Mrp1, Cg-Mrp2 and Cg-NhaP) were characterized in an antiporter-deficient Escherichia coli KNabc strain. Only Cg-Mrp1 was able to effectively complement the Na(+)-sensitive of E. coli KNabc. Cg-Mrp1 exhibited obvious Na(+)(Li(+))/H(+) antiport activities with low apparent Km values of 1.08 mM and 1.41 mM for Na(+) and Li(+), respectively. The Na(+)/H(+) antiport activity of Cg-Mrp1 was optimal in the alkaline pH range. All three antiporters showed detectable K(+)/H(+) antiport activitiy. Cg-NhaP also exhibited Na(+)(Li(+),Rb(+))/H(+) antiport activities but at lower levels of activity. Interestingly, overexpression of Cg-Mrp2 exhibited clear Na(+)(K(+))/H(+) antiport activities. These results suggest that C. glutamicum Na(+)(K(+))/H(+) antiporters may have overlapping roles in coping with salt-alkali and perhaps high-osmolarity stress. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum.

    PubMed

    Takeno, Seiki; Ohnishi, Junko; Komatsu, Tomoha; Masaki, Tatsuya; Sen, Kikuo; Ikeda, Masato

    2007-07-01

    Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O(2)), while no visible colonies were formed in the absence of O(2). However, in the presence of nitrate (NO3-), the organism exhibited limited growth anaerobically with production of nitrite (NO2-), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using L-lysine- and L-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

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

    PubMed Central

    Seibold, Gerd M.; Eikmanns, Bernhard J.

    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 contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways. PMID:23863124

  16. Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum.

    PubMed

    Leßmeier, Lennart; Wendisch, Volker F

    2015-10-16

    Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by experimental evolution in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  19. Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

    PubMed

    Zhang, Chuanzhi; Zhang, Junli; Kang, Zhen; Du, Guocheng; Chen, Jian

    2015-05-01

    Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

  20. A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum.

    PubMed

    Ankri, S; Bouvier, I; Reyes, O; Predali, F; Leblon, G

    1996-07-01

    Brevibacterium linens RBL strain cryptic plasmid pRBL1 (8.0 kb) is described. A region involved in pRBL1 autonomous replication in Corynebacterium glutamicum was identified by insertion and deletion mapping and partially sequenced. This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C. glutamicum) and pAL5000 (Mycobacterium fortuitum). The 310-aa protein also shows significant homology to proteins of pColE5-099 (Shigella sonnei) and pJD1 (Neisseria gonorrhoea). At least one of these proteins, the Rep protein of pColE5-099, is known to be involved in theta replication.

  1. Global Transcriptomic Analysis of the Response of Corynebacterium glutamicum to Vanillin.

    PubMed

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Guo, Chenghao; Ding, Wei; Si, Meiru; Zhang, Yi; Shen, Xihui; Wang, Yao

    2016-01-01

    Lignocellulosic biomass is an abundant and renewable resource for biofuels and bio-based chemicals. Vanillin is one of the major phenolic inhibitors in biomass production using lignocellulose. To assess the response of Corynebacterium glutamicum to vanillin stress, we performed a global transcriptional response analysis. The transcriptional data showed that the vanillin stress not only affected the genes involved in degradation of vanillin, but also differentially regulated several genes related to the stress response, ribosome/translation, protein secretion, and the cell envelope. Moreover, deletion of the sigH or msrA gene in C. glutamicum resulted in a decrease in cell viability under vanillin stress. These insights will promote further engineering of model industrial strains, with enhanced tolerance or degradation ability to vanillin to enable suitable production of biofuels and bio-based chemicals from lignocellulosic biomass.

  2. Global Transcriptomic Analysis of the Response of Corynebacterium glutamicum to Vanillin

    PubMed Central

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Guo, Chenghao; Ding, Wei; Si, Meiru; Zhang, Yi; Shen, Xihui; Wang, Yao

    2016-01-01

    Lignocellulosic biomass is an abundant and renewable resource for biofuels and bio-based chemicals. Vanillin is one of the major phenolic inhibitors in biomass production using lignocellulose. To assess the response of Corynebacterium glutamicum to vanillin stress, we performed a global transcriptional response analysis. The transcriptional data showed that the vanillin stress not only affected the genes involved in degradation of vanillin, but also differentially regulated several genes related to the stress response, ribosome/translation, protein secretion, and the cell envelope. Moreover, deletion of the sigH or msrA gene in C. glutamicum resulted in a decrease in cell viability under vanillin stress. These insights will promote further engineering of model industrial strains, with enhanced tolerance or degradation ability to vanillin to enable suitable production of biofuels and bio-based chemicals from lignocellulosic biomass. PMID:27760214

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

    PubMed

    Eggeling, Lothar; Bott, Michael

    2015-04-01

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

  4. Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Morbach, Susanne; Krämer, Reinhard; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway. PMID:15240254

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

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

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

  6. Impact of different CO2/HCO3- levels on metabolism and regulation in Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Buchholz, Jens; Busche, Tobias; Kalinowski, Jörn; Takors, Ralf

    2013-12-01

    We investigated the growth kinetics and transcriptional responses of Corynebacterium glutamicum in environments with low (pCO2<40 mbar) and high (pCO2 ≥ 300 mbar) CO2/HCO3(-) levels compared to standard conditions. When cultivated at high CO2/HCO3(-)-levels, C. glutamicum showed increased (63%) biomass to substrate yields during the initial growth phase. Other kinetic parameters such as growth rate (μ), specific glucose consumption rate (qS), and selected enzymatic activities of anaplerotic reactions, the pentose phosphate pathway and the tricarboxylic acid cycle were similar to standard conditions. However, microarray hybridization disclosed a complex transcriptional response involving 117 differentially expressed genes. Among those, 60 genes were assigned to the complete DtxR/RipA regulon controlling iron homeostasis in C. glutamicum. Impaired growth of a ΔdtxR mutant at high CO2/HCO3(-) levels validated the relevance of this master regulator to cope with excessive CO2/HCO3(-) availability. At low CO2/HCO3(-) levels, C. glutamicum grew in a bi-level manner with three distinct growth phases. Differential analyses revealed approximately doubled activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase accompanied by the formation of L-alanine and L-valine during the lowest μ occurring in mid-phase of the cultivation. DNA microarray analysis revealed more than 100 differentially expressed genes in growth phase II compared to phase I including almost all thiamin pyrophosphate (TPP) biosynthesis genes, which were significantly up regulated. Concluding, we hypothesize that C. glutamicum counteracts the lack of CO2/HCO3(-) by triggering TPP biosynthesis for increasing the activities of TPP-dependent enzymes involved in CO2 formation. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2012-03-01

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

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

    SciTech Connect

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

    2006-11-01

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

  9. Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production.

    PubMed

    Zhang, Bin; Yu, Miao; Zhou, Ying; Li, Yixue; Ye, Bang-Ce

    2017-09-22

    L-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, L-ornithine production is based on microbial fermentation, and few microbes are used for producing L-ornithine owing to unsatisfactory production titer. In this study, Corynebacterium glutamicum S9114, a high glutamate-producing strain, was developed for L-ornithine production by pathway engineering. First, argF was deleted to block L-ornithine to citrulline conversion. To improve L-ornithine production, ncgl1221 encoding glutamate transporter, argR encoding arginine repressor, and putP encoding proline transporter were disrupted. This base strain was further engineered by attenuating oxoglutarate dehydrogenase to increase L-ornithine production. Plasmid-based overexpression of argCJBD operon and lysine/arginine transport protein LysE was tested to strengthen L-ornithine synthesis and transportation. This resulted in efficient L-ornithine production at a titer of 18.4 g/L. These results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient L-ornithine production and provide new targets for strain development.

  10. Industrial Production of 2,3-Butanediol from the Engineered Corynebacterium glutamicum.

    PubMed

    Yang, Jeongmo; Kim, Borim; Kim, Hyunsu; Kweon, Yuhyeon; Lee, Soojin; Lee, Jinwon

    2015-08-01

    The platform chemical 2,3-butanediol (2,3-BDO) is a valuable product that can be converted into several petroleum-based chemicals via simple chemical reactions. Here, we produced 2,3-BDO with the non-pathogenic and rapidly growing Corynebacterium glutamicum. To enhance the 2,3-BDO production capacity of C. glutamicum, we introduced budA encoding acetolactate decarboxylase from Klebsiella pneumoniae, a powerful 2,3-BDO producer. Additionally, budB (encoding α-acetolactate synthase) and budC (encoding acetoin reductase) were introduced from K. pneumoniae to reinforce the carbon flux in the 2,3-BDO production. Because budC had a negative effect on 2,3-BDO production in C. glutamicum, the budB and budA introduced strain, SGSC102, was selected for 2,3-BDO production, and batch culture was performed at 30 °C, 250 rpm and pH 6.86 with pure glucose, molasses, and cassava powder as carbon substrates. After batch culture, significant amount of 2,3-BDO (18.9 and 12.0 g/L, respectively) was produced from 80 g/L of pure glucose and cassava powder.

  11. Molecular mechanisms and metabolic engineering of glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Hirasawa, Takashi; Kim, Jongpill; Shirai, Tomokazu; Furusawa, Chikara; Shimizu, Hiroshi

    2012-01-01

    Glutamate is a commercially important chemical. It is used as a flavor enhancer and is a major raw material for producing industrially useful chemicals. A coryneform bacterium, Corynebacterium glutamicum, was isolated in 1956 by Japanese researchers as a glutamate-overproducing bacterium and since then, remarkable progress in glutamate production has been made using this microorganism. Currently, the global market for glutamate is over 2.5 million tons per year. Glutamate overproduction by C. glutamicum is induced by specific treatments-biotin limitation, addition of fatty acid ester surfactants such as Tween 40, and addition of β-lactam antibiotics such as penicillin. Molecular biology and metabolic engineering studies on glutamate overproduction have revealed that metabolic flow is significantly altered by these treatments. These studies have also provided insight into the molecular mechanisms underlying these changes. In this chapter, we review our current understanding of the molecular mechanisms of glutamate overproduction in C. glutamicum, and we discuss the advances made by metabolic engineering of this microorganism.

  12. IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum.

    PubMed

    Heider, Sabine A E; Peters-Wendisch, Petra; Beekwilder, Jules; Wendisch, Volker F

    2014-11-01

    Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.

  13. Chromosome segregation impacts on cell growth and division site selection in Corynebacterium glutamicum.

    PubMed

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids.

  14. Chromosome Segregation Impacts on Cell Growth and Division Site Selection in Corynebacterium glutamicum

    PubMed Central

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids. PMID:23405112

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

    PubMed

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

    2016-03-01

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

  16. Metabolic engineering of Corynebacterium glutamicum for the production of L-ornithine.

    PubMed

    Kim, Seo Yun; Lee, Joungmin; Lee, Sang Yup

    2015-02-01

    L-ornithine is a non-essential amino acid for various industrial applications in food industry. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for the production of L-ornithine. First, the proB and argF genes were deleted to block the competitive branch pathway and to block the conversion of L-ornithine to citrulline, respectively. In addition, the argR gene encoding the regulatory repressor of the L-arginine operon was also deleted. The resulting strain produced 230 mg/L of L-ornithine from glucose in flask culture. This base strain was further engineered by the plasmid-based overexpression of the argCJBD genes from C. glutamicum ATCC 21831, which resulted in the production of 7.19 g/L of L-ornithine. To enrich the NADPH pool, the carbon flux was redirected towards the pentose phosphate pathway by changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter. Fed-batch cultivation of this final strain YW06 (pSY223) allowed production of 51.5 g/L of L-ornithine in 40 h with the overall productivity of 1.29 g/L/h. The results obtained in this study demonstrate the possibility of efficiently producing L-ornithine by metabolically engineered C. glutamicum.

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

    PubMed

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

    2007-08-01

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

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

    PubMed

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

    2014-06-01

    In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2014-07-24

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

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

    PubMed

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

    2016-02-01

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

  2. Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

    PubMed

    Radoš, Dušica; Carvalho, Ana Lúcia; Wieschalka, Stefan; Neves, Ana Rute; Blombach, Bastian; Eikmanns, Bernhard J; Santos, Helena

    2015-10-29

    2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1). We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

  3. Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization.

    PubMed

    Kang, Min-Kyoung; Lee, Jungseok; Um, Youngsoon; Lee, Taek Soon; Bott, Michael; Park, Si Jae; Woo, Han Min

    2014-07-01

    Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

    Käß, Friedrich; Junne, Stefan; Neubauer, Peter; Wiechert, Wolfgang; Oldiges, Marco

    2014-01-10

    Corynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. This means the demand for industrial process development is likely to increase. However, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. This leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertain impact on metabolism. Scale-down bioreactors can be applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale. In this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth and intracellular metabolite characteristics. Carbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a futile cycle over extracellular side products and back into oxidative pathways. This

  7. Impact of pulsed electric fields on Corynebacterium glutamicum cell membrane permeabilization.

    PubMed

    Tryfona, Theodora; Bustard, Mark T

    2008-04-01

    The permeability barrier of the microbial cell envelope for substrates and products often causes very low reaction rates of whole cells. Therefore, it is of interest to develop an effective method to reduce this permeability barrier in order to increase product yields. Utilisation of pulse electric fields may improve amino acid release from Corynebacterium glutamicum by up to several orders of magnitude. In particular pulsed electric fields may change the cell/membrane's dielectric properties and induce the release of intracellular metabolites. In this study the parameters for successful electropermeabilization were determined and the viabilities of treated cells were examined. We also found that pulse treated cells not only maintained their viabilities but also their ability to reproduce, post-pulse treatment. Since electropermeabilized cells could maintain both their viabilities and ability to reproduce, we believe that this preliminary data may contribute to the optimization of fermentative production of amino acids and bioprocess enhancement through electropermeabilization and may be beneficial to industrial bioprocesses.

  8. Improved succinate production in Corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system.

    PubMed

    Zhu, Nianqing; Xia, Huihua; Yang, Jiangang; Zhao, Xueming; Chen, Tao

    2014-03-01

    A dual route for anaerobic succinate production was engineered into Corynebacterium glutamicum. The glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. The engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). Further overexpression of succinate exporter, SucE, increased succinate yield to 1.43 mol (mol glucose)(-1). Metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineering succinate export system. Using an anaerobic fed-batch fermentation process, the final strain produced 926 mM succinate (= 109 g l(-1)) with an overall volumetric productivity of 9.4 mM h(-1) and an average yield of 1.32 mol (mol glucose)(-1).

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

    SciTech Connect

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

    2008-11-21

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

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

    PubMed

    Tölzer, Christine; Pal, Sonia; Watzlawick, Hildegard; Altenbuchner, Josef; Niefind, Karsten

    2009-01-01

    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 A resolution.

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

    PubMed Central

    Tölzer, Christine; Pal, Sonia; Watzlawick, Hildegard; Altenbuchner, Josef; Niefind, Karsten

    2009-01-01

    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. PMID:19153452

  12. PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

    PubMed Central

    Costa-Riu, Noelia; Burkovski, Andreas; Krämer, Reinhard; Benz, Roland

    2003-01-01

    The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032ΔporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA. PMID:12896997

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

    PubMed

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

    2016-08-10

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

  14. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

    PubMed

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

    2015-12-01

    ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C5 pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide.

    PubMed

    Witthoff, Sabrina; Mühlroth, Alice; Marienhagen, Jan; Bott, Michael

    2013-11-01

    Methanol is considered an interesting carbon source in "bio-based" microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. The C. glutamicum wild type was able to convert (13)C-labeled methanol to (13)CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δald ΔadhE and Δald ΔmshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.

  16. C1 Metabolism in Corynebacterium glutamicum: an Endogenous Pathway for Oxidation of Methanol to Carbon Dioxide

    PubMed Central

    Witthoff, Sabrina; Mühlroth, Alice

    2013-01-01

    Methanol is considered an interesting carbon source in “bio-based” microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. The C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δald ΔadhE and Δald ΔmshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway. PMID:24014532

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

    PubMed Central

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

    2015-01-01

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

  18. Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production.

    PubMed

    Lai, Shujuan; Zhang, Yun; Liu, Shuwen; Liang, Yong; Shang, Xiuling; Chai, Xin; Wen, Tingyi

    2012-04-01

    L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH(r)). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22 ± 1.41) μmol g(CDM) (-1), which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH(r) improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C(1) units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

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

    PubMed

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

    2013-05-01

    The sustainable production of industrial platform chemicals is one of the great challenges facing the biotechnology field. Ideally, fermentation feedstocks would rather rely on industrial waste streams than on food-based raw materials. Corynebacterium glutamicum was metabolically engineered to produce the bio-nylon precursor 1,5-diaminopentane from the hemicellulose sugar xylose. Comparison of a basic diaminopentane producer strain on xylose and glucose feedstocks revealed a 30% reduction in diaminopentane yield and productivity on the pentose sugar. The integration of in vivo and in silico metabolic flux analysis by (13) C and elementary modes identified bottlenecks in the pentose phosphate pathway and the tricarboxylic acid cycle that limited performance on xylose. By the integration of global transcriptome profiling, this could be specifically targeted to the tkt operon, genes that encode for fructose bisphosphatase (fbp) and isocitrate dehydrogenase (icd), and to genes involved in formation of lysine (lysE) and N-acetyl diaminopentane (act). This was used to create the C. glutamicum strain DAP-Xyl1 icd(GTG) Peftu fbp Psod tkt Δact ΔlysE. The novel producer, designated DAP-Xyl2, exhibited a 54% increase in product yield to 233 mmol mol(-1) and a 100% increase in productivity to 1 mmol g(-1) h(-1) on the xylose substrate. In a fed-batch process, the strain achieved 103 g L(-1) of diaminopentane from xylose with a product yield of 32%. Xylose utilization is currently one of the most relevant metabolic engineering subjects. In this regard, the current work is a milestone in industrial strain engineering of C. glutamicum. See accompanying commentary by Hiroshi Shimizu DOI: 10.1002/biot.201300097. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2007-01-01

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

  1. Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique.

    PubMed

    Pfeifer-Sancar, Katharina; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2013-12-17

    The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation. RNAseq data sets were obtained by two methods, one that focuses on 5'-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3'-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5'-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present. The comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5'-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.

  2. Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique

    PubMed Central

    2013-01-01

    Background The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation. Results RNAseq data sets were obtained by two methods, one that focuses on 5′-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3′-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5′-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present. Conclusions The comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5′-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future. PMID:24341750

  3. Transcriptomic analysis for elucidating the physiological effects of 5-aminolevulinic acid accumulation on Corynebacterium glutamicum.

    PubMed

    Yu, Xiaoli; Jin, Haiying; Cheng, Xuelian; Wang, Qian; Qi, Qingsheng

    2016-11-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways.

  4. Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum.

    PubMed Central

    Hoischen, C; Krämer, R

    1990-01-01

    We showed recently that secretion of glutamate in biotin-limited cells of Corynebacterium glutamicum is mediated by carrier systems in the plasma membrane (C. Hoischen and R. Krämer, Arch. Microbiol. 151:342-347, 1989). In view of the generally accepted hypothesis that glutamate efflux is directly caused by alterations of the membrane, it was necessary to examine the kind of correlation between changes in lipid content and composition of the bacterial membrane and glutamate secretion activity. Two new experimental approaches were used. (i) Changes in lipid content and composition were analyzed in glutamate-producing cells which were forced to switch to nonproducers by addition of biotin in a short-term fermentation. (ii) The time courses of both the fatty acid or phospholipid composition and the efflux activity were analyzed within the first minutes of the switch from high to low secretion activity. The following results were obtained. (i) The time course of the change in fatty acid or phospholipid content and composition was not related to the change in secretion behavior. (ii) There was no specific fatty acid or phospholipid compound which regulated glutamate efflux. (iii) High efflux activity could only be induced when the total lipid content of the membrane was reduced. (iv) Although consistently correlated to high secretion activity, membrane alteration was never a sufficient prerequisite for glutamate efflux in C. glutamicum. PMID:1971623

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

    PubMed Central

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

    2015-01-01

    Summary 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, Ala-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, while 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. Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

    PubMed

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

    2013-01-20

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

  7. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

    PubMed Central

    Eikmanns, Bernhard J

    2011-01-01

    Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions. PMID:22008938

  8. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Eikmanns, Bernhard J

    2011-01-01

    Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Pátek, Miroslav

    2016-01-01

    ABSTRACT 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

  11. Miniaturized and automated adaptive laboratory evolution: Evolving Corynebacterium glutamicum towards an improved d-xylose utilization.

