Advertisement

Molecular and General Genetics MGG

, Volume 241, Issue 3–4, pp 422–430 | Cite as

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

  • Gerold Barth
  • Thomas Scheuber
Original Articles
Received:
Accepted:

Abstract

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.

Key words

Glyoxylate cycle Fungus Yeast Peroxisome, glyoxysome Gene disruption 
This is a preview of subscription content, log in to check access.

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 107:262–266Google Scholar
  2. Ballance DJ, Turner G (1986) Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD). Mol Gen Genet 202:271–275Google Scholar
  3. Barth G (1985) Genetic regulation of isocitrate lyase in the yeast Yarrowia lipolytica. Curr Genet 10:119–124Google Scholar
  4. Barth G, Weber H (1983) Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis. Z Allg Mikrobiol 23:147–157Google Scholar
  5. Barth G, Weber H (1987) Genetic analysis of the gene ICL1 of the yeast Yarrowia lipolytica. Yeast 3:255–262Google Scholar
  6. Beeching JR, Northcote DH (1987) Nucleic acid (cDNA) and amino sequence of isocitrate lyase from castor bean. Plant Mol Biol 8:471–475Google Scholar
  7. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res 7:1513–1523Google Scholar
  8. Bolivar F, Rodriguez RL, Green PJ, Betlach HC, Heynecker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles. Gene 2:95–113Google Scholar
  9. Comai L, Dietrich RA, Maslyar DJ, Baden CS, Harada JJ (1989) Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase in Brassica napus L. Plant Cell 1:293–300Google Scholar
  10. Cryer DR, Eccleshall R, Marmur J (1975) Isolation of yeast DNA, Academic Press, BostonGoogle Scholar
  11. Davidow LS, Apostolakos D, O'Donnell MM, Proctor AR, Ogrydziak DM, Wing RA, Stasko I, DeZeeuw JR (1985) Integrative transformation of the yeast Yarrowia lipolytica. Curr Genet 10:39–48Google Scholar
  12. Dijekema C, Visser J (1987) 13C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism. Biochem Biophys Acta 931:311–319Google Scholar
  13. Domdey H, Apostol B, Lin R-J, Newman A, Brody E, Abelson J (1984) Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39:611–621Google Scholar
  14. El-Mansi EMT, MacKintosh C, Duncan K, Holms WH, Nimmo H (1987) Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308. Biochem J 242:661–665Google Scholar
  15. Fernandez E, Moreno F, Rodicio R (1992) The ICL1 gene from Saccharomyces cerevisiae. Eur J Biochem 204:983–990Google Scholar
  16. Fournier P, Guyaneux L, Chasles M, Gaillardin C (1991) Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica. Yeast 7:25–36Google Scholar
  17. Gainey LDS, Connerton IF, Lewis EH, Turner G, Ballance DJ (1992) Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu3). Curr Genet 21:43–47Google Scholar
  18. Gould SJ, Keller G-A, Schneider M, Howell SH, Garrard Lj, Goodman JM, Distel B, Tabak H, Subramani S (1990) Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J 9:85–90Google Scholar
  19. Johanson RA, Hill JM, McFadden BA (1974a) Isocitrate lyase from Neurospora crassa. I. Purification, kinetic mechanism, and interaction with inhibitors. Biochim Biophys Acta 364:327–340Google Scholar
  20. Johanson RA, Hill JM, McFadden BA (1974b) Isocitrate 1yase from Neurospora crassa. II. Composition, quaternary structure, C-terminus, and active-site modification. Biochim Biophys Acta 364:341–352Google Scholar
  21. Kujau M, Weber H, Barth G (1992) Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase. Yeast 8:193–203Google Scholar
  22. Kushner SR (1978) An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In: Boyer HB, Nicosia S (eds) Genetic engineering. Elsevier/North-Holland, Amsterdam, p 17Google Scholar
  23. Lopez-Boado YS, Herrero P, Fernandez M-T, Fernandez R, Moreno F (1988) Purification of isocitrate lyase from Saccharomyces cerevisiae. Yeast 4:41–46Google Scholar
  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  25. Maloy SR, Nunn WD (1982) Genetic regulation of the glyoxylate shunt in Escherichia coli. J Bacteriol 149:173–180Google Scholar
  26. Matsuoka M, McFadden BA (1988) Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli. J Bacteriol 170:4528–4536Google Scholar
  27. Matsuoka M, Himeno T, Aiba S (1984) Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase. J Bacteriol 157:899–908Google Scholar
  28. Mellon FM, Little PFR, Casselton LA (1987) Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7). Mol Gen Genet 210:352–357Google Scholar
  29. Nimmo HG, Douglas F, Kleanthous C, Campbell DG, MacKintosh C (1989) Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase. Biochem J 261:431–435Google Scholar
  30. Rieul C, Bleicher F, Duclos B, Cortay JC, Cozzone AJ (1988) Nucleotide sequence of the aceA gene coding for isocitrate lyase in Escherichia coli. Nucleic Acids Res 16:5689Google Scholar
  31. Robertson EF, Hoyt JC, Reeves HC (1988) Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. J Biol Chem 263:2477–2482Google Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  33. Turley RB, Choe SM, Trelease RN (1990) Gossypium hirsutum (upland cotton) isocitrate lyase. Unpublished sequence obtained from MIPSX databaseGoogle Scholar
  34. Vanni P, Giachetti E, Pintauti G, McFadden BA (1990) Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol 95B:431–458Google Scholar
  35. Zimmermann R, Neupert W (1980) Biogenesis of glyoxysomes. Synthesis and intracellular transfer of isocitrate lyase. Eur J Biochem 112:225–233Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Gerold Barth
    • 1
  • Thomas Scheuber
    • 1
  1. 1.Department of Microbiology, BiozentrumUniversity of BaselBaselSwitzerland

Personalised recommendations

Cite article

Buy options