Advertisement

Molecular and General Genetics MGG

, Volume 250, Issue 1, pp 97–105 | Cite as

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

  • Patrick Jara
  • Sophie Gilbert
  • Pascal Delmas
  • Jean-Claude Guillemot
  • Mourad Kaghad
  • Pascual Ferrara
  • Gérard Loison
Original Paper

Abstract

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA+)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Key words

Cryphonectria parasitica Acid proteinase eapB/C genes Gene disruption Heterologous protein degradation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Archer DB, MacKenzie DA, Jeenes DJ, Roberts IN (1992) Proteolytic degradation of heterologous proteins expressed inAspergillus niger. Biotechnol Lett 14:357–362Google Scholar
  2. Aronson BD, Lindgren KM, Dunlap JC, Loros JJ (1994) An efficient method for gene disruption inNeurospora crassa. Mol Gen Genet 242:490–494PubMedGoogle Scholar
  3. Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and deletion of the gene encoding Aspergillopepsin A fromAspergillus awamori. Gene 86:153–162PubMedGoogle Scholar
  4. Choi GH, Nuss DL (1992) A viral gene confers hypovirulence-associated traits to the chestnut blight fungus. EMBO J 11:473–477PubMedGoogle Scholar
  5. Churchill ACL, Ciuffetti LM, Hansen DR, Van Etten HD, Van Allen NK (1990) Transformation of the fungal pathogenCryphonectria parasitica with a variety of heterologous plasmids. Curr Genet 17:25–31Google Scholar
  6. Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987) Controlled expression and secretion of bovine chymosin inAspergillus nidulans. Bio/Technology 5:369–376Google Scholar
  7. Hanahan D (1983) Studies on transformation ofEscherichia coli with plasmids. J Mol Biol 166:557PubMedGoogle Scholar
  8. Harkki A, Uusitalo J, Bailey M, Penttilä M, Knowles JKC (1989) A novel fungal expression system: secretion of active calf chymosin from the filamentous fungusTrichoderma reesei. Bio/Technology 7:596–603Google Scholar
  9. von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690PubMedGoogle Scholar
  10. Inoue H, Kimura T, Makabe O, Takahashi K (1991) The gene and deduced protein sequences of the zymogen ofAspergillus niger acid proteinase A. J Biol Chem 29:19484–19489Google Scholar
  11. Jarai G, van den Hombergh H, Buxton FP (1994) Cloning and characterization of thepepE gene ofAspergillus niger encoding a new aspartic protease and regulation ofpepE andpepC. Gene 145:171–178PubMedGoogle Scholar
  12. Jara P, Delmas P, Razanamparany V, Olsen L, Dupin P, Bayol A, Bégueret J, Loison G (1995) Self-cloning in filamentous fungi: application to the construction of endothiapepsin overproducers inCryphonectria parasitica. J Biotechnol 40:111–120PubMedGoogle Scholar
  13. Jaton-Ogay K, Suter M, Crameri R, Falchetto R, Fathi A, Monod M (1992) Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease ofAspergillus fumigatus. FEMS Microbiol Lett 14:917–928Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227:680–685PubMedGoogle Scholar
  15. Maita T, Nagata S, Matsuda G, Maruta S, Oda K, Murao G, Tsuru D (1984) Complete amino acid sequence ofScytalidium lignicolum acid protease B. J Biochem 95:465–475PubMedGoogle Scholar
  16. Mattern IE, van Noort JM, van den Berg P, Archer DB, Roberts IN, van den Hondel CAMJJ (1992) Isolation and characterization of mutants ofAspergillus niger deficient in extracellular proteases. Mol Gen Genet 234:332–336PubMedGoogle Scholar
  17. Monod M, Paris S, Sarfati J, Jaton-Ogay K, Ave P, Latgé JP (1993) Virulence of alkaline protease-deficient mutants ofAspergillus fumigatus FEMS Microbiol Lett 106:39–46PubMedGoogle Scholar
  18. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant ofNeurospora crassa and its use as a dominant selected marker. Mol Cell Biol 6:2452–2461PubMedGoogle Scholar
  19. Osiewacz HD (1994) A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes. Curr Genet 26:87–90PubMedGoogle Scholar
  20. Raeder U, Broda PA (1985) A rapid method for the purification of fungal DNA. Lett Appl Microbiol 1:17–20Google Scholar
  21. Razanamparany V, Jara P, Legoux R, Delmas P, Msayek F, Kaghad M, Loison G (1992) Cloning and mutation of the gene encoding endothiapepsin fromCryphonectria parasitica. Curr Genet 21:455–461PubMedGoogle Scholar
  22. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) Ingel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179PubMedGoogle Scholar
  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  24. Samples DR, Richter RL, Dill CW (1984) Measuring proteolysis in cheddar cheese slurries: comparison of Hull and trinitrobenzene sulfonic acid procedures. J Dairy Sci 67:60–63Google Scholar
  25. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibition. Proc Nat Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  26. Sardinas JL (1966) Milk-curdling enzyme elaborated byEndothia parasitica. US Patent 3:275–453Google Scholar
  27. Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79:544–552PubMedGoogle Scholar
  28. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517PubMedGoogle Scholar
  29. Stemmer WPC, Morris SK (1992) Enzymatic inverse PCR: a restriction site-independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 13:215–220Google Scholar
  30. Takahashi K, Inoue H, Sakai K, Kohama T, Kitahara S, Takishima K, Tanji M, Athauda SBP, Takahashi T, Akanuma H, Mamiya G, Yamasaki M (1991) The primary structure ofAspergillus niger acid proteinase A. J Biol Chem 266:19480–19483PubMedGoogle Scholar
  31. Tsuchiya K, Gomi K, Kitamoto K, Kumagai C, Tamura G (1993) Secretion of calf chymosin from the filamentous fungusAspergillus oryzae. Appl Microbiol Biotechnol 40:327–332PubMedGoogle Scholar
  32. Unkles SE (1992) Gene organisation in industrial filamentous fungi. In: Kinghorn JR, Turner G (eds) Applied molecular genetics of filamentous fungi. Blackie, London, pp 28–53Google Scholar
  33. Ward M (1989) Production of calf chymosin byAspergillus awamori. In: Hershberger CL, Queener SW, Hegeman G (eds) Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington DC, pp 288–294Google Scholar
  34. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains. Nucleotides sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Patrick Jara
    • 1
  • Sophie Gilbert
    • 1
  • Pascal Delmas
    • 1
  • Jean-Claude Guillemot
    • 1
  • Mourad Kaghad
    • 1
  • Pascual Ferrara
    • 1
  • Gérard Loison
    • 1
  1. 1.Centre de Labège, Labège-InnopoleSanofi RechercheLabege CédexFrance

Personalised recommendations