    PubMed

    Radek, Andreas; Tenhaef, Niklas; Müller, Moritz Fabian; Brüsseler, Christian; Wiechert, Wolfgang; Marienhagen, Jan; Polen, Tino; Noack, Stephan

    2017-05-12

    Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCDCc, which is capable of utilizing d-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2evo was obtained, showing a specific growth rate of 0.26h(-1) on d-xylose as sole carbon and energy source. WMB2evo grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved d-xylose utilization. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2015-11-01

    Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    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 overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology. PMID:12406733

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

    PubMed Central

    Liu, Qingbo; Li, Defeng; Hu, Yonglin; Wang, Da-Cheng

    2013-01-01

    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. PMID:23722846

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

    PubMed

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

    2014-10-01

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

  17. Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum.

    PubMed

    Vogt, Michael; Brüsseler, Christian; Ooyen, Jan van; Bott, Michael; Marienhagen, Jan

    2016-11-01

    The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicuml-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37g/l 2-methyl-1-butanol and 2.76g/l 3-methyl-1-butanol in defined medium within 48h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.

  18. Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

    PubMed Central

    Shen, Xi-Hui; Jiang, Cheng-Ying; Huang, Yan; Liu, Zhi-Pei; Liu, Shuang-Jiang

    2005-01-01

    Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified. PMID:16000747

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

    PubMed

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

    2016-01-01

    Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  20. Recent advances in the metabolic engineering of Corynebacterium glutamicum for the production of lactate and succinate from renewable resources.

    PubMed

    Tsuge, Yota; Hasunuma, Tomohisa; Kondo, Akihiko

    2015-03-01

    Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce L- and D-lactate, and succinate from renewable resources.

  1. Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum

    PubMed Central

    Schubert, Karin; Bramkamp, Marc; Frunzke, Julia

    2016-01-01

    ABSTRACT Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum. IMPORTANCE About one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics

  2. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase.

    PubMed Central

    Eikmanns, B J

    1992-01-01

    To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented. Images PMID:1400158

  3. Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products.

    PubMed

    Heider, Sabine A E; Wendisch, Volker F

    2015-08-01

    Corynebacterium glutamicum is the workhorse of biotechnological amino acid production. For more than 50 years amino acid producing strains of this actinomycete have been improved by classical breeding, metabolic engineering and systems and synthetic biology approaches. This review focusses mainly on recent developments on C. glutamicum strain development for non-natural products. Recently, metabolite sensors have accelerated classical strain breeding. Synthetic pathways for access to alternative carbon sources, such as pentoses, and to new products, such as α, ω-amino acids, α, ω-diamines, α-keto acids, isobutanol, carotenoids and terpenes, have been embedded in the central metabolism of C. glutamicum. Furthermore, C. glutamicum is a chassis for new and improved production processes that has been improved in two ways: by rendering it biotin prototrophic and by curing it from its prophage DNA followed by further genome reduction. The first combinations of this chassis approach with production will be highlighted. Although their transfer to industrial scale processes will have to be evaluated, these recent achievements indicate how synthetic biology helps realizing proof-of-principles. Moreover, current and future synthetic biology technology developments hold the promise to explore the full potential of C. glutamicum as production host for value-added chemicals. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. Efficient hydroxyproline production from glucose in minimal media by Corynebacterium glutamicum.

    PubMed

    Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

    2015-02-01

    The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose/isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L(-1) of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose/isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media. © 2014 Wiley Periodicals, Inc.

  7. Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Yamamoto, Shogo; Suda, Masako; Niimi, Satoko; Inui, Masayuki; Yukawa, Hideaki

    2013-11-01

    Microbial production of isobutanol is made difficult by the chemical's high cell toxicity. Corynebacterium glutamicum, inherently one of the more isobutanol-tolerant industrial microorganisms, exhibits unprecedented productivity under oxygen deprivation, potentially allowing for high productivity of such toxic chemicals as isobutanol. Here, we show that development of C. glutamicum strains proficient in isobutanol production depends not only on modulating the activity of 2-keto acid decarboxylase (KDC) and isobutanol dehydrogenase (IBDH) and suppressing by-product formation, but also on optimizing the production process to eschew product inhibition. Isobutanol production under oxygen deprivation reached 343 mM (3.2% v/v) in strain IBU5 expressing kivd (encoding KDC) under the control of ldhA promoter and adhP (encoding IBDH from Escherichia coli MG1655) under the control of gapA promoter. This productivity is double the previously reported best productivity of 1.6% (v/v) and exceeds the 2.5% (v/v) limit beyond which cell growth becomes too severely suppressed. Irrespective, a cumulative 56.5% improvement on yield was possible with the combined effects of disruption of the ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), use of a NAD⁺-specific mutant acetohydroxyacid isomeroreductase (AHAIR), and overexpression of select glycolytic genes. Using oleyl alcohol to continuously extract the isobutanol from reaction mixture and tripling the cell concentration in the reaction mixture to 60 g dry cell/L stretched the yield to 78.1% and volumetric productivity to 981 mM (9.1% v/v). © 2013 Wiley Periodicals, Inc.

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

    PubMed

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

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution

    PubMed Central

    Oide, Shinichi; Gunji, Wataru; Moteki, Yasuhiro; Yamamoto, Shogo; Suda, Masako; Jojima, Toru; Yukawa, Hideaki

    2015-01-01

    Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance. PMID:25595768

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

    PubMed

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

    2014-03-01

    Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application. © 2013 Published by International Metabolic Engineering Society on behalf of International Metabolic Engineering Society.

  11. Thermal and solvent stress cross-tolerance conferred to Corynebacterium glutamicum by adaptive laboratory evolution.

    PubMed

    Oide, Shinichi; Gunji, Wataru; Moteki, Yasuhiro; Yamamoto, Shogo; Suda, Masako; Jojima, Toru; Yukawa, Hideaki; Inui, Masayuki

    2015-04-01

    Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l-isoleucine production.

    PubMed

    Ma, Wenjian; Wang, Jianli; Li, Ye; Hu, Xiaoqing; Shi, Feng; Wang, Xiaoyuan

    2016-11-01

    Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP(+) ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP(+) ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

  13. Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

    PubMed

    Bolten, Christoph J; Schröder, Hartwig; Dickschat, Jeroen; Wittmann, Christoph

    2010-08-01

    In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

  14. Identification and Functional Characterization of Small Alarmone Synthetases in Corynebacterium glutamicum.

    PubMed

    Ruwe, Matthias; Kalinowski, Jörn; Persicke, Marcus

    2017-01-01

    The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP(*)Cg and RelSCg, exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein RelCg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelSCg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelSCg was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP(*)Cg , as well as the slightly differing variant RelPCg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelSCg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in

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

    PubMed Central

    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 diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode. PMID:9045809

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

    PubMed

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

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

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

    SciTech Connect

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

    2008-06-23

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

  18. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  19. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum.

    PubMed

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-11-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5'-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

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

    2016-03-18

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

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

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

    PubMed

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

    2009-08-01

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

  5. Bi-functional cellulases complexes displayed on the cell surface of Corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature.

    PubMed

    Kim, Su Jung; Hyeon, Jeong Eun; Jeon, Sang Duck; Choi, Gi-wook; Han, Sung Ok

    2014-11-01

    Introducing cellulases into Corynebacterium glutamicum leads to the direct degradation of lignocellulosic materials for energy sources. In this study, a cellulase complex containing two cellulolytic enzymes, endoglucanase E (CelE) and β-glucosidase A (BglA), was established to completely degrade cellulose to glucose. The cellulases complexes were displayed on the cell surface of C. glutamicum by using the mechanosensitive channel (Msc) to anchor enzymes in the cytoplasmic membrane. As confirmed by comparison enzyme activities in the cell pellet fraction and supernatant and dual color based immunofluorescence microscopy, the cellulolytic enzymes was successfully associated with the cell surface of C. glutamicum. The displayed cellulases complexes had a synergic effect on the direct conversion of biomass to reducing sugars leading to 3.1- to 6.0-fold increase compared to the conversion by the secreted cellulases complexes. In addition, the displayed cellulases complexes increased the residual activities of cCelE and cBglA at 70°C from 28.3% and 24.3% in the secreted form to 65.1% and 82.8%, respectively. The display of cellulases complexes on the cell surface of C. glutamicum enhances the polysaccharide equivalent and the direct saccharification of low cost biomass via the action of multi-thermostable enzyme complexes. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using Corynebacterium glutamicum immobilized in porous polyurethane filler.

    PubMed

    Shi, Xinchi; Chen, Yong; Ren, Hengfei; Liu, Dong; Zhao, Ting; Zhao, Nan; Ying, Hanjie

    2014-12-01

    An immobilized fermentation system, using cassava bagasse hydrolysate (CBH) and mixed alkalis, was developed to achieve economical succinic acid production by Corynebacterium glutamicum. The C. glutamicum strains were immobilized in porous polyurethane filler (PPF). CBH was used efficiently as a carbon source instead of more expensive glucose. Moreover, as a novel method for regulating pH, the easily decomposing NaHCO3 was replaced by mixed alkalis (NaOH and Mg(OH)2) for succinic acid production by C. glutamicum. Using CBH and mixed alkalis in the immobilized batch fermentation system, succinic acid productivity of 0.42gL(-1)h(-1) was obtained from 35gL(-1) glucose of CBH, which is similar to that obtained with conventional free-cell fermentation with glucose and NaHCO3. In repeated batch fermentation, an average of 22.5gL(-1) succinic acid could be obtained from each batch, which demonstrated the enhanced stability of the immobilized C. glutamicum cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

    PubMed

    Khuat, Hoang Bao Truc; Kaboré, Abdoul Karim; Olmos, Eric; Fick, Michel; Boudrant, Joseph; Goergen, Jean-Louis; Delaunay, Stéphane; Guedon, Emmanuel

    2014-01-01

    The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

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

    PubMed

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

    2016-08-20

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

  9. Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

    PubMed Central

    Srisimarat, Wiraya; Kaulpiboon, Jarunee; Krusong, Kuakarun; Zimmermann, Wolfgang

    2012-01-01

    Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The kcat/Km of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM. PMID:22865069

  10. Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5 platform chemicals 5-aminovalerate and glutarate.

    PubMed

    Rohles, Christina Maria; Gießelmann, Gideon; Kohlstedt, Michael; Wittmann, Christoph; Becker, Judith

    2016-09-13

    The steadily growing world population and our ever luxurious life style, along with the simultaneously decreasing fossil resources has confronted modern society with the issue and need of finding renewable routes to accommodate for our demands. Shifting the production pipeline from raw oil to biomass requires efficient processes for numerous platform chemicals being produced with high yield, high titer and high productivity. In the present work, we established a de novo bio-based production process for the two carbon-5 platform chemicals 5-aminovalerate and glutarate on basis of the lysine-hyperproducing strain Corynebacterium glutamicum LYS-12. Upon heterologous implementation of the Pseudomonas putida genes davA, encoding 5-aminovaleramidase and davB, encoding lysine monooxygenase, 5-aminovalerate production was established. Related to the presence of endogenous genes coding for 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase, 5-aminovalerate was partially converted to glutarate. Moreover, residual L-lysine was secreted as by-product. The issue of by-product formation was then addressed by deletion of the lysE gene, encoding the L-lysine exporter. Additionally, a putative gabT gene was deleted to enhance 5-aminovalerate production. To fully exploit the performance of the optimized strain, fed-batch fermentation was carried out producing 28 g L(-1) 5-aminovalerate with a maximal space-time yield of 0.9 g L(-1) h(-1). The present study describes the construction of a recombinant microbial cell factory for the production of carbon-5 platform chemicals. Beyond a basic proof-of-concept, we were able to specifically increase the production flux of 5-aminovalerate thereby generating a strain with excellent production performance. Additional improvement can be expected by removal of remaining by-product formation and bottlenecks, associated to the terminal pathway, to generate a strain being applicable as centerpiece for a bio

  11. Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

    PubMed

    Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

    In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS(L)), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC(R)), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC(R) (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS(L) (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS(L) mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD660) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

  12. Enhancement of biomolecule transport by electroporation: a review of theory and practical application to transformation of Corynebacterium glutamicum.

    PubMed

    Tryfona, Theodora; Bustard, Mark T

    2006-02-20

    Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.

  13. Media optimization of Corynebacterium glutamicum for succinate production under oxygen-deprived condition.

    PubMed

    Jeon, Jong-Min; Rajesh, Thangamani; Song, Eunjung; Lee, Hyuk-Won; Lee, Hong-Weon; Yang, Yung-Hun

    2013-02-01

    Corynebacterium glutamicum is one of the well-studied industrial strain that is used for the production of nucleotides and amino acids. Recently, it has also been studied as a possible producer of organic acids such as succinic acid, based on its ability to produce organic acids under an oxygen deprivation condition. In this study, we conducted the optimization of medium components for improved succinate production from C. glutamicum under an oxygen deprivation condition by Plackett-Burman design and applied a response surface methodology. A Plackett-Burman design for ten factors such as glucose, ammonium sulfate, magnesium sulfate, potassium phosphate (K2HPO4 and KH2PO4), iron sulfate, manganese sulfate, biotin, thiamine, and sodium bicarbonate was applied to evaluate the effects on succinate production. Glucose, ammonium sulfate, magnesium sulfate, and dipotassium phosphate were found to have significant influence on succinate production, and the optimal concentrations of these four factors were sequentially investigated by the response surface methodology using a Box-Behnken design. The optimal medium components obtained for achieving maximum concentration of succinic acid were as follows: glucose 10 g/l, magnesium sulfate 0.5 g/l, dipotassium phosphate (K2HPO4) 0.75 g/l, potassium dihydrogen phosphate (KH2PO4) 0.5 g/l, iron sulfate 6 mg/l, manganese sulfate 4.2 mg/l, biotin 0.2 mg/l, thiamine 0.2 mg/l, and sodium bicarbonate 100 mM. The parameters that differed from a normal BT medium were glucose changed from 40 g/l to 10 g/l, dipotassium phosphate (K2HPO4) 0.5 g/l changed to 0.75 g/l, and ammonium sulfate ((NH4)2SO4) 7 g/l changed to 0 g/l. Under these conditions, the final succinic acid concentration was 16.3 mM, which is about 1.46 fold higher than the original medium (11.1 mM) at 24 h. This work showed the improvement of succinate production by a simple change of media components deduced from sequential optimization.

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

    PubMed Central

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

  15. Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity.

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

    The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

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

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

    PubMed Central

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-01-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far. PMID:16267288

  19. Pathway identification combining metabolic flux and functional genomics analyses: acetate and propionate activation by Corynebacterium glutamicum.

    PubMed

    Veit, Andrea; Rittmann, Doris; Georgi, Tobias; Youn, Jung-Won; Eikmanns, Bernhard J; Wendisch, Volker F

    2009-03-10

    Corynebacterium glutamicum can utilize acetic acid and propionic acid for growth and amino acid production. Growth on acetate as sole carbon source requires acetate activation by acetate kinase (AK) and phosphotransacetylase (PTA), encoded in the pta-ack operon. Genetic and enzymatic studies showed that these enzymes also catalyze propionate activation and were required for growth on propionate as sole carbon source. However, when glucose was present as a co-substrate strain lacking the AK-PTA pathway was still able to utilize acetate or propionate for growth indicating that an alternative activation pathway exists. As shown by (13)C-labelling experiments, the carbon skeleton of acetate is conserved during activation to acetyl-CoA in this pathway. Metabolic flux analysis during growth on an acetate-glucose mixture revealed that in the absence of the AK-PTA pathway carbon fluxes in glycolysis, the tricarboxylic acid (TCA) cycle and anaplerosis via PEP carboxylase and/or pyruvate carboxylase were increased, while the glyoxylate cycle flux was decreased. DNA microarray experiments identified cg2840 as a constitutively and highly expressed gene putatively encoding a CoA transferase. Purified His-tagged Cg2840 protein was active as CoA transferase interconverting acetyl-, propionyl- and succinyl-moieties as CoA acceptors and donors. Strains lacking both the CoA transferase and the AK-PTA pathway could neither activate acetate nor propionate in the presence or absence of glucose. Thus, when these short-chain fatty acids are co-metabolized with other carbon sources, CoA transferase and the AK-PTA pathway constitute a redundant system for activation of acetate and propionate.

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

    PubMed

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

    2016-07-20

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

  1. Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase.

    PubMed

    Wada, Masaru; Hijikata, Nowaki; Aoki, Ryo; Takesue, Nobuchika; Yokota, Atsushi

    2008-11-01

    We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.

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

    PubMed Central

    2013-01-01

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

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

    PubMed

    Kappelmann, Jannick; Wiechert, Wolfgang; Noack, Stephan

    2016-03-01

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

  4. Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production.

    PubMed

    Diesveld, Ramon; Tietze, Nadine; Fürst, Oliver; Reth, Alexander; Bathe, Brigitte; Sahm, Hermann; Eggeling, Lothar

    2009-01-01

    L-Threonine is an important biotechnological product and Corynebacterium glutamicum is able to synthesize and accumulate this amino acid to high intracellular levels. We here use four exporters of Escherichia coli and show that three of them operate in C. glutamicum, with RhtA and RhtC being the most effective. Whereas RhtA was unspecific, resulting in L-homoserine together with L-threonine excretion, this was not the case with RhtC. Expression of rhtC reduced the intracellular L-threonine concentration from 140 to 11 mM and resulted in maximal excretion rates of 11.2 nmol min(-1) mg(-1) as compared to 2.3 nmol min(-1) mg(-1) obtained without rhtC expression. In combination with an ilvA mutation generated and introduced into the chromosome, an accumulation of up to 54 mM L-threonine was achieved as compared to 21 mM obtained with the ancestor strain. This shows that expression of rhtC is the pivotal point for industrial relevant L-threonine production with C. glutamicum, and might encourage in general the use of heterologous exporters in the field of white biotechnology to make full use of biosynthesis pathways.

  5. Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA.

    PubMed

    Inui, Masayuki; Tsuge, Yota; Suzuki, Nobuaki; Vertès, Alain A; Yukawa, Hideaki

    2005-01-01

    A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

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

    PubMed Central

    2013-01-01

    Background Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. Results Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. Conclusions The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising

  7. Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum.

    PubMed

    Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Guoqiang; Liang, Yong; Wen, Tingyi

    2012-08-01

    Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.

  8. Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets.

    PubMed

    Bott, Michael; Brocker, Melanie

    2012-06-01

    In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m(3) volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species.

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

    PubMed

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

    2004-02-01

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

  10. Identification and Characterization of γ-Aminobutyric Acid Uptake System GabPCg (NCgl0464) in Corynebacterium glutamicum

    PubMed Central

    Zhao, Zhi; Ma, Wen-hua; Zhou, Ning-Yi

    2012-01-01

    Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabPCg) encoded by ncgl0464 was identified and characterized. GabPCg plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabPCg was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na+). The Km and Vmax values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min−1 (mg dry weight [DW])−1, respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min−1 (mg DW)−1, respectively, at pH 7.5. GabPCg has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabPCg deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabPCg represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains. PMID:22307305

  11. Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum.

    PubMed

    Krug, Andreas; Wendisch, Volker F; Bott, Michael

    2005-01-07

    In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose. Here we show that this variation is caused by transcriptional regulation. In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C. glutamicum. Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression. DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C. glutamicum genome. Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from -11 to -28 relative to the transcription start. It thus presumably acts by interfering with the binding of RNA polymerase. The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions. Mutations within this motif inhibited AcnR binding. Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C. glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.

  12. Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Yamamoto, Shogo; Kato, Naoto; Suda, Masako; Vertès, Alain A; Yukawa, Hideaki; Inui, Masayuki

    2015-06-01

    We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.

