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The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression

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Summary

The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5′ and 3′ flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme.

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References

  • Anderson A, Cooper RA (1970) Genetic Mapping of a Locus for Triosephosphate Isomerase on the Genome of Escherichia coli K12. J Gen Microbiol 62:329–334

    Google Scholar 

  • Bachmann BJ (1987) Linkage Map of Escherichia coli K-12, Edition 7. In: Neidthard FC (ed) Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology, vol II. American Society for Microbiology, Washington, DC, pp 807–876

    Google Scholar 

  • Barksdale L (1982) The Genus Corynebacterium. In: Starr MP, Stolp H, Trüper HG, Balous A, Schlegel HG (eds) The Prokaryotes. Springer Verlag, New York, pp 1827–1837

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Cohen SN, Chang ACY, Hsu L (1973) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-plasmid DNA. Proc Natl Acad Sci USA 69:2110–2114

    Google Scholar 

  • Cosloy SD, Oishi M (1973) The nature of the transformation process in Escherichia coli K-12. Mol Gen Genet 124:1–10

    Google Scholar 

  • Follettie MT, Sinskey AJ (1986a) Recombinant DNA Technology for Corynebacterium glutamicum. Food Technol 40:88–94

    Google Scholar 

  • Follettie MT, Sinskey AJ (1986b) Molecular Cloning and Nucleotide Sequence of the Corynebacterium glutamicum pheA Gene. J Bacteriol 167:695–702

    Google Scholar 

  • Follettie MT, Shin HK, Sinskey AJ (1988) Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci. Mol Microbiol 2:53–62

    Google Scholar 

  • Fujita NT, Miwa S, Ishijima K, Izui K, Katsuki H (1984) The Primary Structure of Phosphoenolpyruvate Carboxylase of Escherichia coli. Nucleotide sequence of the ppc Gene and Deduced Amino Acid Sequence. J Biochem 95:909–916

    Google Scholar 

  • Glansdorff N (1965) Topography of cotransducible arginine mutations in Escherichia coli K-12. Genetics 51:167–179

    Google Scholar 

  • Gribskov M, Devereux J, Burgess R (1984) The codon preference plot graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12:539–549

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

    Google Scholar 

  • Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298

    Google Scholar 

  • Holmes DS, Quigley M (1981) A Rapid Boiling Method the Preparation of Bacterial Plasmids. Anal Biochem 114:193–197

    Google Scholar 

  • Izui K, Yoshinaga T, Morikawa M, Katsuki H (1970) Activation of phosphoenolpyruvate carboxylase of Escherichia coli by free fatty acids or their coenzyme A derivatives. Biochem Biophys Res Commun 40:949–956

    Google Scholar 

  • Izui K, Matsuda Y, Kameshita I, Katsuki H, Woods AE (1983) Phosphoenolpyruvate Carboxylase of Escherichia coli. Inhibition by Various Analogs and Homologs of Phosphoenolpyruvate. J Biochem 94:1789–1795

    Google Scholar 

  • Kinoshita S (1985) Glutamic acid bacteria, chapter 5. In: Demain AL, Solomon NA (eds) Biology of Industrial Microorganisms. The Benjamin/Cummings Publishing Company, Loncon, pp 115–142

    Google Scholar 

  • Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Martin JF, Santamaria R, Sandoval H, Del Real G, Mateos LM, Gil JA, Aguilar A (1987) Cloning systems in amino acid-producing Corynebacteria. Bio Technology 5:137–146

    Google Scholar 

  • Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321

    Google Scholar 

  • Miller JH (1972) Experiments of Molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Mizusawa S, Nishimura S, Seela F (1986) Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res 14:1319–1324

    Google Scholar 

  • Mori M, Shiio I (1985a) Purification and some properties of Phosphoenolpyruvate Carboxylase from Brevibacterium flavum and its Aspartate-Overproducing mutant. J. Biochem 97:1119–1128

    Google Scholar 

  • Mori M, Shiio I (1985b) Synergistic Inhibition of Phosphoenolpyruvate Carboxylase by Aspartate and 2-Oxoglutarate in Brevibacterium flavum. J Biochem 98:1621–1630

