1887

Abstract

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of we detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 ± 5 nmol min (mg dry wt)] in cells grown on lactate or pyruvate, and was about two- to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown at low concentrations of biotin (< 5 μg I), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of

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1997-04-01
2021-04-21
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References

  1. Cazzulo J. J., Sundararn T. K., Kornberg H. L. 1970; Properties and regulation of pyruvate carboxylase from Bacillus stearothermophilus.. Proc R Soc Lond B 16:1–19
    [Google Scholar]
  2. Chao Y.-P., Liao J. C. 1993; Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.. Appl Environ Microbiol 59:4261–4265
    [Google Scholar]
  3. Charles A. M., Willer D. W. 1984; Pyruvate carboxylase from Thiobacillus novellus: properties and possible functions.. Can J Microbiol 30:532–539
    [Google Scholar]
  4. Cocaign-Bousquet M., Lindley N. D. 1995; Pyruvate overflow and carbon flux within the central metabolic pathways of Corynebacterium glutamicum during growth on lactate.. Enzyme Microb Techno1 17:260–267
    [Google Scholar]
  5. Cocaign-Bousquet M., Guyonvarch A., Lindley N. D. 1996; Growth rate-dependent modulation of carbon flux through central metabolism and the kinetic consequences for glucose-limited chemostat cultures of Corynebacterium glutamicum.. Appl Environ Microbiol 62:429–436
    [Google Scholar]
  6. Diesterhaft M. D., Freese E. 1973; Role of pyruvate carboxylase, phosphoenolpyruvate carboxykinase and malic enzyme during growth and sporulation of Bacillus subtilis.. J Biol Chem 248:6062–6070
    [Google Scholar]
  7. Eikmanns B. J., Follettie M. T., Griot M. U., Sinskey A. J. 1989; The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression.. Mol Gen Genet 218:330–339
    [Google Scholar]
  8. Eikmanns B. J., Metzger M., Reinscheid D., Kircher M., Sahm H. 1991; Amplification of three theonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains.. Appl Microbiol Biotechnol 34:617–662
    [Google Scholar]
  9. Encarnacion S., Dunn M., Willrns K., Mora J. 1995; Fermentative and aerobic metabolism in Rhizobium etli.. J Bacteriol 177:3058–3066
    [Google Scholar]
  10. Feir H. A., Suzuki I. 1969; Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin-containing carboxylase.. Can J Biochem 47:697–710
    [Google Scholar]
  11. Fisher S. H., Magasanik B. 1984; Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.. J Bacteriol 158:55–62
    [Google Scholar]
  12. Gornall A. G., Bardawill C. J., David M. M. 1949; Determination of serum proteins by means of the biuret reaction.. J BiolChem 177:751–766
    [Google Scholar]
  13. Gubler M., Park S. M., Jetten M., Stephanopoulos G., Sinskey A. J. 1994; Effects of phosphoenolpyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum.. Appl Microbiol Biotechnol 40:857–863
    [Google Scholar]
  14. Gurr J. A., Jones K. M. 1977; Purification and characterization of pyruvate carboxylase from Arthrobacter globiformis.. Arch Biochem Biophys 179:444–455
    [Google Scholar]
  15. Jäger W., Peters-Wendisch P. G., Kalinowski J., Pühler A. 1996; A Corynebacterium glutamicum gene encoding a twodomain protein similar to biotin carboxylases and biotincarboxyl-carrier proteins.. Arch Microbiol 166:76–82
    [Google Scholar]
  16. Jetten M. S. M., Sinskey A. J. 1993; A Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum.. FEMS Microbiol Lett 111:183–188
    [Google Scholar]
  17. Jetten M. S. M., Sinskey A. J. 1995; Purification and properties of oxaloacetate decarboxylase from Corynebacterium glutamicum.. Antonie Leeuwenhoek 67:221–227
    [Google Scholar]
  18. Jetten M. S. M., Pitoc G. A., Follettie M. T., Sinskey A. J. 1994; Regulation of phospho(eno1)-pyruvate- and oxaloacetateconverting enzymes in Corynebacterium glutamicum.. Appl Microbiol Biotechnol 41:47–52
    [Google Scholar]
  19. Kawarnura F., Doi R. H. 1984; Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases.. J Bacteriol 160:442–444
    [Google Scholar]
  20. Kinoshita S. 1985; Glutamic acid bacteria. In Biology of Zndustrial Microorganisms, Edited by A. Demain & N. Solomon. London:. Benjamin/Cummings Publishing115–142