  13. A TatABC-type Tat translocase is required for unimpaired aerobic growth of Corynebacterium glutamicum ATCC13032.

    PubMed

    Oertel, Dan; Schmitz, Sabrina; Freudl, Roland

    2015-01-01

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

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

    PubMed Central

    Oertel, Dan; Schmitz, Sabrina; Freudl, Roland

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Identification of a HAD superfamily phosphatase, HdpA, involved in 1,3-dihydroxyacetone production during sugar catabolism in Corynebacterium glutamicum.

    PubMed

    Jojima, Toru; Igari, Takafumi; Gunji, Wataru; Suda, Masako; Inui, Masayuki; Yukawa, Hideaki

    2012-11-30

    Corynebacterium glutamicum produces 1,3-dihydroxyacetone (DHA) as metabolite of sugar catabolism but the responsible enzyme is yet to be identified. Using a transposon mutant library, the gene hdpA (cgR_2128) was shown to encode a haloacid dehalogenase superfamily member that catalyzes dephosphorylation of dihydroxyacetone phosphate to produce DHA. Inactivation of hdpA led to a drastic decrease in DHA production from each of glucose, fructose, and sucrose, indicating that HdpA is the main enzyme responsible for DHA production from sugars in C. glutamicum. Confirmation of DHA production via dihydroxyacetone phosphatase finally confirms a long-speculated route through which bacteria produce DHA.

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

    PubMed

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

    2014-11-20

    The aerobic soil bacterium Corynebacterium glutamicum ATCC 13032 has a remarkable natural resistance to hydrogen peroxide. A major player in hydrogen peroxide defense is the LysR type transcriptional regulator OxyR, homologs of which are present in a wide range of bacteria. In this study, the global transcriptional response of C. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome DNA microarrays, demonstrating the dynamic reaction of the regulatory networks. Deletion of oxyR resulted in an increased resistance of the C. glutamicum mutant to hydrogen peroxide. By performing DNA microarray hybridizations and RT-qPCR, differentially expressed genes were detected in the mutant. The direct control by OxyR was verified by electrophoretic mobility shift assays for 12 target regions. The results demonstrated that OxyR in C. glutamicum acts as a transcriptional repressor under non-stress conditions for a total of 23 genes. The regulated genes encode proteins related to oxidative stress response (e.g. katA), iron homeostasis (e.g. dps) and sulfur metabolism (e.g. suf cluster). Besides the regulator of the suf cluster, SufR, OxyR regulated the gene cg1695 encoding a putative transcriptional regulator, indicating the role of OxyR as a master regulator in defense against oxidative stress. Using a modified DNase footprint approach, the OxyR-binding sites in five target promoter regions, katA, cydA, hemH, dps and cg1292, were localized and in each upstream region at least two overlapping binding sites were found. The DNA regions protected by the OxyR protein are about 56bp in length and do not have evident sequence similarities. Still, by giving an insight in the H2O2 stimulon and extending the OxyR regulon this study considerably contributes to the understanding of the response of C. glutamicum to hydrogen peroxide-mediated oxidative stress. Copyright © 2014. Published by Elsevier B.V.

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

    PubMed Central

    Santha, Sangilimadan; Pandaranayaka, Eswari P. J.; Rosen, Barry P.; Thiyagarajan, Saravanamuthu

    2011-01-01

    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. PMID:22139180

  20. Modeling and optimization of glutamic acid production using mixed culture of Corynebacterium glutamicum NCIM2168 and Pseudomonas reptilivora NCIM2598.

    PubMed

    Kumar, Rajaram Shyam; Moorthy, Innasi Muthu Ganesh; Baskar, Rajoo

    2013-01-01

    In this study, a hybrid system of response surface methodology followed by genetic algorithm has been adopted to optimize the production medium for L-glutamic acid fermentation with mixed cultures of Corynebacterium glutamicum and Pseudomonas reptilovora. The optimal combination of media components for maximal production of L-glutamic acid was found to be 49.99 g L(-1) of glucose, 10 g L(-1) of urea, 18.06% (v/v) of salt solution, and 4.99% (v/v) of inoculum size. The experimental glutamic acid yield at optimum condition was 19.69 g L(-1), which coincided well to the value predicted by the model (19.61 g L(-1)). Using this methodology, a nonlinear regression model was developed for the glutamic acid production. The model was validated statistically and the determination coefficient (R (2)) was found to be 0.99.

  1. Investigation of phosphorylation status of OdhI protein during penicillin- and Tween 40-triggered glutamate overproduction by Corynebacterium glutamicum.

    PubMed

    Kim, Jongpill; Hirasawa, Takashi; Saito, Masaki; Furusawa, Chikara; Shimizu, Hiroshi

    2011-07-01

    Glutamate overproduction by Corynebacterium glutamicum is triggered by treatment with penicillin or Tween 40 and is accompanied by a decrease in 2-oxoglutarate dehydrogenase complex (ODHC) activity. We have reported that de novo synthesis of OdhI, which inhibits ODHC activity by interacting specifically with the E1o subunit of ODHC (OdhA), is induced by penicillin, and that odhI overexpression induces glutamate overproduction in the absence of any triggers for glutamate overproduction. In this study, to determine the function of OdhI in glutamate overproduction by C. glutamicum, changes in OdhI levels and phosphorylation status during penicillin- and Tween 40-induced glutamate overproduction were examined by western blot. The synthesis of both unphosphorylated and phosphorylated OdhI was increased by addition of Tween 40 or penicillin and the levels of unphosphorylated OdhI, which can inhibit ODHC activity, was significantly higher than those of phosphorylated OdhI, which is unable to inhibit ODHC activity. Meanwhile, the OdhA levels were maintained throughout the culture. These results indicate that OdhI synthesis is induced by additions of penicillin and Tween 40 and most synthesized OdhI is unphosphorylated, resulting in the decrease in ODHC activity and glutamate overproduction. Similarly, in the odhI-overexpressing strain, both unphosphorylated and phosphorylated OdhI were synthesized, while the levels of OdhA were nearly constant throughout culture. Our results suggest that high level of unphosphorylated OdhI regulates glutamate overproduction by C. glutamicum.

  2. In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15N Nuclear Magnetic Resonance

    PubMed Central

    Tesch, M.; de Graaf, A. A.; Sahm, H.

    1999-01-01

    Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type. PMID:10049869

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

    PubMed Central

    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 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

  4. Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant

    PubMed Central

    Mishra, Arun K.; Klein, Christina; Gurcha, Sudagar S.; Alderwick, Luke J.; Babu, Ponnusamy; Hitchen, Paul G.; Morris, Howard R.; Dell, Anne

    2008-01-01

    The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac1PIM2) resulting in the accumulation of Ac1PIM1 and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed ‘Cg-LM-A’) and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed ‘Cg-LM-B’. Further chemical analyses established the lipoglycan possessed an α-d-glucopyranosyluronic acid-(1 → 3)-glycerol (GlcAGroAc2) based anchor which was then further glycosylated by 8–22 mannose residues, with Man12–20GlcAGroAC2 molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-α by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity. PMID:18421567

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

    PubMed

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

    2012-06-15

    Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2012-01-13

    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. 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. 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.

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

    PubMed Central

    2012-01-01

    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. PMID:22243621

  8. Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum

    PubMed Central

    Huang, Jinhai; Wu, Yao; Wu, Wenjun; Zhang, Ye; Liu, Dehua; Chen, Zhen

    2017-01-01

    Production of 1,3-propanediol (1,3-PDO) from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process. PMID:28176878

  9. Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source - a membrane proteome-centric view.

    PubMed

    Haussmann, Ute; Qi, Su-Wei; Wolters, Dirk; Rögner, Matthias; Liu, Shuang-Jiang; Poetsch, Ansgar

    2009-07-01

    The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with (15)N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate-grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope-labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake.

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

    PubMed

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

    2016-04-15

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

  11. Gene expression profiling of Corynebacterium glutamicum during Anaerobic nitrate respiration: induction of the SOS response for cell survival.

    PubMed

    Nishimura, Taku; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-03-01

    The gene expression profile of Corynebacterium glutamicum under anaerobic nitrate respiration revealed marked differences in the expression levels of a number of genes involved in a variety of cellular functions, including carbon metabolism and respiratory electron transport chain, compared to the profile under aerobic conditions using DNA microarrays. Many SOS genes were upregulated by the shift from aerobic to anaerobic nitrate respiration. An elongated cell morphology, similar to that induced by the DivS-mediated suppression of cell division upon cell exposure to the DNA-damaging reagent mitomycin C, was observed in cells subjected to anaerobic nitrate respiration. None of these transcriptional and morphological differences were observed in a recA mutant strain lacking a functional RecA regulator of the SOS response. The recA mutant cells additionally showed significantly reduced viability compared to wild-type cells similarly grown under anaerobic nitrate respiration. These results suggest a role for the RecA-mediated SOS response in the ability of cells to survive any DNA damage that may result from anaerobic nitrate respiration in C. glutamicum.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2016-01-01

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

  14. Corynebacterium glutamicum Chassis C1*: Building and Testing a Novel Platform Host for Synthetic Biology and Industrial Biotechnology.

    PubMed

    Baumgart, Meike; Unthan, Simon; Kloß, Ramona; Radek, Andreas; Polen, Tino; Tenhaef, Niklas; Müller, Moritz Fabian; Küberl, Andreas; Siebert, Daniel; Brühl, Natalie; Marin, Kay; Hans, Stephan; Krämer, Reinhard; Bott, Michael; Kalinowski, Jörn; Wiechert, Wolfgang; Seibold, Gerd; Frunzke, Julia; Rückert, Christian; Wendisch, Volker F; Noack, Stephan

    2017-08-30

    Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with d-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.

  15. Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum.

    PubMed

    Huang, Jinhai; Wu, Yao; Wu, Wenjun; Zhang, Ye; Liu, Dehua; Chen, Zhen

    2017-02-08

    Production of 1,3-propanediol (1,3-PDO) from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process.

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

    PubMed

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

    2015-11-20

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

  17. Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity.

    PubMed

    Ruan, Yili; Zhu, Linjiang; Li, Qi

    2015-12-01

    To improve the transformation efficiency of Corynebacterium glutamicum cells with heterogenous plasmid DNA and single-strand DNA (ssDNA) using a methodology based on electro-transformation. A semicomplex hypertonic medium was selected with addition of glycine and DL-threonine to weaken cell walls and addition of Tween 80 and isonicotinic acid hydrazide to increase cytoplasmic membrane fluidity. Their contents were optimized by response surface methodology. Cell growth, electro-transformation buffer, and transformation protocol were also optimized. Temporary heating inactivation of the host restriction enzyme showed a significant effect. Finally, a high transformation efficiency of 3.57 ± 0.13 × 10(7) cfu/μg DNA of plasmid and 1.05 × 10(6) Str (R) cfu per 10(9) viable cells with a ssDNA was achieved. The results shed light on the application in functional genomics and genome editing of C. glutamicum.

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

    PubMed Central

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

    2013-01-01

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

  19. Maltose uptake by the novel ABC transport system MusEFGK2I causes increased expression of ptsG in Corynebacterium glutamicum.

    PubMed

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W; Krämer, Reinhard; Seibold, Gerd M

    2013-06-01

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

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

    PubMed

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

    2012-07-01

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

  1. The ncgl1108 (PheP (Cg)) gene encodes a new L-Phe transporter in Corynebacterium glutamicum.

    PubMed

    Zhao, Zhi; Ding, Jiu-Yuan; Li, Tang; Zhou, Ning-Yi; Liu, Shuang-Jiang

    2011-06-01

    Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP(Cg), was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP (Cg), which is located upstream of the genetic cluster (ncgl1110 ~ ncgl1113) for resorcinol catabolism, was identified as a new L-Phe specific transporter from C. glutamicum RES167. The disruption of pheP (Cg) resulted in RES167∆ncgl1108, and this mutant showed decreased growth on L-Phe (as nitrogen source) but not on L-Tyr or L-Trp. Uptake assays with unlabeled and (14)C-labeled L-Phe and L-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in L-Phe uptake than RES167. Expression of pheP (Cg) in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 restored their ability to uptake for L-Phe and growth on L-Phe. The uptake of L-Phe was not inhibited by nine amino acids but by L-Tyr. The K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 for L-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min(-1) (mg DW)(-1), respectively, which are different from K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg)) for L-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min(-1) (mg DW)(-1)]. In conclusion, this PheP(Cg) is a new L-Phe transporter in C. glutamicum.

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-03-09

    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.

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

    PubMed

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

    2014-09-01

    Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2gL(-1)h(-1), and a final titer of 88gL(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Yukawa, Hideaki

    2014-01-01

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

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

    PubMed

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

    2016-05-01

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

  8. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  9. The three-component system EsrISR regulates a cell envelope stress response in Corynebacterium glutamicum.

    PubMed

    Kleine, Britta; Chattopadhyay, Ava; Polen, Tino; Pinto, Daniela; Mascher, Thorsten; Bott, Michael; Brocker, Melanie; Freudl, Roland

    2017-09-18

    When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria. This article is protected by copyright. All rights reserved. © 2017 John Wiley & Sons Ltd.

  10. Graded Response of the Multifunctional 2-Cysteine Peroxiredoxin, CgPrx, to Increasing Levels of Hydrogen Peroxide in Corynebacterium glutamicum.

    PubMed

    Si, Meiru; Wang, Tietao; Pan, Junfeng; Lin, Jinshui; Chen, Can; Wei, Yahong; Lu, Zhiqiang; Wei, Gehong; Shen, Xihui

    2017-01-01

    Eukaryotic typical 2-cysteine (Cys) peroxiredoxins (Prxs) are multifunctional proteins subjected to complex regulation and play important roles in oxidative stress resistance, hydrogen peroxide (H2O2) signaling modulation, aging, and cancer, but the information on the biochemical functions and regulation mechanisms of prokaryotic atypical 2-Cys Prxs is largely lacking. In this study, we show that at low peroxide concentrations, the atypical 2-Cys Prx in Corynebacterium glutamicum (CgPrx) mainly exists as monomers and displays thioredoxin (Trx)-dependent peroxidase activity. Moderate oxidative stress causes reversible S-mycothiolation of the H2O2-sensing Cys63 residue, which keeps CgPrx exclusively in dimer form with neither peroxidase nor chaperone activity. Then, the increased levels of H2O2 could act as a messenger to oxidize the redox-sensitive regulator hydrogen peroxide-inducible gene activator, leading to activation of expression of the more efficient mycothiol peroxidase and catalase to eliminate excessive peroxide. If oxidative stress is too severe, the H2O2-sensing Cys63 becomes hyperoxidized to sulfonic acid, which irreversibly inactivates the peroxidase activity, and most of CgPrx will be converted to multimeric chaperones for salvage of damaged proteins. We demonstrate for the first time that atypical 2-Cys CgPrx acts as both a Trx-dependent peroxidase and a molecular chaperone and plays a regulatory role in modulating the peroxide-mediated signaling cascades. These results reveal that CgPrx functions as a multifunctional protein crucial for adapting appropriate responses to different levels of oxidative challenge in C. glutamicum. Antioxid. Redox Signal. 26, 1-14.

  11. A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum.

    PubMed

    Chai, Xin; Shang, Xiuling; Zhang, Yu; Liu, Shuwen; Liang, Yong; Zhang, Yun; Wen, Tingyi

    2016-11-16

    Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role. Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WTΔpyk1Δpyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn(2+) and K(+) and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WTΔpyk1 strain produced 60.27 ± 1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation. Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen

  12. Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress.

    PubMed

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

    2014-02-01

    Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Transcriptomics revealed the strong upregulation of the disulfide stress σ(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives.

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

    PubMed Central

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

    2014-01-01

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

  14. Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

    PubMed

    Feng, Jun; Quan, Yufen; Gu, Yanyan; Liu, Fenghong; Huang, Xiaozhong; Shen, Haosheng; Dang, Yulei; Cao, Mingfeng; Gao, Weixia; Lu, Xiaoyun; Wang, Yi; Song, Cunjiang; Wang, Shufang

    2017-05-22

    Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher

  15. Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

    PubMed Central

    Binder, Dennis; Frohwitter, Jonas; Mahr, Regina; Bier, Claus; Grünberger, Alexander; Loeschcke, Anita; Peters-Wendisch, Petra; Kohlheyer, Dietrich; Pietruszka, Jörg; Frunzke, Julia; Jaeger, Karl-Erich

    2016-01-01

    ABSTRACT Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-β-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum. In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCE Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous

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

    PubMed

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

    2012-06-15

    Genomic context analysis in actinobacteria revealed that biotin biosynthesis and transport (bio) genes are co-localized in several genomes with a gene encoding a transcription regulator of the TetR protein family, now named BioQ. Comparative analysis of the upstream regions of bio genes identified the common 13-bp palindromic motif TGAAC-N3-GTTAC as candidate BioQ-binding site. To verify the role of BioQ in controlling the transcription of bio genes, a deletion in the bioQ coding region (cg2309) was constructed in Corynebacterium glutamicum ATCC 13032, resulting in the mutant strain C. glutamicum IB2309. Comparative whole-genome DNA microarray hybridizations and subsequent expression analyses by real-time reverse transcriptase PCR revealed enhanced transcript levels of all bio genes in C. glutamicum IB2309, when compared with the wild-type strain ATCC 13032. Accordingly, the BioQ protein of C. glutamicum acts as a repressor of ten genes that are organized in four transcription units: bioA-bioD, cg2884-cg2883, bioB-cg0096-cg0097, and bioY-bioM-bioN. DNA band shift assays with an intein-tagged BioQ protein demonstrated the specific binding of the purified protein to DNA fragments containing the candidate BioQ-binding sites, which were located within the mapped promoter regions of bioA, cg2884, bioB, and bioY. These data confirmed the direct regulatory role of BioQ in the control of biotin biosynthesis and transport genes in C. glutamicum. Differential expression of bio genes in C. glutamicum IB2309 was moreover complemented by bioQ genes cloned from other corynebacterial genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle.

    PubMed

    Claes, Wilfried A; Pühler, Alfred; Kalinowski, Jörn

    2002-05-01

    Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the C. glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that C. glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C. glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C. glutamicum prpD2B2C2 genes by propionate as an additional carbon source. Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C. glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.

  18. Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

    PubMed Central

    Carpinelli, Jorge; Krämer, Reinhard; Agosin, Eduardo

    2006-01-01

    Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content. PMID:16517642

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    2012-01-01

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

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

    PubMed Central

    Zhang, Dalong; Guan, Dan; Liang, Jingbo; Guo, Chunqian; Xie, Xixian; Zhang, Chenglin; Xu, Qingyang; Chen, Ning

    2014-01-01

    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. PMID:25763057

  4. Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production.

    PubMed

    Nakamura, Jun; Hirano, Seiko; Ito, Hisao; Wachi, Masaaki

    2007-07-01

    Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.

  5. Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum.

    PubMed

    Uy, Davin; Delaunay, Stéphane; Goergen, Jean-Louis; Engasser, Jean-Marc

    2005-05-01

    Corynebacterium glutamicum 2262 strain, when triggered for glutamate excretion, experiences a rapid decrease in growth rate and increase in glutamate efflux. In order to gain a better quantitative understanding of the factors controlling the metabolic transition, the fermentation dynamics was investigated for a temperature-sensitive strain cultivated in batch and glucose-limited continuous cultures. For non-excreting cells at 33 degrees C, increasing the growth rate resulted in strong increases in the central metabolic fluxes, but the intracellular glutamate level, the oxoglutarate dehydrogenase complex (ODHC) activity and the flux distribution at the oxoglutarate node remained essentially constant. When subjected to a temperature rise to 39 degrees C, at both high- and low-metabolic activities, the bacteria showed a rapid attenuation in ODHC activity and an increase from 28% to more than 90% of the isocitrate dehydrogenase flux split towards glutamate synthesis. Simultaneously to the reduction in growth rate, the cells activated a high capacity export system capable of expelling the surplus of synthesized glutamate.

