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Applied Microbiology and Biotechnology

, Volume 34, Issue 5, pp 617–622 | Cite as

Amplification of three threonine biosynthesis genes inCorynebacterium glutamicum and its influence on carbon flux in different strains

  • Bernhard J. Eikmanns
  • Markus Metzger
  • Dieter Reinscheid
  • Manfred Kircher
  • Hermann Sahm
Applied Genetics and Regulation

Summary

The hom-thrB operon (homoserine dehydrogenase/homoserine kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13 032 and the homFBR (homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as homFBR-thrB operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of homoserine dehydrogenase, homoserine kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum, amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thrB resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of homFBR-thrB as well as of homFBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor homoserine, respectively. Overexpression of thrC alone or in combination with that of homFBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested.

Keywords

Threonine Recombinant Strain Carbon Flux Corynebacterium Shuttle Vector 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Bell SC, Turner JM (1976) Bacterial catabolism of threonine; threonine degradation initiated by l-threonine-NAD+ oxidoreductase. Biochem J 156:449–458Google Scholar
  2. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  3. 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 72:248–254Google Scholar
  4. 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 69:2110–2114Google Scholar
  5. Cremer J, Treptow C, Eggeling L, Sham H (1988) Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. J Gen Microbiol 134:3221–3229Google Scholar
  6. Follettie MT, Sinskey AJ (1986) Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene. J Bacteriol 167:695–702Google Scholar
  7. Follettie MT, Shin HK, Sinskey AJ (1988) Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci. Mol Microbiol 2:53–62Google Scholar
  8. Han KS, Archer JAC, Sinskey AJ (1990) The molecular structure of the Corynebacterium glutamicum threonine synthase gene. Mol Microbiol 4:1693–1702Google Scholar
  9. Ishida M, Yoshino E, Makihara R, Sato K, Enei H, Nakamori S (1989) Improvement of an l-threonine producer derived from Brevibacterium flavum using threonine operon of Escherichia coli K-12. Agric Biol Chem 53:2269–2271Google Scholar
  10. Katsumata R, Mizukami T, Kikuchi Y, Kino K (1986) Threonine production by the lysine producing strain of Corynebacterium glutamicum with amplified threonine biosynthetic operon. In: Alacevic M, Hranueli D, Toman Z (eds) Genetics of industrial microorganisms B. Pliva, Zagreb, pp 217–226Google Scholar
  11. Kinoshita S (1985) Glutamic acid bacteria. In: Demain AL, Solomon NA (eds) Biology of industrial microorganisms. Benjammin/Cummings Publishing Company, London, pp 115–142Google Scholar
  12. Kleemann A, Leuchtenberger W, Hoppe B, Tanner H (1985) Amino acids. In: Bartholomé E, Biekert E, Hellmann H (eds) Ullmann's encyclopedia of industrial chemistry A2. VCH-Verlagsgesellschaft, Weinheim, pp 57–97Google Scholar
  13. Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 63:607–613Google Scholar
  14. Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206Google Scholar
  15. Liebl W, Bayerl A, Schein B, Stillner U, Schleifer KH (1989) High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett 65:299–304Google Scholar
  16. Martin JF, Santamaria R, Sandoval H, Real G del, Mateos LM, Gil JA, Aguilar A (1987) Cloning system in amino acid producing corynebacteria. Bio Technology 5:137–146Google Scholar
  17. Mateos LM, Real G del, Aguilar A, Martin JF (1987a) Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum. Mol Gen Genet 206:361–367Google Scholar
  18. Mateos LM, Real G del, Aguilar A, Martin JF (1987b) Nucleotide sequence of the homoserine dehydrogenase (thrA) gene of Brevibacterium lactofermentum. Nucleic Acids Res 15:10598Google Scholar
  19. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.Google Scholar
  20. Miyajima R, Shiio I (1970) Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum; III. Properties of homoserine dehydrogenase. J Biochem 68:311–319Google Scholar
  21. Miyajima R, Shiio I (1971) Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum; IV. Repression of the enzymes in threonine biosynthesis. Agric Biol Chem 35:424–430Google Scholar
  22. Miyajima R, Otsuka SI, Shiio I (1968) Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum; I. Inhibition by amino acids of the enzymes in threonine biosynthesis. J Biochem 63:139–148Google Scholar
  23. Morinaga Y, Takagi H, Ishida M, Miwa K, Sato T, Nakamori S, Sano K (1987) Threonine production by coexistence of cloned genes coding homoserine dehydrogenase and homoserine kinase in Brevibacterium lactofermentum. Agric Biol Chem 51:93–100Google Scholar
  24. 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 2:53–62Google Scholar
  25. Sano K, Shiio I (1970) Microbial production of l-lysine. III. Production by mutants resistant to S-(2-aminoethyl)-l-cysteine. J Gen Appl Microbiol 16:373–391Google Scholar
  26. Shames SL, Wedler FC (1984) Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by l-aspartate semialdehyde. Arch Biochem Biophys 235:359–370Google Scholar
  27. Shiio I, Miyajima R (1969) Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem 65:849–859Google Scholar
  28. Shiio I, Miyajima R, Nakamori S (1970) Homoserine dehydrogenase genetically desensitized to the feedback inhibition in Brevibacterium flavum. J Biochem 68:859–866Google Scholar
  29. Skarstedt MT, Greer SB (1973) Threonine synthetase of Bacillus subtilis. J Biol Chem 248:1032–1044Google Scholar
  30. Theze J, Saint-Girons I (1974) Threonine locus of Escherichia coli K-12. Genetic structure and evidence for an operon. J Bacteriol 118:990–998Google Scholar
  31. Tosaka O, Ishihara M, Morinaga Y, Takinami K (1979) Mode of conversion of asparto β-semialdehyde to l-threonine and l-lysine in Brevibacterium lactofermentum. Agric Biol Chem 43:265–270Google Scholar
  32. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Bernhard J. Eikmanns
    • 1
  • Markus Metzger
    • 1
  • Dieter Reinscheid
    • 1
  • Manfred Kircher
    • 2
  • Hermann Sahm
    • 1
  1. 1.Institut für Biotechnologie 1 des Forschungszentrums Jülich GmbHJülichFederal Republic of Germany
  2. 2.Degussa AGHalle-KünsebeckFederal Republic of Germany

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