    [Google Scholar]
  21. Kornberg H. L. 1966; Anaplerotic sequences and their role in metabolism. In Essays in Biochemistry, Edited by P.N. Campbell & G.P. Greville. New York:. Academic Press 2:1–31
    [Google Scholar]
  22. Krulwich T. A., Pelliccione N. J. 1979; Catabolic pathways of coryneforms, nocardias and mycobacteria.. Ann Rev Microbiol 33:95–111
    [Google Scholar]
  23. Liebl W. 1991; The genus Corynebacterium-nonmedical. In The Prokaryotes, Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York:. Springer-Verlag II:1157–1171
    [Google Scholar]
  24. Liebl W., Schein B. 1990; Isolation of restriction deficient mutants of Corynebacterium glutamicum. In DECHEMA Biotechnology Conferences, Edited by D. Behrens & P. Krämer. Weinheim:. Verlag Chemie 4:323–327
    [Google Scholar]
  25. Milrad de Forchetti S. R., Cazzulo J. J. 1976; Some properties of the pyruvate carboxylase from Pseudomonas fluorescens.. J Gen Microbiol 93:75–81
    [Google Scholar]
  26. Modak H. V., Kelly D. J. 1995; Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography.. Microbiology 141:2619–2618.
    [Google Scholar]
  27. 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]
  28. 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]
  29. O’Brien R. W., Chuang D. T., Taylor B. L., Utter M. F. 1977; Novel enzymic machinery for the metabolism of oxaloacetate, phosphoenolpyruvate and pyruvate in Pseudomonas citronellolis.. J Biol chem 252:1257–1263
    [Google Scholar]
  30. Ozaki H., Shiio I. 1969; Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum.. J Biochem 66:297–311
    [Google Scholar]
  31. Payne J., Morris J. G. 1969; Pyruvate carboxylase in Rhodopseudomonas sphaeroides.. J Gen Microbiol 59:97–101
    [Google Scholar]
  32. Peters-Wendisch P. G., Eikmanns B. J., Thierbach G., Bachmann B, Sahm H. 1993; Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production.. FEMS Microbiol Lett 112:269–274
    [Google Scholar]
  33. Peters-Wendisch P. G., Wendisch V. F., de Graaf A. A., Eikmanns B. J., Sahm H. 1996; C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylasedeficient Corynebacterium glutamicum.. Arch Microbiol 165:387–396
    [Google Scholar]
  34. Ruiz-Amil M., deTorrontegui G., Palacian E., Catalina L., Losada M. 1965; Properties and functions of yeast pyruvate carboxylase.. J Biol Chem 240:3485–3492
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY:. Cold Spring Harbor Laboratory.
    [Google Scholar]
  36. Schobert P., Bowien B. 1984; Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus.. J Bacteriol 159:167–172
    [Google Scholar]
  37. Schrumpf B., Eggeling L., Sahm H. 1992; Isolation and prominent characteristics of an l-lysine hyperproducing strain of Corynebacterium glutamicum.. Appl Microbiol Biotechnol 37:566–571
    [Google Scholar]
  38. Scrutton M. C., Taylor B. L. 1974; Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP.. Arch Biochem Biophys 164:641–654
    [Google Scholar]
  39. Scrutton M. C., Young M. R. 1972; Pyruvate carboxylase. In The Enzymes, Edited by P. D. Boyer. . New York: Academic Press. 6:1–35
    [Google Scholar]
  40. Seubert W., Weicker H. 1969; Pyruvate carboxylase from Pseudomonas.. Methods Enzymol 13:258–262
    [Google Scholar]
  41. Sundaram T. K. 1973; Physiological role of pyruvate carboxylase in a thermophilic bacillus.. J Bacteriol 113:549–557
    [Google Scholar]
  42. 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]
  43. Vallino J.J., Stephanopoulos G. 1993; Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction.. Biotechnol Bioeng 41:633–646
    [Google Scholar]
  44. Willison J.C. 1988; Pyruvate and acetate metabolism in the photosynthetic bacterium Rhodobacter capsulatus.. J Gen Microbiol 134:2429–2439
    [Google Scholar]
  45. Wood H. D., Utter M. F. 1965; The role of CO2 fixation in metabolism. In Essays in Biochemistry . 2:1–27 Edited by P. N. Campbell & G. D. Greville. New York: Academic Press.
    [Google Scholar]
  46. Young M. R., Tolbert B., Utter M. F. 1969; Pyruvate carboxylase from Saccharomyces cerevisiae.. Methods Enzymol 13:250–258
    [Google Scholar]
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