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

    PubMed

    Peifer, Susanne; Barduhn, Tobias; Zimmet, Sarah; Volmer, Dietrich A; Heinzle, Elmar; Schneider, Konstantin

    2012-10-24

    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. Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol g(CDW)⁻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 g(CDW)⁻¹). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol g(CDW)⁻¹) derived from IMP degradation. 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.

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

    PubMed Central

    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 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 α2β2-type heterotetrameric structure made up of two α subunits and two β 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 α subunit and β subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of α2β2-type AK but also the mechanism of concerted inhibition in CgAK. PMID:20573952

  8. Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Diaminopimelate epimerase (DapF) is one of the crucial enzymes involved in l-lysine biosynthesis, where it converts l,l-diaminopimelate (l,l-DAP) into d,l-DAP. DapF is also considered as an attractive target for the development of antibacterial drugs. Here, we report the crystal structure of DapF from Corynebacterium glutamicum (CgDapF). Structures of CgDapF obtained under both oxidized and reduced conditions reveal that the function of CgDapF is regulated by redox-switch modulation via reversible disulfide bond formation between two catalytic cysteine residues. Under oxidized condition, two catalytic cysteine residues form a disulfide bond; these same cysteine residues exist in reduced form under reduced condition. Disulfide bond formation also induces a subsequent structural change in the dynamic catalytic loop at the active site, which results in open/closed conformational change at the active site. We also determined the crystal structure of CgDapF in complex with its product d,l-DAP, and elucidated how the enzyme recognizes its substrate l,l-DAP as a substrate. Moreover, the structure in complex with the d,l-DAP product reveals that CgDapF undergoes a large open/closed domain movement upon substrate binding, resulting in a completely buried active site with the substrate bound. PMID:28176858

  9. Functional expression of plant-derived O-methyltransferase, flavanone 3-hydroxylase, and flavonol synthase in Corynebacterium glutamicum for production of pterostilbene, kaempferol, and quercetin.

    PubMed

    Kallscheuer, Nicolai; Vogt, Michael; Bott, Michael; Marienhagen, Jan

    2017-09-20

    Plant polyphenols receive significant attention due to their anti-oxidative and health-promoting properties, and several microorganisms are currently engineered towards producing these valuable compounds. Previously, Corynebacterium glutamicum has been engineered for synthesizing polyphenol core structures such as the stilbene resveratrol and the (2S)-flavanone naringenin. Decoration of these compounds by O-methylation or hydroxylation would provide access to polyphenols of even higher commercial interest. In this study, introduction of a heterologous O-methyltransferase into a resveratrol-producing C. glutamicum strain allowed synthesis of 42mg/L (0.16mM) of the di-O-methylated pterostilbene from p-coumaric acid. A prerequisite for reaching this product titer was a fusion of O-methyltransferase with the maltose-binding protein of Escherichia coli lacking its signal peptide, thereby increasing the solubility of the O-methyltransferase. Furthermore, expression of heterologous dioxygenase genes in (2S)-flavanone-producing C. glutamicum strains enabled the production of flavanonols and flavonols starting from the phenylpropanoids p-coumaric acid and caffeic acid. For the flavonols kaempferol and quercetin, maximum product titers of 23mg/L (0.08mM) and 10mg/L (0.03mM) could be achieved, respectively. The obtained results demonstrate that C. glutamicum is a suitable host organism for the production of more complex plant polyphenols. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Promiscuous activity of (S,S)-butanediol dehydrogenase is responsible for glycerol production from 1,3-dihydroxyacetone in Corynebacterium glutamicum under oxygen-deprived conditions.

    PubMed

    Jojima, Toru; Igari, Takafumi; Moteki, Yasuhiro; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-02-01

    Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum. Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by (S,S)-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S-acetoin and (S,S)-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.

  11. Molecular Identification of the Urea Uptake System and Transcriptional Analysis of Urea Transporter- and Urease-Encoding Genes in Corynebacterium glutamicum

    PubMed Central

    Beckers, Gabriele; Bendt, Anne K.; Krämer, Reinhard; Burkovski, Andreas

    2004-01-01

    The molecular identification of the Corynebacterium glutamicum urea uptake system is described. This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation. Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes. The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C. glutamicum. The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation. To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified. PMID:15516578

  12. Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA.

    PubMed

    Brocker, Melanie; Schaffer, Steffen; Mack, Christina; Bott, Michael

    2009-06-01

    In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacterium Corynebacterium glutamicum was studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded by citH belongs to the citrate-Mg(2+):H(+)/citrate-Ca(2+):H(+) symporter family, whereas the permease encoded by the tctCBA operon is a member of the tripartite tricarboxylate transporter family. The expression of citH or tctCBA in Escherichia coli enabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinant E. coli strains indicated that CitH is active with Ca(2+) or Sr(2+) but not with Mg(2+) and that TctABC is active with Ca(2+) or Mg(2+) but not with Sr(2+). We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, the C. glutamicum wild type grew best when the minimal medium was supplemented with CaCl(2) but that MgCl(2) and SrCl(2) also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression of citH and tctCBA was activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates. C. glutamicum CitAB is the first member of this family studied in Actinobacteria.

  13. Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR.

    PubMed

    Uhde, Andreas; Brühl, Natalie; Goldbeck, Oliver; Matano, Christian; Gurow, Oksana; Rückert, Christian; Marin, Kay; Wendisch, Volker F; Krämer, Reinhard; Seibold, Gerd M

    2016-08-15

    Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the

  14. Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

    PubMed Central

    Uhde, Andreas; Brühl, Natalie; Goldbeck, Oliver; Matano, Christian; Gurow, Oksana; Rückert, Christian; Marin, Kay; Wendisch, Volker F.; Krämer, Reinhard

    2016-01-01

    ABSTRACT Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCE Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator

  15. Structure of AmtR, the global nitrogen regulator of Corynebacterium glutamicum, in free and DNA-bound forms.

    PubMed

    Palanca, Carles; Rubio, Vicente

    2016-03-01

    Corynebacterium glutamicum is a bacterium used for industrial amino acid production, and understanding its metabolic pathway regulation is of high biotechnological interest. Here, we report crystal structures of AmtR, the global nitrogen regulator of C. glutamicum, in apo (2.25-Å and 2.65-Å resolution) and DNA-bound (3-Å resolution) forms. These structures reveal an all-α homodimeric TetR family regulator composed of a helix-turn-helix-hosting N-terminal DNA-binding domain and a C-terminal dimerization domain. AmtR has several unique structural features that appear to be invariant among AmtR proteins, which may be related to its regulation by the nitrogen-sensing trimeric protein GlnK rather than by small-molecule effectors. As compared with other TetR family members, AmtR has an extra C-terminal helix, a large extended external loop that resembles the flexible tranducer T-loop of GlnK in sequence, and a large open cavity towards the intersubunit region that changes shape upon DNA binding. The marked kinking of helix 4 decreases in the DNA-bound form. The binding of one AmtR dimer to its DNA operator involves not only the insertion of helices 3 and 3' in adjacent turns of the double-helix major groove, but also the anchoring of 19-residue, arginine-rich and proline-rich N-terminal extensions to two external minor grooves. Electrophoretic mobility shift assays with a deletion mutant reveal that the 19-residue extension is crucial for AmtR binding to DNA. N-extension anchoring explains the flanking by AT sequences of the recognized target DNA sequence core. The significance of these findings for the entire TetR family of regulators and for GlnK regulation of AmtR is discussed. The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 5DXZ (native AmtR), 5DY1 (SeMet-AmtR), and 5DY0 (AmtR·DNA)]. © 2016 Federation of European Biochemical Societies.

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

    PubMed Central

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

    2016-01-01

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

  17. High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.

    PubMed

    Reimer, Lorenz C; Spura, Jana; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known

  18. The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum

    PubMed Central

    Brune, Iris; Werner, Hendrikje; Hüser, Andrea T; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

    2006-01-01

    Background The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum. Results A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network. Conclusion This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron

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

    PubMed Central

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

    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 coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h. PMID:22138982

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

    PubMed

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

    2015-07-20

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

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

    PubMed

    Taniguchi, Hironori; Wendisch, Volker F

    2015-01-01

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

  2. Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate.

    PubMed

    Litsanov, Boris; Brocker, Melanie; Bott, Michael

    2012-05-01

    Previous studies have demonstrated the capability of Corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat Δpqo Δpta-ackA ΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase gene pyc(P458S) into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the fdh gene from Mycobacterium vaccae, coding for an NAD(+)-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gap accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

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

    PubMed

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

    2015-02-01

    For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application. © 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made.

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

    PubMed Central

    Taniguchi, Hironori; Wendisch, Volker F.

    2015-01-01

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

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

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  6. Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.

    PubMed

    Nampoothiri, K M; Hoischen, C; Bathe, B; Möckel, B; Pfefferle, W; Krumbach, K; Sahm, H; Eggeling, L

    2002-01-01

    L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.

  7. Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

    PubMed Central

    Chao, Hongjun

    2014-01-01

    Corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of genDFM and genKH operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three gen operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located −13 to +8 bp upstream of the genDFM promoter, which was involved in negative regulation of genDFM transcription. The GlxR binding site R-KHx01 (located between positions −11 to +5) was upstream of the genKH promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to genR promoter to repress its expression, was found within a footprint extending from positions −71 to −91 bp. These results reveal that GlxR represses the transcription of all three gen operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR. PMID:24795375

  8. ScrB (Cg2927) is a sucrose-6-phosphate hydrolase essential for sucrose utilization by Corynebacterium glutamicum.

    PubMed

    Engels, Verena; Georgi, Tobias; Wendisch, Volker F

    2008-12-01

    Corynebacterium glutamicum can grow on a variety of carbohydrates from which glucose, fructose and sucrose are taken up and phosphorylated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Here, we show that cg2927 (scrB) encodes sucrose-6-phosphate hydrolase. The purified His-tagged protein hydrolyzed sucrose-6-phosphate and sucrose, but not sucrose-6'-phosphate. The Km value for sucrose was 190 mM while the Km for sucrose-6-phosphate was much lower, 0.04 mM. Sucrose-6-phosphate hydrolase activity was stimulated by MgSO4 and fructose-6-phosphate and was inhibited by MnCl2, CaCl2, CuSO4 and ZnSO4. A scrB deletion mutant could not grow on sucrose as the sole carbon source. In addition, growth in the absence of scrB was severely decreased when sucrose was present in addition to glucose, fructose or acetate, suggesting that higher intracellular concentrations of sucrose-6-phosphate are toxic. Transcriptional start sites in the cg2929-cg2928-scrB-ptsS locus could be revealed upstream of cg2929 and upstream of the sucrose-specific PTS gene ptsS. Of these, only ptsS showed increased expression when grown in the presence of sucrose, which was due to control by the transcriptional regulator SugR. The sucrose-6-phosphate hydrolase activity, however, was increased two- to threefold during growth in fructose- or sucrose-containing media, regardless of the presence or absence of SugR.

  9. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

    PubMed

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas; Dautin, Nathalie

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

  10. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

    PubMed Central

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated. PMID:28199365

  11. Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology.

    PubMed

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

    2013-10-01

    The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

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

    PubMed Central

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

    2013-01-01

    The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology. PMID:23892752

  13. Corynebacterium glutamicum RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5' maturation of 5S rRNA.

    PubMed

    Maeda, Tomoya; Wachi, Masaaki

    2012-02-01

    Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the -1 site of the 5' end of 5S rRNA. However, 3' maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5' maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.

  14. Identification of an α(1→6) mannopyranosyltransferase (MptA), involved in Corynebacterium glutamicum lipomanann biosynthesis, and identification of its orthologue in Mycobacterium tuberculosis

    PubMed Central

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

    2007-01-01

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

  15. Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

    PubMed

    Porco, Antonietta; Gamero, Elida E; Mylonás, Elena; Istúriz, Tomás

    2008-01-01

    Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

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

    PubMed

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

    2007-09-01

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

  17. Heat shock proteome analysis of wild-type Corynebacterium glutamicum ATCC 13032 and a spontaneous mutant lacking GroEL1, a dispensable chaperone.

    PubMed

    Barreiro, Carlos; González-Lavado, Eva; Brand, Sven; Tauch, Andreas; Martín, Juan F

    2005-02-01

    Proteome analysis of Corynebacterium glutamicum ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of C. glutamicum, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, IsCg1, that was integrated at position 327 downstream of the translation start codon of the groEL1 gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in C. glutamicum. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate groEL1 and groEL2 genes is analyzed.

  18. Heat Shock Proteome Analysis of Wild-Type Corynebacterium glutamicum ATCC 13032 and a Spontaneous Mutant Lacking GroEL1, a Dispensable Chaperone

    PubMed Central

    Barreiro, Carlos; González-Lavado, Eva; Brand, Sven; Tauch, Andreas; Martín, Juan F.

    2005-01-01

    Proteome analysis of Corynebacterium glutamicum ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of C. glutamicum, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, IsCg1, that was integrated at position 327 downstream of the translation start codon of the groEL1 gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in C. glutamicum. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate groEL1 and groEL2 genes is analyzed. PMID:15659666

  19. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-02

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  20. Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum

    PubMed Central

    Graf, Simone; Fedotovskaya, Olga; Kao, Wei-Chun; Hunte, Carola; Ädelroth, Pia; Bott, Michael; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Complex III in C. glutamicum has an unusual di-heme cyt. c1 and it co-purifies with complex IV in a supercomplex. Here, we investigated the kinetics of electron transfer within this supercomplex and in the cyt. aa3 alone (cyt. bc1 was removed genetically). In the reaction of the reduced cyt. aa3 with O2, we identified the same sequence of events as with other A-type oxidases. However, even though this reaction is associated with proton uptake, no pH dependence was observed in the kinetics. For the cyt. bc1-cyt. aa3 supercomplex, we observed that electrons from the c-hemes were transferred to CuA with time constants 0.1–1 ms. The b-hemes were oxidized with a time constant of 6.5 ms, indicating that this electron transfer is rate-limiting for the overall quinol oxidation/O2 reduction activity (~210 e−/s). Furthermore, electron transfer from externally added cyt. c to cyt. aa3 was significantly faster upon removal of cyt. bc1 from the supercomplex, suggesting that one of the c-hemes occupies a position near CuA. In conclusion, isolation of the III-IV-supercomplex allowed us to investigate the kinetics of electron transfer from the b-hemes, via the di-heme cyt. c1 and heme a to the heme a3-CuB catalytic site of cyt. aa3. PMID:27682138

  1. Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum.

    PubMed

    Dong, Xunyan; Zhao, Yue; Hu, Jinyu; Li, Ye; Wang, Xiaoyuan

    2016-11-01

    The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The two-component signal transduction system CopRS of Corynebacterium glutamicum is required for adaptation to copper-excess stress.

    PubMed

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

    2011-01-01

    Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu(2+) was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress.

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

    PubMed

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

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    van Ooyen, Jan

    2015-01-01

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

  5. Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

    PubMed Central

    Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

    2014-01-01

    The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains. PMID:24465669

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

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

  9. Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum.

    PubMed

    Albersmeier, Andreas; Pfeifer-Sancar, Katharina; Rückert, Christian; Kalinowski, Jörn

    2017-09-10

    The genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cDNA library enriched for native 5' transcript ends, is ideally suited for the analysis of promoters. Here, the transcriptome of Corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5'-ends of native transcripts. Total RNA samples were harvested from the exponential phase of growth, therefore the study mainly addressed promoters recognized by the main house-keeping sigma factor σ(A). The identification of 2454 transcription start sites (TSS) allowed the detailed analysis of most promoters recognized by σ(A) and furthermore enabled us to form different promoter groups according to their location relative to protein-coding regions. These groups included leaderless transcripts (546 promoters), short-leadered (<500 bases) transcripts (917), and long-leadered (>500 bases) transcripts (173) as well as intragenic (557) and antisense transcripts (261). All promoters and the individual groups were searched for information, e.g. conserved residues and promoter motifs, and general design features as well as group-specific preferences were identified. A purine was found highly favored as TSS, whereas the -1 position was dominated by pyrimidines. The spacer between TSS and -10 region were consistently 6-7 bases and the -10 promoter motif was generally visible, whereas a recognizable -35 region was only occurring in a smaller fraction of promoters (7.5%) and enriched for leadered and antisense transcripts but depleted for leaderless transcripts. Promoters showing an extended -10 region were especially frequent in case of non-canonical -10 motifs (45.5%). Two bases downstream of the -10 core region, a G was conserved, exceeding 40% abundance in most groups. This fraction reached 74.6% for a group of putative σ(B)-dependent promoters

  10. Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

    PubMed

    Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki; Inui, Masayuki

    2017-03-01

    Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this

  11. Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum

    PubMed Central

    Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki

    2016-01-01

    ABSTRACT Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3′-to-5′ exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3′ maturation of 4.5S RNA in C. glutamicum. The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3′ maturation of 4.5S RNA. Primer extension analysis also revealed that the 5′ mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex. IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3′ maturation of the SRP RNA (4

  12. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

    PubMed Central

    Mormann, Sascha; Lömker, Alexander; Rückert, Christian; Gaigalat, Lars; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2006-01-01

    Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L

  13. Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol.

    PubMed

    Radoš, Dušica; Turner, David L; Catarino, Teresa; Hoffart, Eugenia; Neves, Ana Rute; Eikmanns, Bernhard J; Blombach, Bastian; Santos, Helena

    2016-12-01

    The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA (Cg) was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by (1)H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V max values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min(-1) mg protein(-1), and K m values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butA (Cg) ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA (Cg) (from 0.30 ± 0.03 to 0.004 ± 0.001 μmol min(-1) mg protein(-1)), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.

  14. Metabolic profile of 1,5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor inhomogeneities.

    PubMed

    Limberg, Michael H; Schulte, Julia; Aryani, Tita; Mahr, Regina; Baumgart, Meike; Bott, Michael; Wiechert, Wolfgang; Oldiges, Marco

    2017-03-01

    Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD(+) recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD(+) regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc.

  15. Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays.

    PubMed

    Jochmann, Nina; Kurze, Anna-Katharina; Czaja, Lisa F; Brinkrolf, Karina; Brune, Iris; Hüser, Andrea T; Hansmeier, Nicole; Pühler, Alfred; Borovok, Ilya; Tauch, Andreas

    2009-05-01

    The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter-operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

  16. NdnR is an NAD-responsive transcriptional repressor of the ndnR operon involved in NAD de novo biosynthesis in Corynebacterium glutamicum.

    PubMed

    Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2012-04-01

    The Corynebacterium glutamicum ndnR gene, which is chromosomally located in a gene cluster involved in NAD de novo biosynthesis, negatively regulates expression of the cluster genes, i.e. nadA, nadC, nadS and ndnR itself. Although ndnR encodes a member of the recently identified NrtR family of transcriptional regulators, whether or not the NdnR protein directly regulates these NAD biosynthesis genes remains to be verified. Here, two NdnR binding sites in the promoter region of the ndnR-nadA-nadC-nadS operon in C. glutamicum were confirmed by in vitro DNA binding assay and analysis of in vivo expression of the chromosomally integrated ndnR promoter-lacZ reporter fusion. Electrophoretic mobility shift assay revealed that the NdnR protein binds to the 5'-upstream region of ndnR, and that the binding is significantly enhanced by NAD. Mutation in two 21 bp NdnR binding motifs in the ndnR promoter region inhibited the binding of NdnR in vitro. The mutation also enhanced the promoter activity in cells cultured in the presence of nicotinate, which is utilized in NAD biosynthesis, resulting in the loss of the repression in response to an exogenous NAD precursor; this is consistent with the effect of deletion of ndnR reported in our previous study. These results indicate that NAD acts as a co-repressor for the NdnR protein that directly regulates the ndnR operon involved in NAD de novo biosynthesis; the NAD-NdnR regulatory system likely plays an important role in the control of NAD homeostasis in C. glutamicum.

  17. Functional architecture and global properties of the Corynebacterium glutamicum regulatory network: Novel insights from a dataset with a high genomic coverage.

