Advertisement

Archives of Microbiology

, Volume 156, Issue 6, pp 439–443 | Cite as

High level expression of isocitrate lyase gene of n-alkane-utilizing yeast Candida tropicalis in Sacchromyces cerevisiae

  • Keinosuke Oda
  • Haruyuki Atomi
  • Mitsuyoshi Ueda
  • Jun Kondo
  • Yutaka Teranishi
  • Atsuo Tanaka
Original Papers
  • 19 Downloads

Abstract

The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.

Key words

Candida tropicalis Saccharomyces cerevisiae Peroxisomes Isocitrate lyase GAL7 promoter High level expression 

Abbreviations

ICL

isocitrate lyase

SDS-PAGE

sodium dodecylsulfate-polyacrylamide gel electrophoresis

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Atomi H, Ueda M, Hikida M, Hishida T, Teranishi Y, Tanaka A (1990) Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization. J Biochem 107: 262–266CrossRefGoogle Scholar
  2. Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102–1106PubMedGoogle Scholar
  3. Frevert J, Kindl H (1978) Plant microbody proteins — Purification and glycoprotein nature of glyoxysomal isocitrate lyase from cucumber cotyledons. Eur J Biochem 92: 35–43CrossRefGoogle Scholar
  4. Gould SJ, Keller GA, Hosken N, Wilkinson J, Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108: 1657–1664CrossRefGoogle Scholar
  5. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153: 163–168PubMedPubMedCentralGoogle Scholar
  6. Lazarow PB, Fujiki Y (1985) Biogenesis of peroxisomes. Annu Rev Cell Biol 1: 489–530CrossRefGoogle Scholar
  7. Miyazawa S, Osumi T, Hashimoto T, Ohno K, Miura S, Fujiki Y (1989) Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase residues at the carboxy terminus. Mol Cell Biol 9: 83–91CrossRefGoogle Scholar
  8. Nabeshima S, Tanaka A, Fukui S (1977) Effect of carbon sources on the level of glyoxylate cycle enzymes in n-alkane-utilizable yeasts. Agric Biol Chem 41: 275–279Google Scholar
  9. Okada H, Ueda M, Sugaya T, Atomi H, Mozaffar S, Hishida T, Teranishi Y, Okazaki K, Takechi T, Kamiryo T, Tanaka A (1987) Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources. Eur J Biochem 170: 105–110CrossRefGoogle Scholar
  10. Osumi M, Miwa N, Teranishi Y, Tanaka A, Fukui S (1974) Ultrastructure of Candida yeasts grown on n-alkanes. Appearance of microbodies and its relationship to high catalase activity. Arch Microbiol 99: 181–201CrossRefGoogle Scholar
  11. Osumi M, Fukuzumi F, Teranishi Y, Tanaka A, Fukui S (1975) Development of microbodies in Candida tropicalis during incubation in a n-alkane medium. Arch Microbiol 103: 1–11CrossRefGoogle Scholar
  12. Shimada H, Fukasawa T, Ishimura Y (1989) Expression of bovine myoglobin cDNA as a functionally active holoprotein in Saccharomyces cerevisiae. J Biochem 105: 417–422CrossRefGoogle Scholar
  13. Tajima M, Nogi Y, Fukasawa T (1985) Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1: 67–77CrossRefGoogle Scholar
  14. Tajima M, Nogi Y, Fukasawa T (1986) Duplicate upstream activation sequence in the promoter region of the Saccharomyces cerevisiae GAL7 gene. Mol Cell Biol 6: 246–256CrossRefGoogle Scholar
  15. Tanaka A, Osumi M, Fukui S (1982) Peroxisomes of alkane-grown yeast. Fundamental and practical aspects. Ann N Y Acad Sci 386: 183–199CrossRefGoogle Scholar
  16. Uchida M, Ueda M, Matsuki T, Okada H, Tanaka A, Fukui S (1986) Properties of isocitrate lyase from an alkane-utilizable yeast, Candida tropicalis. Agric Biol Chem 50: 127–134Google Scholar
  17. Ueda M, Mozaffar S, Atomi H, Osumi M, Tanaka A (1989) Characterization of peroxisomes in an n-alkane-utilizable yeast, Candida tropicalis, grown on glucose and propionate. J Ferment Bioeng 68: 411–416CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Keinosuke Oda
    • 1
  • Haruyuki Atomi
    • 1
  • Mitsuyoshi Ueda
    • 1
  • Jun Kondo
    • 2
  • Yutaka Teranishi
    • 2
  • Atsuo Tanaka
    • 1
  1. 1.Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of EngineeringKyoto UniversityKyotoJapan
  2. 2.Research CenterMitsubishi Kasei CorporationYokohama, KanagawaJapan

Personalised recommendations