    Google Scholar 

  • Mori M, Shiio I (1986) Multiple Interaction of Fructose 1,6-Bisphosphate and Other Effectors on Phosphoenolpyruvate Carboxylase from Brevibacterium flavum and its Aspartate-Producing Mutant. Agric Biol Chem 50:2605–2614

    Google Scholar 

  • Ozaki H, Shiio I (1969) Regulation of the TCA and Glyoxylate Cycles in Brevibacterium flavum. J Biochem 66:297–311

    Google Scholar 

  • Pahel G, Bloom FR, Tyler B (1979) Deletion Mapping of the polA-metB Region of the Escherichia coli Chromosome. J Bacteriol 138:653–656

    Google Scholar 

  • Peoples OP, Liebl W, Bodis M, Maeng PJ, Follettie MT, Archer JA, Sinskey AJ (1988) Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon. Mol Microbiol 2:63–72

    Google Scholar 

  • Pichersky E, Gottlieb LD, Hess JF (1984) Nucleotide sequence of the triosephosphate isomerase gene of Escherichia coli. Mol Gen Genet 195:314–320

    Google Scholar 

  • Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251

    Google Scholar 

  • Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Shiio I, Ujigawa K (1978) Enzymes of the Glutamate and Aspartate Synthetic Pathways in a Glutamate-Producing Bacterium, Brevibacterium flavum. J Biochem 84:647–657

    Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Google Scholar 

  • Staden R (1982) Automation of computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res 10:4731–4751

    Google Scholar 

  • Staden R (1984) Graphic methods to determine the function of nucleic acid sequences. Nucleic Acids Res 12:521–538

    Google Scholar 

  • Teraoka H, Izui K, Katsuki H (1974) Phosphoenolpyruvate Carboxylase of Escherichia coli. Multiple Conformational States Elicited by Allosteric Effectors. Biochemistry 13:5121–5128

    Google Scholar 

  • Tosaka O, Morioka H, Takinami K (1979) The Role of Biotindependent Pyruvate Carboxylase in L-Lysine Production. Agric Biol Chem 43:1513–1519

    Google Scholar 

  • Utter MF, Kohlenbrander HM (1972) Formation of Oxaloacetate by CO2 Fixation on Phosphoenolpyruvate. In: Boyer PD (ed) The Enzymes, vol VI. Academic Press, New York, pp 117–170

    Google Scholar 

  • Vanderwinkel E, Liard P, Ramos F, Wiame JM (1963) Genetic control of the regulation of isocitritase and malate synthase in Escherichia coli K-12. Biochem Biophys Res Commun 12:157–162

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Wood HD, Utter MF (1965) The Role of CO2 Fixation in Metabolism. In: Campbell PN, Greville GD (eds) Essays in Biochemistry. Academic Press, New York, pp 1–27

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Google Scholar 

  • Yeh P, Sicard AM, Sinskey AJ (1988) Nucleotide sequence of the lysA gene of Corynebacterium glutamicum and possible mechanisms for modulation of its expression. Mol Gen Genet 212:112–119

    Google Scholar 

  • Yoshihama H, Higashiro K, Rao EA, Akedo M, Shanabruch WG, Follettie MT, Walker GC, Sinskey AJ (1985) Cloning Vector System for Corynebacterium glutamicum. J Bacteriol 162:591–597

    Google Scholar 

  • Yoshinaga F, Nakamori F (1983) Production of Amino Acids. In: Herrmann KM, Somerville RL (eds) Amino Acids Biosynthesis and Genetic Regulation. Addison-Wesley Publishing Company, London, pp 405–430

    Google Scholar 

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Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, 02139, Cambridge, MA, USA

    Bernhard J. Eikmanns, Max T. Follettie, Martin U. Griot & Anthony J. Sinskey

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  1. Bernhard J. Eikmanns
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  2. Max T. Follettie
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  3. Martin U. Griot
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  4. Anthony J. Sinskey
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Communicated by J. Lengeler

Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany

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Eikmanns, B.J., Follettie, M.T., Griot, M.U. et al. The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression. Molec Gen Genet 218, 330–339 (1989). https://doi.org/10.1007/BF00331286

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  • DOI: https://doi.org/10.1007/BF00331286

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