    PubMed

    Freyre-González, Julio A; Tauch, Andreas

    2017-09-10

    Corynebacterium glutamicum is a Gram-positive, anaerobic, rod-shaped soil bacterium able to grow on a diversity of carbon sources like sugars and organic acids. It is a biotechnological relevant organism because of its highly efficient ability to biosynthesize amino acids, such as l-glutamic acid and l-lysine. Here, we reconstructed the most complete C. glutamicum regulatory network to date and comprehensively analyzed its global organizational properties, systems-level features and functional architecture. Our analyses show the tremendous power of Abasy Atlas to study the functional organization of regulatory networks. We created two models of the C. glutamicum regulatory network: all-evidences (containing both weak and strong supported interactions, genomic coverage=73%) and strongly-supported (only accounting for strongly supported evidences, genomic coverage=71%). Using state-of-the-art methodologies, we prove that power-law behaviors truly govern the connectivity and clustering coefficient distributions. We found a non-previously reported circuit motif that we named complex feed-forward motif. We highlighted the importance of feedback loops for the functional architecture, beyond whether they are statistically over-represented or not in the network. We show that the previously reported top-down approach is inadequate to infer the hierarchy governing a regulatory network because feedback bridges different hierarchical layers, and the top-down approach disregards the presence of intermodular genes shaping the integration layer. Our findings all together further support a diamond-shaped, three-layered hierarchy exhibiting some feedback between processing and coordination layers, which is shaped by four classes of systems-level elements: global regulators, locally autonomous modules, basal machinery and intermodular genes. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level

    PubMed Central

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-01-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698

  19. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

    PubMed

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-03-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

  20. Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum.

    PubMed

    Lubitz, Dorit; Jorge, João M P; Pérez-García, Fernando; Taniguchi, Hironori; Wendisch, Volker F

    2016-10-01

    L-arginine is a semi-essential amino acid with application in cosmetic, pharmaceutical, and food industries. Metabolic engineering strategies have been applied for overproduction of L-arginine by Corynebacterium glutamicum. LysE was the only known L-arginine exporter of this bacterium. However, an L-arginine-producing strain carrying a deletion of lysE still accumulated about 10 mM L-arginine in the growth medium. Overexpression of the putative putrescine and cadaverine export permease gene cgmA was shown to compensate for the lack of lysE with regard to L-arginine export. Moreover, plasmid-borne overexpression of cgmA rescued the toxic effect caused by feeding of the dipeptide Arg-Ala to lysE-deficient C. glutamicum and argO-deficient Escherichia coli strains. Deletion of the repressor gene cgmR improved L-arginine titers by 5 %. Production of L-lysine and L-citrulline was not affected by cgmA overexpression. Taken together, CgmA may function as an export system not only for the diamine putrescine and cadaverine but also for L-arginine. The major export system for L-lysine and L-arginine LysE may also play a role in L-citrulline export since production of L-citrulline was reduced when lysE was deleted and improved by 45 % when lysE was overproduced.

  1. Elucidation of a protein-protein interaction network involved in Corynebacterium glutamicum cell wall biosynthesis as determined by bacterial two-hybrid analysis.

    PubMed

    Jankute, Monika; Byng, Charlotte V; Alderwick, Luke J; Besra, Gurdyal S

    2014-10-01

    Mycobacterium species have a highly complex and unique cell wall that consists of a large macromolecular structure termed the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. This complex is essential for growth, survival and virulence of the human pathogen Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. The closely related species Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes and proteins involved in mAGP synthesis. This study examines the construction of a protein-protein interaction network for the major cell wall component arabinogalactan in C. glutamicum based on the use of a bacterial two-hybrid system. We have identified twenty-four putative homotypic and heterotypic protein interactions in vivo. Our results demonstrate an association between glycosyltransferases, GlfT1 and AftB, and interaction between the sub-units of decaprenylphosphoribose epimerase, DprE1 and DprE2. These analyses have also shown that AftB interacts with AftA, which catalyzes the addition of the first three arabinose units onto the galactan chain. Both AftA and AftB associate with other arabinofuranosyltransferases, including Emb and AftC, that elongate and branch the arabinan domain. Moreover, a number of proteins involved in arabinogalactan biosynthesis were shown to form dimers or multimers. These findings provide a useful recourse for understanding the biosynthesis and function of the mycobacterial cell wall, as well as providing new therapeutic targets.

  2. Phosphatase activity of the histidine kinases ensures pathway specificity of the ChrSA and HrrSA two-component systems in Corynebacterium glutamicum.

    PubMed

    Hentschel, Eva; Mack, Christina; Gätgens, Cornelia; Bott, Michael; Brocker, Melanie; Frunzke, Julia

    2014-06-01

    The majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. The Gram-positive soil bacterium Corynebacterium glutamicum contains two homologous two-component systems (TCS) involved in the haem-dependent regulation of gene expression. Whereas the HrrSA system is crucial for utilization of haem as an alternative iron source, ChrSA is required to cope with high toxic haem levels. In this study, we analysed the interaction of HrrSA and ChrSA in C. glutamicum. Growth of TCS mutant strains, in vitro phosphorylation assays and promoter assays of P(hrtBA) and P(hmuO) fused to eyfp revealed cross-talk between both systems. Our studies further indicated that both kinases exhibit a dual function as kinase and phosphatase. Mutation of the conserved glutamine residue in the putative phosphatase motif DxxxQ of HrrS and ChrS resulted in a significantly increased activity of their respective target promoters (P(hmuO) and P(hrtBA) respectively). Remarkably, phosphatase activity of both kinases was shown to be specific only for their cognate response regulators. Altogether our data suggest the phosphatase activity of HrrS and ChrS as key mechanism to ensure pathway specificity and insulation of these two homologous systems.

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

    PubMed

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

    2016-06-01

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

  4. Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

    PubMed Central

    Komati Reddy, Gajendar; Lindner, Steffen N.

    2015-01-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  5. Increased glucose utilization and cell growth of Corynebacterium glutamicum by modifying the glucose-specific phosphotransferase system (PTS(Glc)) genes.

    PubMed

    Xu, Jianzhong; Zhang, Junlan; Liu, Dongdong; Zhang, Weiguo

    2016-12-01

    The phosphoenolpyruvate:glucose phosphotransferase system (PTS(Glc)) is the major pathway of glucose uptake in Corynebacterium glutamicum. This study investigated glucose consumption rate, cell growth, and metabolite changes resulting from modification of PTS(Glc). The classical l-lysine producer C. glutamicum XQ-8 exhibited low glucose consumption, cell growth, and l-lysine production rates, whereas these parameters were significantly increased during cultivating on glucose plus maltose, through inactivation of SugR, or by overexpression of PTS(Glc) genes. XQ-8sugR::cat/pDXW-8-ptsI exhibited the highest increase in glucose consumption, growth rate, and l-lysine production, followed by XQ-8sugR::cat/pDXW-8-ptsG. However, overexpression of ptsH had little effect on the above-mentioned factors. Although co-overexpression of ptsGHI led to the highest glucose consumption, growth rate, and final l-lysine production; the l-lysine production rate was lower than that of XQ-8sugR::cat/pDXW-8-ptsIH. In fed-batch fermentation, XQ-8sugR::cat/pDXW-8-ptsIH had a higher growth rate of 0.54 h(-1) to a dry cell mass of 66 g·L(-1) after 16 h, and had a higher l-lysine production rate of 159.2 g·L(-1) after 36 h. These results indicate that modification of the sugar transport systems improves amino acid production, especially for mutants obtained by repeated physical and (or) chemical mutagenesis. However, modification of these systems needs to be performed on a case-by-case basis.

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

    PubMed Central

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

    2014-01-01

    Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using 13C-labeled glucose and 13C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H+:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation. PMID:24610842

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

    PubMed Central

    Lange, Julian; Watzlawick, Hildegard; Kalinowski, Jörn; Altenbuchner, Josef

    2014-01-01

    Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10−6 s−1, with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator. PMID:25535273

  8. Expression analysis of the csp-like genes from Corynebacterium glutamicum encoding homologs of the Escherichia coli major cold-shock protein cspA.

    PubMed

    Kim, Wan-Soo; Park, Soo-Dong; Lee, Seok-Myung; Kim, Younhee; Kim, Pil; Lee, Heung-Shick

    2007-08-01

    Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of 30 degrees C but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to 15 degrees C, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at 20 degrees C, but not at 30 degrees C. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.

  9. Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH.

    PubMed

    Seletzky, Juri M; Noak, Ute; Fricke, Jens; Welk, Eike; Eberhard, Werner; Knocke, Christof; Büchs, Jochen

    2007-11-01

    Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model. (c) 2007 Wiley Periodicals, Inc.

  10. Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

    PubMed

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

    2014-05-01

    Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (~5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.

  11. Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct.

    PubMed

    Kubota, Takeshi; Watanabe, Akira; Suda, Masako; Kogure, Takahisa; Hiraga, Kazumi; Inui, Masayuki

    2016-11-01

    para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of

  12. Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH.

    PubMed

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Flechoso, Fabio; Martín, Juan F

    2006-01-01

    Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7.0-9.0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9.0. Growth still occurred at pH 9.5 but at a reduced rate. The expression of the pH-regulated F0 F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7.5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9.0. The same occurred with a 1.2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0 F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0 F1 operon, was expressed at a lower level than the polycistronic 7.5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0 F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative sigma factor of C. glutamicum, whereas the -35 and -10 boxes of P-atp2 fitted the consensus sequence for sigma(H)-recognized Mycobacterium tuberculosis promoters C(C)/(G)GG(A)/(G)AC 17-22 nt (C)/(G)GTT(C)/(G), known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0 F1 operon is highly expressed at alkaline pH, probably using a sigma (H) RNA polymerase.

  13. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

  14. Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination.

    PubMed

    Höppner, Astrid; Schomburg, Dietmar; Niefind, Karsten

    2013-11-01

    Quinate dehydrogenase (QDH) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (NADH) and is involved in the catabolic quinate metabolism required for the degradation of lignin. The enzyme is a member of the family of shikimate/quinate dehydrogenases (SDH/QDH) occurring in bacteria and plants. We characterized the dual-substrate quinate/shikimate dehydrogenase (QSDH) from Corynebacterium glutamicum (CglQSDH) kinetically and revealed a clear substrate preference of CglQSDH for quinate compared with shikimate both at the pH optimum and in a physiological pH range, which is a remarkable contrast to closely related SDH/QDH enzymes. With respect to the cosubstrate, CglQSDH is strictly NAD(H) dependent. These substrate and cosubstrate profiles correlate well with the details of three atomic resolution crystal structures of CglQSDH in different functional states we report here: with bound NAD+ (binary complex) and as ternary complexes with NADH plus either shikimate or quinate. The CglQSDH-NADH-quinate structure is the first complex structure of any member of the SDH/QDH family with quinate. Based on this novel structural information and systematic sequence and structure comparisons with closely related enzymes, we can explain the strict NAD(H) dependency of CglQSDH as well as its discrimination between shikimate and quinate.

  15. Target Genes, Consensus Binding Site, and Role of Phosphorylation for the Response Regulator MtrA of Corynebacterium glutamicum ▿ †

    PubMed Central

    Brocker, Melanie; Mack, Christina; Bott, Michael

    2011-01-01

    The two-component signal transduction system consisting of the sensor kinase MtrB and the response regulator MtrA is highly conserved in corynebacteria and mycobacteria. Whereas mtrA of Mycobacterium tuberculosis was reported to be essential, we recently succeeded in creating ΔmtrAB and ΔmtrA deletion mutants of Corynebacterium glutamicum and provided evidence that mepA and nlpC, both encoding putative cell wall peptidases, are directly repressed by MtrA, whereas proP and betP, both encoding carriers for compatible solutes, are directly activated by MtrA. In the present study, novel MtrA target genes were identified, including mepB, encoding another putative cell wall peptidase. The repressor or activator functions of MtrA correlate with the distance between the MtrA binding site and the transcriptional start site. From the identified binding sites within 20 target promoters, a 19-bp MtrA consensus motif was derived which represents a direct repeat of 8 base pairs separated by 3 base pairs. Gene expression of a strain containing MtrA with a D53N mutation instead of wild-type MtrA resembled that of a ΔmtrA mutant, indicating that MtrA is active in its phosphorylated form. This result was confirmed by electrophoretic mobility shift assays with phosphorylated MtrA which showed an increased binding affinity. PMID:21183673

  16. The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins.

    PubMed

    Mahne, Martina; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2006-06-01

    Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.

  17. The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum

    PubMed Central

    Gaigalat, Lars; Schlüter, Jan-Philip; Hartmann, Michelle; Mormann, Sascha; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2007-01-01

    Background The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. Results Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6-P

  18. Transcriptional regulation of the vanillate utilization genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor.

    PubMed

    Morabbi Heravi, Kambiz; Lange, Julian; Watzlawick, Hildegard; Kalinowski, Jörn; Altenbuchner, Josef

    2015-03-01

    Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10(-6) s(-1), with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Translation efficiency of antiterminator proteins is a determinant for the difference in glucose repression of two β-glucoside phosphotransferase system gene clusters in Corynebacterium glutamicum R.

    PubMed

    Tanaka, Yuya; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-01-01

    Corynebacterium glutamicum R has two β-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bglA-bglG and bglF2-bglA2-bglG2. Previously, we reported that whereas β-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the β-glucoside-dependent induction of the bglF cluster. The glucose repression of bglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon of bglG with AUG was effective in relieving the glucose repression of bglF and bglG. Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose.

  20. Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation.

    PubMed

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

    2006-12-01

    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 >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.

  1. GenR, an IclR-Type Regulator, Activates and Represses the Transcription of gen Genes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum

    PubMed Central

    Chao, Hongjun

    2013-01-01

    The genes required for 3-hydroxybenzoate and gentisate catabolism in Corynebacterium glutamicum are closely clustered in three operons. GenR, an IclR-type regulator, can activate the transcription of genKH and genDFM operons in response to 3-hydroxybenzoate and gentisate, and it can repress its own expression. Footprinting analyses demonstrated that GenR bound to four sites with different affinities. Two GenR-binding sites (DFMn01 and DFMn02) were found to be located between positions −41 and −84 upstream of the −35 and −10 regions of the genDFM promoter, which was involved in positive regulation of genDFM transcription. The GenR binding site R-KHn01 (located between positions −47 and −16) overlapped the −35 region of the genKH promoter sequence and is involved in positive regulation of its transcription. The binding site R-KHn02, at which GenR binds to its own promoter, was found within a footprint extending from position −44 to −67. It appeared to be involved in negative regulation of the activity of the genR promoter. A consensus motif with a 5-bp imperfect palindromic sequence [ATTCC-N7(5)-GGAAT] was identified among all four GenR binding sites and found to be necessary to GenR regulation through site-directed mutagenesis. The results reveal a new regulatory function of the IclR family in the catabolism of aromatic compounds. PMID:23354754

  2. Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

    PubMed

    Shin, Kyung-Chul; Sim, Dong-Hyun; Seo, Min-Ju; Oh, Deok-Kun

    2016-11-02

    The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

  3. The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

    PubMed Central

    Townsend, Philip D.; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A.; Cole, Stewart T.; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J.; Pohl, Ehmke

    2014-01-01

    The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition. PMID:25469635

  4. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    PubMed

    Townsend, Philip D; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A; Cole, Stewart T; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J; Pohl, Ehmke

    2014-01-01

    The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  5. Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy.

    PubMed

    Oberreuter, Helene; Charzinski, Joachim; Scherer, Siegfried

    2002-05-01

    The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

  6. Identification of a novel alpha(1-->6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant.

    PubMed

    Mishra, Arun K; Alderwick, Luke J; Rittmann, Doris; Wang, Cindy; Bhatt, Apoorva; Jacobs, William R; Takayama, Kuni; Eggeling, Lothar; Besra, Gurdyal S

    2008-06-01

    Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C(16)/C(18:1)-(alpha-D-glucopyranosyluronic acid)-(1-->3)-glycerol (GlcAGroAc(2)) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) and Man(1)GlcAGroAc(2) were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[(14)C]Man established that C. glutamicum synthesized a novel alpha(1-->6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac(1)PIM(2) and Man(1)GlcAGroAc(2) respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core alpha(1-->6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac(1)PI[(14)C]M(2) and [(14)C]Man(1)GlcAGroAc(2) primers respectively. Use of the acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C(8) in an in vitro cell-free assay confirmed NCgl1505 as an alpha(1-->6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroalpha(1-->6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.

  7. Identification of a novel α(1→6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant

    PubMed Central

    Mishra, Arun K; Alderwick, Luke J; Rittmann, Doris; Wang, Cindy; Bhatt, Apoorva; Jacobs, William R; Takayama, Kuni; Eggeling, Lothar; Besra, Gurdyal S

    2008-01-01

    Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C16/C18:1-(α-D-glucopyranosyluronic acid)-(1→3)-glycerol (GlcAGroAc2) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac1PIM2) and Man1GlcAGroAc2 were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[14C]Man established that C. glutamicum synthesized a novel α(1→6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac1PIM2 and Man1GlcAGroAc2 respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core α(1→6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac1PI[14C]M2 and [14C]Man1GlcAGroAc2 primers respectively. Use of the acceptor α-D-Manp-(1→6)-α-D-Manp-O-C8 in an in vitro cell-free assay confirmed NCgl1505 as an α(1→6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroα(1→6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria. PMID:18452585

  8. Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Becker, Judith; Wittmann, Christoph

    2013-01-01

    In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on L-lysine formation and analysis of systemic consequences.

    PubMed

    Hochheim, Jennifer; Kranz, Angela; Krumbach, Karin; Sokolowsky, Sascha; Eggeling, Lothar; Noack, Stephan; Bocola, Marco; Bott, Michael; Marienhagen, Jan

    2017-02-01

    To explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular L-lysine accumulation by this bacterium. The analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing L-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance L-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM L-lysine with a yield of 0.17 g/g (L-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high L-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower L-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on L-lysine formation. MurE is an attractive target to achieve high L-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated L-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers.

  10. The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

    PubMed Central

    Rückert, Christian; Milse, Johanna; Albersmeier, Andreas; Koch, Daniel J; Pühler, Alfred; Kalinowski, Jörn

    2008-01-01

    Background Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. Results Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. Conclusion CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules. PMID:18854009

  11. Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values

    PubMed Central

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Sola-Landa, Alberto; Martín, Juan F

    2013-01-01

    Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F0F1-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F0F1 ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (−35)TTGGAT…18nt…GTTA(−10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium. PMID:23298179

  12. Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values.

    PubMed

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Sola-Landa, Alberto; Martín, Juan F

    2013-03-01

    Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F(0)F(1)-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F(0) F(1) ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (-35)TTGGAT…18nt…GTTA(-10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium.

  13. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    PubMed

    Podder, M S; Majumder, C B

    2016-11-05

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  14. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668 mg/g for As(III) and 2651.675 mg/g for As(V) at 30 °C temperature and 220 min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  15. Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum.

    PubMed

    Brunger, Axel T; Das, Debanu; Deacon, Ashley M; Grant, Joanna; Terwilliger, Thomas C; Read, Randy J; Adams, Paul D; Levitt, Michael; Schröder, Gunnar F

    2012-04-01

    Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.

  16. Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum

    PubMed Central

    Brunger, Axel T.; Das, Debanu; Deacon, Ashley M.; Grant, Joanna; Terwilliger, Thomas C.; Read, Randy J.; Adams, Paul D.; Levitt, Michael; Schröder, Gunnar F.

    2012-01-01

    Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence. PMID:22505259

  17. Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

    PubMed Central

    2009-01-01

    Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense. PMID:20025733

  18. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources.

    PubMed

    Wendisch, Volker F; Brito, Luciana Fernandes; Gil Lopez, Marina; Hennig, Guido; Pfeifenschneider, Johannes; Sgobba, Elvira; Veldmann, Kareen H

    2016-09-20

    Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Multilocus sequence typing identifies evidence for recombination and two distinct lineages of Corynebacterium diphtheriae.

    PubMed

    Bolt, Frances; Cassiday, Pamela; Tondella, Maria Lucia; Dezoysa, Aruni; Efstratiou, Androulla; Sing, Andreas; Zasada, Aleksandra; Bernard, Kathryn; Guiso, Nicole; Badell, Edgar; Rosso, Marie-Laure; Baldwin, Adam; Dowson, Christopher

    2010-11-01

    We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

  20. Effect of Polyhydroxybutyrate (PHB) storage on L-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation.

    PubMed

    Xu, Meijuan; Qin, Jingru; Rao, Zhiming; You, Hengyi; Zhang, Xian; Yang, Taowei; Wang, Xiaoyuan; Xu, Zhenghong

    2016-01-19

    Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5. C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain. Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L

  1. Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

    PubMed Central

    Hodgson, A L; Tachedjian, M; Corner, L A; Radford, A J

    1994-01-01

    An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector. Images PMID:7960105

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

    PubMed Central

    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 gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures. PMID:18757581

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

    PubMed Central

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

    2009-01-01

    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 an about twofold-lower Vmax; and (iii) insensitivity against the inhibitors l-valine, l-isoleucine, and l-leucine (10 mM each). Introduction of the modified AHAS into the l-lysine producers C. glutamicum DM1729 and DM1933 increased l-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased l-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to l-valine, l-isoleucine, and l-leucine auxotrophy and to further-improved l-lysine production. In batch fermentations, C. glutamicum DM1729 ΔilvB produced about 85% more l-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 ΔilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the l-lysine biosynthetic pathway. PMID:19047397

  4. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    PubMed

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  5. The IclR-Type Transcriptional Repressor LtbR Regulates the Expression of Leucine and Tryptophan Biosynthesis Genes in the Amino Acid Producer Corynebacterium glutamicum▿

    PubMed Central

    Brune, Iris; Jochmann, Nina; Brinkrolf, Karina; Hüser, Andrea T.; Gerstmeir, Robert; Eikmanns, Bernhard J.; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

    2007-01-01

    The transcriptional regulator Cg1486 of Corynebacterium glutamicum ATCC 13032 is a member of the IclR protein family and belongs to the conserved set of regulatory proteins in corynebacteria. A defined deletion in the cg1486 gene, now designated ltbR (leucine and tryptophan biosynthesis regulator), led to the mutant strain C. glutamicum IB1486. According to whole-genome expression analysis by DNA microarray hybridizations, transcription of the leuB and leuCD genes encoding enzymes of the leucine biosynthesis pathway was enhanced in C. glutamicum IB1486 compared with the wild-type strain. Moreover, the genes of the trpEGDCFBA operon involved in tryptophan biosynthesis of C. glutamicum showed an enhanced expression in the cg1486 mutant strain. Bioinformatics pattern searches in the upstream regions of the differentially expressed genes revealed the common 12-bp motif CA(T/C)ATAGTG(A/G)GA that is located downstream of the −10 region of the mapped promoter sequences. DNA band shift assays with a streptavidin-tagged LtbR protein demonstrated the specific binding of the purified protein to 40-mers containing the 12-bp motif localized in front of leuB, leuC, and trpE, thereby confirming the direct regulatory role of LtbR in the expression of the leucine and tryptophan biosynthesis pathway genes of C. glutamicum. Genes homologous with ltbR were detected upstream of the leuCD genes in almost all sequenced genomes of bacteria belonging to the taxonomic class Actinobacteria. The ltbR-like genes of Corynebacterium diphtheriae, Corynebacterium jeikeium, Mycobacterium bovis, and Bifidobacterium longum were cloned and shown to complement the deregulation of leuB, leuCD, and trpE gene expression in C. glutamicum IB1486. PMID:17259312

  6. Reduced Folate Supply as a Key to Enhanced l-Serine Production by Corynebacterium glutamicum▿

    PubMed Central

    Stolz, Michael; Peters-Wendisch, Petra; Etterich, Helga; Gerharz, Tanja; Faurie, Robert; Sahm, Hermann; Fersterra, Holger; Eggeling, Lothar

    2007-01-01

    The amino acid l-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of l-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on Ptac and lacIQ are unstable, acquiring mutations in lacIQ, for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum ΔsdaA strain (pserACB) accumulates only traces of l-serine, with the C. glutamicum ΔpabABCΔsdaA strain (pserACB), l-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM l-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM l-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial l-serine production is available. PMID:17142381

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

    PubMed

    Kim, Ju-Sim; Holmes, Randall K

    2012-01-01

    Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Δcat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H(2)O(2). In C. diphtheriae C7(β), both catalase activity and cat transcription decreased ~2-fold during transition from exponential growth to early stationary phase. Prototypic OxyR in Escherichia coli senses oxidative stress and it activates katG transcription and catalase production in response to H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat. OxyR from C. diphtheriae and C. glutamicum have 52% similarity with E. coli OxyR and contain homologs of the two cysteine residues involved in H(2)O(2) sensing by E. coli OxyR. In-frame ΔoxyR deletion mutants of C. diphtheriae C7(β), C. diphtheriae NCTC13129, and C. glutamicum were much more resistant than their parental wild type strains to growth inhibition by H(2)O(2). In the C. diphtheriae C7(β) ΔoxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(β) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in ΔoxyR mutants of C. diphtheriae and C. glutamicum and decreased their H(2)O(2) resistance to the level of their parental strains. Gel-mobility shift, DNaseI footprint, and primer extension assays showed that purified OxyR from C. diphtheriae C7(β) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position -55 to -10 with respect to the +1 nucleotide in the cat ORF. These results demonstrate that OxyR from C. diphtheriae or C. glutamicum functions as a transcriptional repressor of the cat gene by a mechanism that is independent of oxidative stress induced by H(2)O(2).

  8. Characterization of OxyR as a Negative Transcriptional Regulator That Represses Catalase Production in Corynebacterium diphtheriae

    PubMed Central

    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 H2O2. 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 H2O2. In contrast, exposure of C. diphtheriae C7(β) to H2O2 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 H2O2 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 H2O2. 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 H2O2 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 H2O2. PMID:22438866

  9. Structural characteristics of the Corynebacterium lilium bacteriophage CL31.

    PubMed Central

    Trautwetter, A; Blanco, C; Sicard, A M

    1987-01-01

    Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected. Images PMID:3033280

  10. pH fluctuations imperil the robustness of C. glutamicum to short term oxygen limitation.

    PubMed

    Limberg, Michael H; Joachim, Mathias; Klein, Bianca; Wiechert, Wolfgang; Oldiges, Marco

    2017-10-10

    The presence of complex gradients for, e.g., nutrients, oxygen or pH in industrial scale fed batch processes are a major challenge for process performance. To consider such impact of scale-up during laboratory scale process development, scale-down bioreactor simulation, i.e. mimicking inhomogeneous conditions, became the method of choice. However, most scale-down studies simulate combined inhomogeneities of more than one parameter, so that the impact of the individual parameters remains unclear. The presented scale down study addresses this challenge by separating the influence of glucose, pH and oxygen fluctuations in terms of their specific impact in a well-established two compartment scale down device. This was carried out for an 1,5-diaminopentane production process using the industrial production host Corynebacterium glutamicum. Strikingly, oxygen depletion alone showed no effect on the process performance while changes of only one pH unit in acidic as well as alkaline direction reduced the biomass and product formation. Even more pronounced phenotypes up to -13% of μ and -39% of YX/S were observed, when an oscillatory acidic pH shift was combined with dissolved oxygen fluctuations. These losses are accompanied by a missing regulation of fermentative pathways. In conclusion, large-scale C. glutamicum processes seem to be most sensitive to pH variation. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

    PubMed

    Käß, Friedrich; Hariskos, Ioanna; Michel, Andrea; Brandt, Hans-Jürgen; Spann, Robert; Junne, Stefan; Wiechert, Wolfgang; Neubauer, Peter; Oldiges, Marco

    2014-06-01

    Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. L-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, L-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C. glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.

  12. [Corynebacterium ulcerans pulmonary infection].

    PubMed

    Thouvenin, Maxime; Beilouny, Bassam; Badell, Edgar; Guiso, Nicole

    2016-01-01

    Corynebacterium ulcerans is a bacterium able to infect humans by inducing a disease close to diphtheria. We describe the case of a 83-year-old patient hospitalized as a matter of urgency in intensive care for which C. ulcerans was isolated in pure culture in its bronchial samples. Even if the isolate was not secreting toxin in vitro, it possesses the tox gene which motivated the use of specific antitoxin serum. After two months of intensive care the patient went out of the service. It is about a remarkable case of clinicobiologic collaboration.

  13. [Cloning, expression and sequence analysis of DS I gene in Corynebacterium pekinense AS1.299 and PD-67].

    PubMed

    Zhang, Chunhua; Zhao, Zhi; Zhang, Yingzi; Wang, Yu; Ding, Jiuyuan

    2008-11-01

    3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54;DS) is the key enzyme in tryptophan synthesis pathway. Cloning DS I gene from Corynebacterium pekinense and expression of DS I gene might facilitate testing the existence and function of DS I in Corynebacterium pekinense. According to the homology between Corynebacterium glutamicum ATCC13032 and Corynebacterium pekinense, we designed a pair of PCR primers to clone the DS I gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant DS I gene was expressed in C. pekinense PD-67 by subcloning the the PCR fragment into plasmid pAK6. Analysis of PCR fragments revealed that they contained the whole DS I gene. There was no base change all over the structure genes and regulatory sequences between C. pekinense AS1.299 and PD-67. An internal promoter was found in the upstream of the DS I gene from C. pekinense and it functioned in E. coli 3257. The DS I gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of DS I in C. pekinense PD-67 (pAD1) was much higher than that of the control strain C. pekinense PD-67(pAK6). This is the first report that DS I gene existed in Corynebaterium Pekinense, The amplification of the specific activity of DS I is expected to increase L-tryptophan accumulation of C. pekinense PD-67.

  14. The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ermCX.

    PubMed

    Tauch, A; Kassing, F; Kalinowski, J; Pühler, A

    1995-09-01

    Analysis of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium xerosis M82B revealed that the erythromycin resistance gene, ermCX, is located on a 4524-bp composite transposable element, Tn5432. The ends of Tn5432 are identical, direct repeats of an insertion sequence, designated IS1249, encoding a putative transposase of the IS256 family. IS1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. The nucleotide sequence of the 1754-bp Tn5432 central region is 99% identical to the previously sequenced erythromycin resistance region of the Corynebacterium diphtheriae plasmid pNG2. It encodes the erythromycin resistance gene, ermCX, and an ORF homologous to the amino-terminal end of the transposase of IS31831 from Corynebacterium glutamicum. Transposons with regions flanking the insertion sites were recovered from the C. glutamicum chromosome by a plasmid rescue technique. Insertion of Tn5432 created 8-bp target site duplications. A Tn5432-induced isoleucine/valine-auxotrophic mutant was found to carry the transposon in the 5' region of the ilvBNC cluster; in pTP10 the transposon is inserted in a region similar to replication and partitioning functions of the Enterococcus faecalis plasmid pAD1 and the Agrobacterium tumefaciens plasmid pTAR.

  15. Corynebacterium ulcerans cutaneous diphtheria.

    PubMed

    Moore, Luke S P; Leslie, Asuka; Meltzer, Margie; Sandison, Ann; Efstratiou, Androulla; Sriskandan, Shiranee

    2015-09-01

    We describe the case of a patient with cutaneous diphtheria caused by toxigenic Corynebacterium ulcerans who developed a right hand flexor sheath infection and symptoms of sepsis such as fever, tachycardia, and elevated C-reactive protein, after contact with domestic cats and dogs, and a fox. We summarise the epidemiology, clinical presentation, microbiology, diagnosis, therapy, and public health aspects of this disease, with emphasis on improving recognition. In many European countries, C ulcerans has become the organism commonly associated with cutaneous diphtheria, usually seen as an imported tropical disease or resulting from contact with domestic and agricultural animals. Diagnosis relies on bacterial culture and confirmation of toxin production, with management requiring appropriate antimicrobial therapy and prompt administration of antitoxin, if necessary. Early diagnosis is essential for implementation of control measures and clear guidelines are needed to assist clinicians in managing clinical diphtheria. This case was a catalyst to the redrafting of the 2014 national UK interim guidelines for the public health management of diphtheria, released as final guidelines in March, 2015. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker.

    PubMed

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

    1995-02-01

    The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.

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

    PubMed Central

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  18. Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis.

    PubMed

    Sagong, Hye-Young; Kim, Kyung-Jin

    2016-02-01

    Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP(+) and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme.

  19. Corynebacterium mooreparkense, a later heterotypic synonym of Corynebacterium variabile.

    PubMed

    Gelsomino, Roberto; Vancanneyt, Marc; Snauwaert, Cindy; Vandemeulebroecke, Katrien; Hoste, Bart; Cogan, Timothy M; Swings, Jean

    2005-05-01

    Strains of a Gram-positive bacterium were isolated from the Irish smear-ripened cheese Gubbeen, and assigned to a new species, Corynebacterium mooreparkense, in 2001. During a further study on the same cheese, no additional isolates from this species could be found. Instead, multiple isolates of its nearest phylogenetic neighbour, Corynebacterium variabile, were found. A first screening with rep-PCR and SDS-PAGE pointed to a similarity between C. mooreparkense and C. variabile. Following this peculiar result, attempts were made to collect all type strains deposited at different culture collections and all strains described by Brennan et al. [Int J Syst Evol Microbiol (2001) 51, 843-852]. Subsequently, 16S rRNA gene sequencing and DNA-DNA hybridizations were performed. All C. mooreparkense strains had a 16S rRNA gene sequence similarity of at least 99.5 % with C. variabile and the DNA-DNA relatedness was 95 %. On the basis of these results, it is concluded that C. mooreparkense is a later heterotypic synonym of C. variabile.

  20. Cystic neutrophilic granulomatous mastitis associated with Corynebacterium including Corynebacterium kroppenstedtii.

    PubMed

    Johnstone, Kate J; Robson, Jennifer; Cherian, Sarah G; Wan Sai Cheong, Jenny; Kerr, Kris; Bligh, Judith F

    2017-06-01

    Granulomatous (lobular) mastitis is a rare inflammatory breast disease affecting parous reproductive-aged women. Once considered idiopathic, there is growing evidence of an association with corynebacteria infection, especially in the setting of a distinct histological pattern termed cystic neutrophilic granulomatous mastitis (CNGM). We describe 15 cases with histological features either confirming (n = 12) or suggesting (n = 3) CNGM, and concurrent microbiological evidence of Corynebacterium species. The organism was detected by culture or 16S rRNA gene sequencing of specimens obtained at surgery or fine needle aspiration. In seven cases, Gram-positive organisms were seen within vacuolated spaces. Speciation was performed in nine cases, with Corynebacterium kroppenstedtii subsequently identified. These cases provide further evidence in support of this association and in doing so highlight the importance of recognising these histological clues as well as the limitations of Gram stain and microbiological culture in detecting this previously under-recognised disease process. Copyright © 2017 Royal College of Pathologists of Australasia. All rights reserved.

  1. Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

    PubMed Central

    Norouzy, Amir; Schulz, Robert; Nau, Werner M.; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

    2013-01-01

    Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. PMID:24116064

  2. Toxigenic Corynebacterium ulcerans in human and non-toxigenic Corynebacterium diphtheriae in cat.

    PubMed

    Detemmerman, L; Rousseaux, D; Efstratiou, A; Schirvel, C; Emmerechts, K; Wybo, I; Soetens, O; Piérard, D

    2013-10-01

    Corynebacterium diphtheriae and Corynebacterium ulcerans are rarely isolated from clinical samples in Belgium. A case of toxigenic C. ulcerans in a woman is described, which confirms that this pathogen is still present. During investigation of the patient's cats, only a non-toxigenic toxin-bearing C. diphtheriae strain was detected.

  3. Draft Genome Sequence of Corynebacterium kefirresidentii SB, Isolated from Kefir

    PubMed Central

    Blasche, Sonja; Kim, Yongkyu

    2017-01-01

    ABSTRACT The genus Corynebacterium includes Gram-positive species with a high G+C content. We report here a novel species, Corynebacterium kefirresidentii SB, isolated from kefir grains collected in Germany. Its draft genome sequence was remarkably dissimilar (average nucleotide identity, 76.54%) to those of other Corynebacterium spp., confirming that this is a unique novel species. PMID:28912312

  4. Characterization of Corynebacterium species in macaques

    PubMed Central

    Venezia, Jaime; Cassiday, Pamela K.; Marini, Robert P.; Shen, Zeli; Buckley, Ellen M.; Peters, Yaicha; Taylor, Nancy; Dewhirst, Floyd E.; Tondella, Maria L.

    2012-01-01

    Bacteria of the genus Corynebacterium are important primary and opportunistic pathogens. Many are zoonotic agents. In this report, phenotypic (API Coryne analysis), genetic (rpoB and 16S rRNA gene sequencing), and physical methods (MS) were used to distinguish the closely related diphtheroid species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, and to definitively diagnose Corynebacterium renale from cephalic implants of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques used in cognitive neuroscience research. Throat and cephalic implant cultures yielded 85 isolates from 43 macaques. Identification by API Coryne yielded C. ulcerans (n = 74), Corynebacterium pseudotuberculosis (n = 2), C. renale or most closely related to C. renale (n = 3), and commensals and opportunists (n = 6). The two isolates identified as C. pseudotuberculosis by API Coryne required genetic and MS analysis for accurate characterization as C. ulcerans. Of three isolates identified as C. renale by 16S rRNA gene sequencing, only one could be confirmed as such by API Coryne, rpoB gene sequencing and MS. This study emphasizes the importance of adjunct methods in identification of coryneforms and is the first isolation of C. renale from cephalic implants in macaques. PMID:22723254

  5. Complete Genome Sequence and Analysis of the Multiresistant Nosocomial Pathogen Corynebacterium jeikeium K411, a Lipid-Requiring Bacterium of the Human Skin Flora

    PubMed Central

    Tauch, Andreas; Kaiser, Olaf; Hain, Torsten; Goesmann, Alexander; Weisshaar, Bernd; Albersmeier, Andreas; Bekel, Thomas; Bischoff, Nicole; Brune, Iris; Chakraborty, Trinad; Kalinowski, Jörn; Meyer, Folker; Rupp, Oliver; Schneiker, Susanne; Viehoever, Prisca; Pühler, Alfred

    2005-01-01

    Corynebacterium jeikeium is a “lipophilic” and multidrug-resistant bacterial species of the human skin flora that has been recognized with increasing frequency as a serious nosocomial pathogen. Here we report the genome sequence of the clinical isolate C. jeikeium K411, which was initially recovered from the axilla of a bone marrow transplant patient. The genome of C. jeikeium K411 consists of a circular chromosome of 2,462,499 bp and the 14,323-bp bacteriocin-producing plasmid pKW4. The chromosome of C. jeikeium K411 contains 2,104 predicted coding sequences, 52% of which were considered to be orthologous with genes in the Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae genomes. These genes apparently represent the chromosomal backbone that is conserved between the four corynebacteria. Among the genes that lack an ortholog in the known corynebacterial genomes, many are located close to transposable elements or revealed an atypical G+C content, indicating that horizontal gene transfer played an important role in the acquisition of genes involved in iron and manganese homeostasis, in multidrug resistance, in bacterium-host interaction, and in virulence. Metabolic analyses of the genome sequence indicated that the “lipophilic” phenotype of C. jeikeium most likely originates from the absence of fatty acid synthase and thus represents a fatty acid auxotrophy. Accordingly, both the complete gene repertoire and the deduced lifestyle of C. jeikeium K411 largely reflect the strict dependence of growth on the presence of exogenous fatty acids. The predicted virulence factors of C. jeikeium K411 are apparently involved in ensuring the availability of exogenous fatty acids by damaging the host tissue. PMID:15968079

  6. Nutritional requirements of Corynebacterium pyogenes.

    PubMed

    Fraga, A M; Reddy, C A

    1982-08-01

    The nutritional requirements of Corynebacterium pyogenes (strains C100, 5, and 1909), a commonly encountered animal pathogen, were determined in this study. A semidefined medium (SDM) containing glucose, HCO3-, hemin, charcoal-treated Trypticase, and a defined mixture of purines and pyrimidines, amino acids, and minerals which supported optimal growth of C. pyogenes was employed in all nutritional studies. Adenine and uracil were required for optimal growth of strains 5 and C100 but were not required for strain 1909. Riboflavin and nicotinic acid were required for good growth of all three strains; biotin and thiamin were stimulatory but did not appear to be required for growth. Hemin and NaHCO3 were stimulatory for growth, whereas lipoic acid and Tween 80 were neither stimulatory nor required for growth. The replacement of Trypticase with a specific peptide fraction (obtained by fractionation of Trypticase on Sephadex G-25) rich in dipeptides gave growth comparable to that in SDM, indicating a peptide requirement for the growth of C. pyogenes. It was of considerable interest that growth comparable to that in SDM was obtained when Trypticase was replaced by inositol (1 microgram/ml of SDM).

  7. Nutritional requirements of Corynebacterium pyogenes.

    PubMed Central

    Fraga, A M; Reddy, C A

    1982-01-01

    The nutritional requirements of Corynebacterium pyogenes (strains C100, 5, and 1909), a commonly encountered animal pathogen, were determined in this study. A semidefined medium (SDM) containing glucose, HCO3-, hemin, charcoal-treated Trypticase, and a defined mixture of purines and pyrimidines, amino acids, and minerals which supported optimal growth of C. pyogenes was employed in all nutritional studies. Adenine and uracil were required for optimal growth of strains 5 and C100 but were not required for strain 1909. Riboflavin and nicotinic acid were required for good growth of all three strains; biotin and thiamin were stimulatory but did not appear to be required for growth. Hemin and NaHCO3 were stimulatory for growth, whereas lipoic acid and Tween 80 were neither stimulatory nor required for growth. The replacement of Trypticase with a specific peptide fraction (obtained by fractionation of Trypticase on Sephadex G-25) rich in dipeptides gave growth comparable to that in SDM, indicating a peptide requirement for the growth of C. pyogenes. It was of considerable interest that growth comparable to that in SDM was obtained when Trypticase was replaced by inositol (1 microgram/ml of SDM). PMID:6288763

  8. Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese.

    PubMed

    Brennan, N M; Brown, R; Goodfellow, M; Ward, A C; Beresford, T P; Simpson, P J; Fox, P F; Cogan, T M

    2001-05-01

    Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).

  9. Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum▿†

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-01-01

    Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant. PMID:21665967

  10. First Complete Genome Sequence of Corynebacterium riegelii

    PubMed Central

    Greninger, Alexander L.; Streithorst, Jessica

    2017-01-01

    ABSTRACT Here, we report the first complete genome sequence of Corynebacterium riegelii strain PUDD_83A45, isolated from the urine of a patient with urinary tract infection. The genome measured 2.56 Mb and contained no plasmid. PMID:28360160

  11. Propionibacterium, Corynebacterium, Mycobacterium and Lepra bacilli.

    PubMed

    Barksdale, L; Kim, K S

    1984-01-01

    Evidence is presented which suggests that certain key markers of lepra bacilli reside collectively in Proprionibacterium acnes, Corynebacterium tuberculostearicum and Mycobacterium leprae. The unrestricted replication of Mycobacterium leprae depends most probably upon the presence of an immune-deficiency-inducing viral agent or possibly on the combined effects of the organisms considered.

  12. Novel Corynebacterium diphtheriae in Domestic Cats

    PubMed Central

    Cassiday, Pamela K.; Bernard, Kathryn A.; Bolt, Frances; Steigerwalt, Arnold G.; Bixler, Danae; Pawloski, Lucia C.; Whitney, Anne M.; Iwaki, Masaaki; Baldwin, Adam; Dowson, Christopher G.; Komiya, Takako; Takahashi, Motohide; Hinrikson, Hans P.; Tondella, Maria L.

    2010-01-01

    Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae. PMID:20350389

  13. A fatal case of urosepsis due to Corynebacterium riegelii.

    PubMed

    Aygun, Gokhan; Midilli, Kenan; Cilingir, Hatice; Yilmaz, Mesut; Kutukcu, Aysegul; Eker, Engin

    2013-01-01

    Corynebacterium species other than Corynebacterium diphtheriae rarely cause infections in human but rather reside in flora, however they have been reported to cause opportunistic infections in both immunocompromised and immunecompetent patients. Here we report for the first time a case of an elderly female patient presenting with a fatal urosepsis caused by a recently defined pathogen, Corynebacterium riegelii, identified on second day after patient hospitalization leading to a progressive worsening and death of the patient on 6th day.

  14. Corynebacterium ulcerans, an emerging human pathogen.

    PubMed

    Hacker, Elena; Antunes, Camila A; Mattos-Guaraldi, Ana L; Burkovski, Andreas; Tauch, Andreas

    2016-09-01

    While formerly known infections of Corynebacterium ulcerans are rare and mainly associated with contact to infected cattle, C. ulcerans has become an emerging pathogen today. In Western Europe, cases of respiratory diphtheria caused by C. ulcerans have been reported more often than infections by Corynebacterium diphtheria, while systemic infections are also increasingly reported. Little is known about factors that contribute to host colonization and virulence of this zoonotic pathogen. Research in this field has received new impetus by the publication of several C. ulcerans genome sequences in the past years. This review gives a comprehensive overview of the basic knowledge of C. ulcerans, as well as the recent advances made in the analysis of putative virulence factors.

  15. Characterization of strains of Corynebacterium bovis.

    PubMed Central

    Brooks, B W; Barnum, D A

    1984-01-01

    The biochemical and morphological characteristics of 104 strains of Corynebacterium bovis isolated from bovine milk samples and the C. bovis reference strain were found to be uniform. Valuable criteria for identification were presence of catalase and oxidase, production of acid from glucose and fructose and a requirement for enriched basal media. Six strains of human and three strains of bovine origin were found to be inconsistent with the reference strain. PMID:6722650

  16. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... devices that consist of antisera conjugated with a fluorescent dye used to identify Corynebacterium...

  17. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... devices that consist of antisera conjugated with a fluorescent dye used to identify Corynebacterium...

  18. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... devices that consist of antisera conjugated with a fluorescent dye used to identify Corynebacterium...

  19. Effect of Tween 40 and DtsR1 on L-arginine overproduction in Corynebacterium crenatum.

    PubMed

    Chen, Minliang; Chen, Xuelan; Wan, Fang; Zhang, Bin; Chen, Jincong; Xiong, Yonghua

    2015-08-12

    L-Glutamate is an important precursor in the L-arginine (L-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and dtsR1 disruption, have been widely used to induce L-glutamate overproduction in Corynebacterium glutamicum. In this study, a novel strategy for L-Arg overproduction through Tween 40 trigger and ΔdtsR1 mutant were proposed in Corynebacterium crenatum. Corynebacterium crenatum mutant (CCM01) was selected as a host strain, whose argR was lethal via mutagenesis screening, the proB gene was knocked out, and argB was replaced by argB M4 (E19R, H26E, D311R, and D312R) to release L-Arg feedback resistance. After Tween 40 trigger in the logarithmic period, L-Arg production increased from 15.22 to 17.73 g/L in CCM01 strain. When NCgl1221 and dtsR1 disruption (CCM03), L-Arg production drastically increased to 27.45 g/L and then further to 29.97 g/L after Tween 40 trigger. Moreover, the specific activity of α-oxoglutarate dehydrogenase complex (ODHC) decreased, whereas the regeneration of NADP(+)/NADPH significantly increased after dtsR1 disruption and Tween 40 trigger. Results of real-time PCR showed that the transcriptional levels of odhA, sucB, and lpdA (encoding three subunits of the ODHC complex) were downregulated after Tween 40 trigger or dtsR1 disruption. By contrast, zwf transcription (encoding glucose-6-phosphate dehydrogenase) showed no significant difference among CCM01, CCM02 (ΔNCgl1221), and CCM03 (ΔNCgl1221ΔdtsR1) strains without Tween 40 trigger but evidently increased by 5.50 folds after Tween 40 trigger. A novel strategy for L-Arg overproduction by dtsR1 disruption and Tween 40 trigger in C. crenatum was reported. Tween 40 addition exhibited a bifunctional mechanism for L-Arg overproduction, including reduced ODHC activity and enhanced NADPH pools accumulation by downregulated dtsR1 expression and upregulated zwf expression, respectively.

  20. The killing of macrophages by Corynebacterium ulcerans.

    PubMed

    Hacker, Elena; Ott, Lisa; Schulze-Luehrmann, Jan; Lührmann, Anja; Wiesmann, Veit; Wittenberg, Thomas; Burkovski, Andreas

    2016-01-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen.

  1. Cutaneous infections due to Corynebacterium diphtheriae

    PubMed Central

    Cockcroft, W. H.; Boyko, W. J.; Allen, D. E.

    1973-01-01

    Toxigenic Corynebacterium diphtheriae was grown from skin lesions of 44 indigent patients seen at the emergency or out-patient departments of this hospital, 43 of them within the last 16 months of the study period. In all cases staphylococci or hemolytic streptococci were also present in the wounds. An increase in the incidence of clinical diphtheria occurred in the few months preceding and overlapping the period of recognition of the cutaneous infections. The gravis strains, which accounted for the majority of the infections, were sensitive to erythromycin and to penicillin, but were relatively resistant to cloxacillin. PMID:4632361

  2. Analysis of Corynebacterium vaginale by an Immunodiffusion Technique

    PubMed Central

    Smaron, Mary F.; Vice, John L.

    1974-01-01

    An immunodiffusion technique was employed to study the antigenic relationship of Corynebacterium vaginale isolates, vaginal diphtheroids, and members of the genera Corynebacterium and Lactobacillus. Antisera were prepared against C. vaginale ATCC strain 14018 grown diphasically and on blood agar plates and were tested against extracts of organisms prepared by sonication. Ouchterlony analysis demonstrated that all of the isolates of C. vaginale examined possess a common antigenic determinant. No antigenic relationship was detected between C. vaginale and members of the genera Corynebacterium or Lactobacillus. This study also demonstrated that alterations in the cultural conditions can cause variations in the antigenic composition of C. vaginale. Images PMID:4132956

  3. [Breast cancer treated by antibiotherapy? Granulomatous mastitis with Corynebacterium].

    PubMed

    Buhler, J; Grignon, Y; Gallon, F

    2015-09-01

    Granulomatous mastitis is a rare disease, often associated with Corynebacterium infection. It raises the problem of diagnosis of breast tumor with a fast evolution and inflammatory character. We report two cases of granulomatous mastitis with Corynebacterium. It concerns the clinical and radiological description, followed by the therapeutic alternatives and future of the patients. The clinical presentation is variable. The treatment consists in a surgical procedure of resection. The medical treatment based of corticosteroids also proves efficient. The association between Corynebacterium presence and this pathology seems frequent and needs a specific bacteriological search.

  4. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen.

    PubMed

    Díez-Aguilar, M; Ruiz-Garbajosa, P; Fernández-Olmos, A; Guisado, P; Del Campo, R; Quereda, C; Cantón, R; Meseguer, M A

    2013-06-01

    The purpose of the study was to describe the microbiological and clinical features of ten cases of lower respiratory tract infection due to Corynebacterium striatum, Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum. Respiratory samples were recovered from hospitalised patients who were diagnosed of pneumonia and exacerbations of chronic respiratory infections. The samples were Gram-stained and seeded on conventional bacterial growing media. Bacteria were identified by matrix-assisted linear desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested by the disk diffusion method. All patients presented an acute respiratory onset, most of them in the context of an underlying disease and/or immunosuppression. In all patients, the microscopical examination of Gram-stained respiratory samples showed numerous polymorphonuclear cells and Gram-positive bacilli, suggestive of the Corynebacterium morphotype. A pure culture growth of Corynebacterium was obtained in the majority (72 %) of samples. The conclusions are that non-diphtheriae Corynebacterium species are an emerging cause of respiratory infection among patients with chronic respiratory disease and/or immunosuppression, and cannot always be considered as mere colonisers. The microorganism's predominance in Gram-stained purulent respiratory samples together with abundant growth in the culture is the key for the microbiological diagnosis.

  5. Complete genome sequence, lifestyle, and multi-drug resistance of the human pathogen Corynebacterium resistens DSM 45100 isolated from blood samples of a leukemia patient.

    PubMed

    Schröder, Jasmin; Maus, Irena; Meyer, Katja; Wördemann, Stephanie; Blom, Jochen; Jaenicke, Sebastian; Schneider, Jessica; Trost, Eva; Tauch, Andreas

    2012-04-23

    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. 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. The detected gene repertoire of C. resistens DSM 45100 provides insights into the lipophilic lifestyle and virulence functions of this newly recognized pathogen. Plasmid pJA144188 revealed a

  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

    PubMed Central

    2012-01-01

    Background Corynebacterium resistens was initially recovered from human infections and recognized as a new coryneform species that is highly resistant to antimicrobial agents. Bacteremia associated with this organism in immunocompromised patients was rapidly fatal as standard minocycline therapies failed. C. resistens DSM 45100 was isolated from a blood culture of samples taken from a patient with acute myelocytic leukemia. The complete genome sequence of C. resistens DSM 45100 was determined by pyrosequencing to identify genes contributing to multi-drug resistance, virulence, and the lipophilic lifestyle of this newly described human pathogen. Results The genome of C. resistens DSM 45100 consists of a circular chromosome of 2,601,311 bp in size and the 28,312-bp plasmid pJA144188. Metabolic analysis showed that the genome of C. resistens DSM 45100 lacks genes for typical sugar uptake systems, anaplerotic functions, and a fatty acid synthase, explaining the strict lipophilic lifestyle of this species. The genome encodes a broad spectrum of enzymes ensuring the availability of exogenous fatty acids for growth, including predicted virulence factors that probably contribute to fatty acid metabolism by damaging host tissue. C. resistens DSM 45100 is able to use external L-histidine as a combined carbon and nitrogen source, presumably as a result of adaptation to the hitherto unknown habitat on the human skin. Plasmid pJA144188 harbors several genes contributing to antibiotic resistance of C. resistens DSM 45100, including a tetracycline resistance region of the Tet W type known from Lactobacillus reuteri and Streptococcus suis. The tet(W) gene of pJA144188 was cloned in Corynebacterium glutamicum and was shown to confer high levels of resistance to tetracycline, doxycycline, and minocycline in vitro. Conclusions The detected gene repertoire of C. resistens DSM 45100 provides insights into the lipophilic lifestyle and virulence functions of this newly recognized

  7. [Pneumonia caused byCorynebacterium pseudodiphtheriticum].

    PubMed

    Furiasse, Daniela; Gasparotto, Ana M; Monterisi, Aída; Castellano, Gabriela; Rocchi, Marta

    Microorganisms of the genera Corynebacterium, specie pseudodiphtheriticum are a part of the indigenous microbiota of human skin and oropharinx. Nevertheless in recent decades these bacilli are emerging as opportunistic pathogens causing clinically significant infections in patients with previous compromise. We report the case of a 76 years old female patient, with a history of hypertension, hypothyroidism, type 2 diabetes and chronic renal failure, who presented pneumonia during their stay at the intensive care unit. The induced sputum revealed a representative sample with monomicrobial gram positive pleomorphic coryneform rods (Gram stain) and cultures demonstrated the presence of C. pseudodiphtheriticum as the only bacteria recovered. The pacient received an empirical third generation cephalosporin medication with a succesfull recovery.

  8. Active Transport of Alcohol in Corynebacterium acetophilum

    PubMed Central

    Murooka, Yoshikatsu; Harada, Tokuya

    1974-01-01

    The transport of alcohols was studied in Corynebacterium acetophilum, which was isolated as a strain growing well on acetate and ethanol. The transport of ethanol was found to be inducible by ethanol, n-propanol, n-butanol, and acetate, whereas transport of methanol occurred by noninducible passive diffusion. The entry of ethanol into the cells occurred against a concentration gradient and showed saturation kinetics with two Km values of 2.4 × 10−5 M and 6.0 × 10−5 M. Uptake of ethanol was inhibited by sodium azide, sodium cyanide, 2,4-dinitrophenol, and p-chloromercuribenzoate. The transport of ethanol was competitively inhibited by normal alcohols, but not by iso- or tert-alcohols. From these studies, we concluded that an inducible active alcohol transport system mediates the entry of ethanol, n-propanol, or n-butanol into the cells of C. acetophilum. Images PMID:4821092

  9. The killing of macrophages by Corynebacterium ulcerans

    PubMed Central

    Hacker, Elena; Ott, Lisa; Schulze-Luehrmann, Jan; Lührmann, Anja; Wiesmann, Veit; Wittenberg, Thomas; Burkovski, Andreas

    2016-01-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen. PMID:26632348

  10. Corynebacterium jeikeium bacteremia in a hemodialyzed patient.

    PubMed

    Ifantidou, Athina M; Diamantidis, Michael D; Tseliki, Georgia; Angelou, Argiri S; Christidou, Photini; Papa, Anna; Pentilas, Demetrius

    2010-09-01

    Corynebacterium jeikeium, frequently encountered in clinical specimens, is part of the normal skin flora. Nevertheless, a few cases of C. jeikeium bacteremia followed by severe clinical manifestations have been reported. C. jeikeium has been reported to cause endocarditis, septicemia, meningitis, pneumonia and osteomyelitis, along with soft tissue and trauma infections. Herein we describe a case of C. jeikeium bacteremia in Greece. The isolation of a coryneform bacterium from a clinical specimen should not immediately be considered a superinfection by the skin flora. Clinical and laboratory investigations are essential in order to evaluate such cases before applying appropriate treatment. On the other hand, the association of coryneform bacteria and disease should be critically investigated, with a thorough identification of the strain, ideally beyond the classical methods, at a specialized center.

  11. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species

    PubMed Central

    Ramsey, Matthew M.; Freire, Marcelo O.; Gabrilska, Rebecca A.; Rumbaugh, Kendra P.; Lemon, Katherine P.

    2016-01-01

    Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe–microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species. PMID:27582729

  12. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species.

    PubMed

    Ramsey, Matthew M; Freire, Marcelo O; Gabrilska, Rebecca A; Rumbaugh, Kendra P; Lemon, Katherine P

    2016-01-01

    Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.

  13. [Screening of dissolved oxygen induced promoters in Corynebacterium crenatum and functional verification].

    PubMed

    Zhang, Bohui; Xu, Meijuan; Rao, Zhiming; Xu, Zhenghong

    2013-09-04

    Purpose of this work was to screen promoters which were induced by dissolved oxygen in Corynebacterium crenatum SYPA5-5. Based on the genomic information of Corynebacterium, we identified target proteins which were the highest expression level of one copy of 27 known proteins from our earlier study. Based on the upstream sequence of the coding gene sequences, we amplified two promoters, named P-tkt and P-fum. The tac promoter of recombinant pDXW-8-cat and pDXW-8-gfp were replaced by the new promoters, and the recombinant plasmids were transformed into E. coli JM109 and C. crenatum SYPA5-5 respectively. At different dissolved oxygen level, we compared the function of promoters by the expression of chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP). Results show that we identified 2 target proteins, which were transketolase and fumarate hydratase. At the high dissolved oxygen level the CAT activity of E. coli JM109/pDXW-Ptkt-cat and C. crenatum SYPA5-5/pDXW-Ptkt-cat were 3.032 U/mg and 1.987 U/mg, which were 2.8-fold and 3.2-fold than that in the low dissolved oxygen, respectively. We got similar results by using a 5-L fermentor. The P-tkt promoter induced by high dissolved oxygen was suitable for fermentation to produce amino acids and beneficial to improve ability of synthesis and metabolism of amino acids at high dissolved oxygen level.

  14. Transitional forms of Corynebacterium acnes in disease.

    PubMed

    Zierdt, C H; Wertlake, P T

    1969-02-01

    A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term "transitional" was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form.

  15. Transitional Forms of Corynebacterium acnes in Disease

    PubMed Central

    Zierdt, C. H.; Wertlake, P. T.

    1969-01-01

    A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term “transitional” was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form. Images PMID:4886295

  16. Inherited resistance to Corynebacterium kutscheri in mice.

    PubMed Central

    Hirst, R G; Wallace, M E

    1976-01-01

    An analysis of the factors responsible for inherited resistance to Corynebacterium kutscheri was undertaken. Various inbred mouse strains were examined; these included the Swiss Lynch and C57Bl/l mice, their F1 and F2 progeny, and the progeny of the F1 backcrossed to each parent strain. Two modes of inherited resistance are described. An examination suggested that resistance as measured by the mean lethal dose of C. kutscheri was under polygenic control and was inherited continuously. However, the efficiency with which C. kutscheri was eliminated by the mononuclear phagocyte cells of the liver over 3 days differed markedly among strains. A genetic analysis of this mononuclear phagocyte microbicidal efficiency (MPME) in Swiss Lynch and C57Bl/6 mice was undertaken. The trait, MPME, was present, but did not segregate, in the F1 progeny or in the progeny of the backcross to the resistant C57Bl/6 parent; this was clear evidence of dominance. Moreover, MPME segregated in a ratio of 1:1 in the progeny of the backcross to the sensitive Swiss Lynch parent and in a ratio of 3:1 in the F2 progeny. It was concluded that MPME was inherited discontinuously and was controlled by a single dominant autosomal gene (or closely linked group); the recessive allele was assigned the gene symbol ack. Linkage experiments showed there to be no association between the ack locus and any of the immune-response genes. PMID:971958

  17. Corynebacterium pseudotuberculosis infection in Israeli dairy cattle.

    PubMed Central

    Yeruham, I.; Elad, D.; Friedman, S.; Perl, S.

    2003-01-01

    Two forms of Corynebacterium pseudotuberculosis infection in Israeli dairy cattle herds during a survey period of 13 years (1989-2001) are described. The more common form, which was diagnosed in 45 herds, was characterized by ulcerative granulomatous lesions which occurred either sporadically--in 26 herds (with a morbidity rate of up to 5%)--or in an epidemic course in 19 herds. Most (80.6%) of the affected animals were cows; the rest were first-calving cows (16.2%) and heifers (3.2%). The morbidity occurred mostly during the summer months. The ulcerative granulomatous lesions appeared in three clinical forms: cutaneous, mastitic and visceral. Mixed forms were also observed. The morbidity rate was 6.4% and the culling rate reached 16.3% of the affected animals. Most of the strains of C. pseudotuberculosis which were isolated from the abscesses in the cutaneous form of the disease and from milk samples failed to reduce nitrate. A decrease in milk production (6%) and an increase in bulk-milk somatic cell count were noted. Necrotic and ulcerative dermatitis on the heel of the foot occurred in an epidemic course in heifers in only two herds during the winter months, with morbidity rates of 7.5 and 76.2%, respectively. C. pseudotuberculosis isolates from skin lesions and from the soil did reduce nitrate. Clinical, epizootiological and microbiological aspects of the infection are described. PMID:14596537

  18. Septic arthritis in a native knee due to Corynebacterium striatum.

    PubMed

    Molina Collada, Juan; Rico Nieto, Alicia; Díaz de Bustamante Ussia, Macarena; Balsa Criado, Alejandro

    2017-03-07

    We describe a case of septic arthritis in a native knee due to Corynebacterium striatum, gram-positive bacilli that are usually commensal organisms of skin and mucosal membranes, but are seldom implicated in native septic arthritis. An 84-year-old man with Corynebacterium striatum septic arthritis of his native left knee and no response to conventional antibiotic therapy. Thus, the patient was allowed to take dalbavancin for compassionate use, with an excellent clinical outcome. This case emphasizes de role of Corynebacterium striatum in native joint infections and highlights the importance of early detection and appropriate treatment in improving the clinical outcome. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

  19. Oxidation of Secondary Alcohols by Extracts of a Corynebacterium

    PubMed Central

    Rosenberg, Eugene; Holmes, Paul

    1965-01-01

    Rosenberg, Eugene (University of California, Los Angeles), and Paul Holmes. Oxidation of secondary alcohols by extracts of a Corynebacterium. J. Bacteriol. 89:1212–1216. 1965.—A Corynebacterium was isolated from soil by use of the enrichment culture technique. This Corynebacterium oxidized and grew on the chemically synthesized secondary alcohol, 3-tetrahydrofuranol. Extracts of this organism contained at least two different nicotinamide adenine dinucleotide-requiring soluble secondary alcohol dehydrogenases. These enzymes were distinguished by the relative rates at which they oxidized thymidylic acid and 3-tetrahydrofuranol. In addition to their substrate specificity, the two enzymes differed in pH optima and thermal stability. Also, the 3-tetrahydrofuranol dehydrogenase was induced by 3-tetrahydrofuranol, whereas the thymidylic acid dehydrogenase was constitutive. PMID:14292988

  20. Corynebacterium spp. in dogs and cats with otitis externa and/or media: a retrospective study.

    PubMed

    Henneveld, Kerstin; Rosychuk, Rodney A W; Olea-Popelka, Francisco J; Hyatt, Doreene R; Zabel, Sonja

    2012-01-01

    The role of Corynebacterium spp. in the pathogenesis of canine and feline otitis externa/media and their appropriate antimicrobial therapy are unclear. The objectives of this study were to (1) better establish the pathogenicity of Corynebacterium spp. in otitis utilizing reported criteria and by assessing clinical response to antibiotic therapy and (2) to determine the antimicrobial susceptibility patterns of Corynebacterium spp. associated with otitis. The study was retrospective, targeting cultures positive for Corynebacterium spp. Corynebacterium spp. were part of mixed microbial populations in 79/81 cultures. Corynebacterium spp. pathogenicity was highly questionable because of their almost invariable presence with other microbes and the observation that Corynebacterium spp. usually disappear from the ear with resolution of other infections, even when the Corynebacterium spp. are resistant to the prescribed antibiotic(s). However, 2/81 cultures came from two canine ears wherein Corynebacterium spp. may have been pathogenic. Antimicrobial sensitivities for Corynebacterium spp. were available for 54 isolates. Most isolates were susceptible to chloramphenicol (53/54), amikacin (50/54), tetracycline (50/54), gentamicin (46/54), and enrofloxacin (32/54). Among those antibiotics available in otic products, gentamicin and enrofloxacin would be rational choices for the empirical, topical therapy of Corynebacterium spp.

  1. Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli.

    PubMed

    Lacmata, Stephen Tamekou; Kuiate, Jules-Roger; Ding, Yamei; Xian, Mo; Liu, Huizhou; Boudjeko, Thaddée; Feng, Xinjun; Zhao, Guang

    2017-01-01

    Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12.

  2. Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli

    PubMed Central

    Lacmata, Stephen Tamekou; Kuiate, Jules-Roger; Ding, Yamei; Xian, Mo; Liu, Huizhou; Boudjeko, Thaddée; Feng, Xinjun; Zhao, Guang

    2017-01-01

    Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12. PMID:28253372

  3. Application of granular activated carbon/MnFe₂O₄ composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies.

    PubMed

    Podder, M S; Majumder, C B

    2016-01-15

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG(0), ΔH(0) and ΔS(0) revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

  4. Application of granular activated carbon/MnFe2O4 composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-01-01

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG0, ΔH0 and ΔS0 revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. Native valve endocarditis due to Corynebacterium group JK.

    PubMed

    Moffie, B G; Veenendaal, R A; Thompson, J

    1990-12-01

    We report a case of a 32-yr-old woman on chronic intermittent haemodialysis, who developed endocarditis due to a Corynebacterium group JK, involving both the native aortic and mitral valves. Despite a four-week treatment with vancomycin, an aortic root abscess developed. The diagnosis was confirmed on autopsy.

  7. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria belonging to the genus Corynebacterium and provides epidemiological information on diseases caused by these.... However, many other types of corynebacteria form part of the normal flora of the human respiratory tract...

  8. Early prosthetic valve endocarditis caused by Corynebacterium kroppenstedtii.

    PubMed

    Hagemann, Jürgen Benjamin; Essig, Andreas; Herrmann, Manuel; Liebold, Andreas; Quader, Mohamed Abo

    2015-12-01

    Corynebacterium (C.) kroppenstedtii is a rarely detected agent of bacterial infections in humans. Here, we describe the first case of prosthetic valve endocarditis caused by C. kroppenstedtii. Application of molecular methods using surgically excised valve tissue was a cornerstone for the establishment of the microbiological diagnosis, which is crucial for targeted antimicrobial treatment.

  9. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3140...

  10. Corynebacterium pseudotuberculosis liver abscess in a mature alpaca (Lama pacos)

    PubMed Central

    Sprake, Philippa; Gold, Jenifer R.

    2012-01-01

    A mature female alpaca was evaluated for weight loss and a 10-day history of anorexia, diarrhea, abdominal distension, and ventral edema. Ultrasonography revealed a hepatic mass, culture of which identified Corynebacterium pseudotuberculosis. This is the first reported case of an internal caseous lymphadenitis lesion resulting in clinical disease in a camelid. PMID:23024384

  11. Caseous lymphadenitis caused by Corynebacterium ulcerans in the dromedary camel.

    PubMed Central

    Tejedor, M T; Martin, J L; Lupiola, P; Gutierrez, C

    2000-01-01

    Caseous lymphadenitis that affected the dorsal and ventral superficial lymph nodes in the left cervicothoracic region of a young dromedary camel is described. The agent isolated was Corynebacterium ulcerans. To our knowledge, this is the first description of purulent lymphadenitis caused by C. ulcerans in a species belonging to the Camelidae. Images p127-a PMID:10723599

  12. Assessing the Genotypic Differences between Strains of Corynebacterium pseudotuberculosis biovar equi through Comparative Genomics

    PubMed Central

    Ramos, Rommel T. J.; Veras, Adonney A. O.; Pinheiro, Kenny C.; Benevides, Leandro J.; Edman, Judy M.; Spier, Sharon J.; Azevedo, Vasco; Silva, Artur

    2017-01-01

    Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as “pigeon fever” which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at

  13. Assessing the Genotypic Differences between Strains of Corynebacterium pseudotuberculosis biovar equi through Comparative Genomics.

    PubMed

    Baraúna, Rafael A; Ramos, Rommel T J; Veras, Adonney A O; Pinheiro, Kenny C; Benevides, Leandro J; Viana, Marcus V C; Guimarães, Luís C; Edman, Judy M; Spier, Sharon J; Azevedo, Vasco; Silva, Artur

    2017-01-01

    Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as "pigeon fever" which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at

  14. Human clinical isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans collected in Canada from 1999 to 2003 but not fitting reporting criteria for cases of diphtheria.

    PubMed

    Dewinter, Leanne M; Bernard, Kathryn A; Romney, Marc G

    2005-07-01

    A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

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

    PubMed

    Sangal, Vartul; Hoskisson, Paul A

    2016-09-01

    Diphtheria is a debilitating disease caused by toxigenic Corynebacterium diphtheriae strains and has been effectively controlled by the toxoid vaccine, yet several recent outbreaks have been reported across the globe. Moreover, non-toxigenic C. diphtheriae strains are emerging as a major global health concern by causing severe pharyngitis and tonsillitis, endocarditis, septic arthritis and osteomyelitis. Molecular epidemiological investigations suggest the existence of outbreak-associated clones with multiple genotypes circulating around the world. Evolution and pathogenesis appears to be driven by recombination as major virulence factors, including the tox gene and pilus gene clusters, are found within genomic islands that appear to be mobile between strains. The number of pilus gene clusters and variation introduced by gain or loss of gene function correlate with the variable adhesive and invasive properties of C. diphtheriae strains. Genomic variation does not support the separation of C. diphtheriae strains into biovars which correlates well with findings of studies based on multilocus sequence typing. Genomic analyses of a relatively small number of strains also revealed a recombination driven diversification of strains within a sequence type and indicate a wider diversity among C. diphtheriae strains than previously appreciated. This suggests that there is a need for increased effort from the scientific community to study C. diphtheriae to help understand the genomic diversity and pathogenicity within the population of this important human pathogen.

  16. Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae.

    PubMed

    Kim, Seonghun; Oh, Doo-Byoung; Kwon, Ohsuk; Kang, Hyun Ah

    2010-04-01

    Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.

  17. [Effect of Corynebacterium non diphtheriae on functional activity and apoptosis of macrophages].

    PubMed

    Kharseeva, G G; Voronina, N A; Tiukavkina, S Iu

    2014-01-01

    Determine the ability of Corynebacterium non diphtheriae to induce phagocytosis and apoptosis of macrophages and evaluate regulatory effect of nuetrophilokines (NPK) induced by Corynebacterium non diphtheriae on these processes. The ability of Corynebacterium non diphtheriae, isolated from upper respiratory tract, skin and urogenital tract (UGT) were studied for the ability to induce phagocytosis and apoptosis of mice macrophages (MP; in vitro during staining by May-Grunwald with additional staining by Romanowsky-Giemsa) before and after the addition of NPK induced by Corynebacterium non diphtheriae. Phagocytic index (PI) was the same for all the Corynebacterium non diphtheriae species, phagocytic number (PN) and index of phagocytosis completion (IPC)--were minimal relative to corynebacteria isolated from UGT. All the studied corynebacteria species induced MP apoptosis; the most pronounced apoptogenic effect was detected in Corynebacterium pseudotuberculosis isolated from UGT. NPK increased PN against corynebacteria isolated from the studied biotopes, IPC--only during studies of corynebacteria isolated from skin. The effect of NPK resulted in a reduction of apoptogenic effect for almost all the Corynebacterium non diphtheriae, regardless of the isolation location. A pronounced apoptogenic effect and insufficiency of phagocytosis processes induced by corynebacteria are the means of realization of Corynebacterium non diphtheriae pathogenic effect. NPK use is possible for immune correction of immune deficiency conditions developing against the background of diseases determined by Corynebacterium non diphtheriae.

  18. Corynebacterium tapiri sp. nov. and Corynebacterium nasicanis sp. nov., isolated from a tapir and a dog, respectively.

    PubMed

    Baumgardt, Sandra; Loncaric, Igor; Kämpfer, Peter; Busse, Hans-Jürgen

    2015-11-01

    Two Gram-stain-positive bacterial isolates, strain 2385/12T and strain 2673/12T were isolated from a tapir and a dog's nose, respectively. The two strains were rod to coccoid-shaped, catalase-positive and oxidase-negative. The highest 16S rRNA gene sequence similarity identified Corynebacterium singulare CCUG 37330T (96.3% similarity) as the nearest relative of strain 2385/12T and suggested the isolate represented a novel species. Corynebacterium humireducens DSM 45392T (98.7% 16S rRNA gene sequence similarity) was identified as the nearest relative of strain 2673/12T. Results from DNA-DNA hybridization with the type strain of C. humireducens demonstrated that strain 2673/12T also represented a novel species. Strain 2385/12T showed a quinone system consisting predominantly of menaquinones MK-8(H2) and MK-9(H2) whereas strain 2673/12T contained only MK-8(H2) as predominant quinone. The polar lipid profiles of the two strains showed the major compounds phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. Phosphatidylinositol was identified as another major lipid in 2673/12T whereas it was only found in moderate amounts in strain 2385/12T. Furthermore, moderate to minor amounts of phosphatidylinositol-mannoside, β-gentiobiosyl diacylglycerol and variable counts of several unidentified lipids were detected in the two strains. Both strains contained corynemycolic acids. The polyamine patterns were characterized by the major compound putrescine in strain 2385/12T and spermidine in strain 2673/12T. In the fatty acid profiles, predominantly C18:1ω9c and C16:0 were detected. The two strains are distinguishable from each other and the nearest related established species of the genus Corynebacterium phylogenetically and phenotypically. In conclusion, two novel species of the genus Corynebacterium are proposed, namely Corynebacterium tapiri sp. nov. (type strain, 2385/12T = CCUG 65456T = LMG 28165T) and Corynebacterium nasicanis sp. nov. (type

  19. Corynebacterium urealyticum: a comprehensive review of an understated organism.

    PubMed

    Salem, Nagla; Salem, Lamyaa; Saber, Sally; Ismail, Ghada; Bluth, Martin H

    2015-01-01

    Corynebacterium urealyticum is a Gram positive, slow-growing, lipophilic, multi-drug resistant, urease positive micro-organism with diphtheroid morphology. It has been reported as an opportunistic nosocomial pathogen and as the cause of a variety of diseases including but not limited to cystitis, pyelonephritis, and bacteremia among others. This review serves to describe C. urealyticum with respect to its history, identification, laboratory investigation, relationship to disease and treatment in order to allow increased familiarity with this organism in clinical disease.

  20. Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in foals.

    PubMed Central

    Takai, S; Ohkura, H; Watanabe, Y; Tsubaki, S

    1986-01-01

    Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in newborn foals for 12 weeks after birth were investigated on two horse breeding farms. R. equi was found in the feces of foals during week 1 of life. The greatest numbers of R. equi were present in the feces of foals during the first 8 weeks of their lives, which coincides with the age when foals are most liable to be exposed to R. equi. PMID:3700632

  1. Corynebacterium pseudodiphtheriticum keratitis and conjunctivitis: a case report.

    PubMed

    Li, A; Lal, S

    2000-02-01

    A case of keratitis and conjunctivitis in an 86-year-old man caused by Corynebacterium pseudodiphtheriticum is reported. Corynebacteria are uncommon causes of ocular surface infections. However, the presence of corneal and conjunctival epithelial defects in an immunocompromised patient can result in severe infection by a commensal organism such as C. pseudodiphtheriticum. The significance of a positive culture in these settings should not be overlooked.

  2. Gangrenous dermatitis caused by Corynebacterium ulcerans in Richardson ground squirrels.

    PubMed

    Olson, M E; Goemans, I; Bolingbroke, D; Lundberg, S

    1988-08-01

    Gangrenous dermatitis caused by Corynebacterium ulcerans developed in 63 of 350 wild Richardson ground squirrels (Spermophilus richardsonii). Six squirrels died of toxemia and/or septicemia, but 57 responded to topical and parenteral administration of antibiotics. The epizo-otic was believed to be associated with fighting; infected and carrier ground squirrels most likely transmitted the C ulcerans through bite wounds. Individuals handling ground squirrels should be cautioned that C ulcerans may produce a diphtheria-like disease in human beings.

  3. [Significant bacteremias by Corynebacterium amycolatum: an emergent pathogen].

    PubMed

    Oteo, J; Aracil, B; Ignacio Alós, J; Luis Gómez-Garcés, J

    2001-03-01

    Corynebacterium sp. is an extremely varied genus which includes little known species and of which only Corynebacterium diphteriae, Corynebacterium urealyticum and Corynebacterium jeikeium are considered indisputable pathogens. Other species, such as C. amycolatum are at present being reconsidered as causative agents in infectious pathologies, partly on account of our greater aquaintance and improved identification techniques for these microorganisms and partly on account of the growing number of immunocompromised patients in whom all their pathogenic capacity is usually able to develope. We present 3 cases of significant bacteremia by C. amycolatum. Bacterial isoliations from blood culture were obtained using the Vital Systems. Identification was performed by means of Gran stain, colony morphology, the results of numerous biochemical tests (including the Api Coryne systems), the behaviour of the strains against the vibriostatic agent O/129 and the antibiotic susceptibility pattern obtained with the E-test. The three isolates of C. amycolatum were obtained from patients after a lenghtly hospitalization, multi-instrumentation and who had severe underlying disease. All three presented with concomitant isolates of C. amycolatum from other sites: sputum, wound and catheter respectively, which could explain the origin of the bacteremia. Colony morphology, antibiotic susceptibility patterns, resistance to the vibriostatic agent O/129 and the results of the biochemical test carried out were similar to those previously describe in the literature. C. amycolatum should be born in mind as a agent responsable for significant and severe pathology in this type of patient. In addition, it as certain specific characteristics which assits in its identification in the normal micr

  4. Draft genome sequence of Corynebacterium diphtheriae biovar intermedius NCTC 5011.

    PubMed

    Sangal, Vartul; Tucker, Nicholas P; Burkovski, Andreas; Hoskisson, Paul A

    2012-09-01

    We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.

  5. Corynebacterium propinquum: A Rare Cause of Prosthetic Valve Endocarditis.

    PubMed

    Jangda, Umair; Upadhyay, Ankit; Bagheri, Farshad; Patel, Nilesh R; Mende