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Molecular and General Genetics MGG

, Volume 234, Issue 2, pp 332–336 | Cite as

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases

  • Ineke E. Mattern
  • Johannes M. van Noort
  • Paul van den Berg
  • David B. Archer
  • Ian N. Roberts
  • Cees A. M. J. J. van den Hondel
Short Communications

Summary

In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated. The major protease, which is responsible for 80–85% of the total activity, is aspergillopepsin A, a protein of ca. 43 kDa, the activity of which is inhibited by pepstatin. In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin. Several protease-deficient mutants were obtained by in vivo UV mutagenesis. In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy. In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted. In three UV-induced mutants, aspergillopepsin A is also missing. One of these mutants, AB 1.18, is mutated in the pepA gene, which is located on chromosome I. One of the other mutants, AB1.13, which has only 1–2 % of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B. The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene. Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII. Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.

Key words

Aspergillus niger Extracellular proteases Protease-deficient mutants Parasexual analysis Aspergillopepsin A Heterologous protein degradation 

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References

  1. Archer DB, Jeenes DJ, Mackenzie DA, Brightwell G, Lambert N, Lowe G, Radford SE, Dobson CM (1990) Hen egg white lysozyme expressed in, and secreted from, Aspergillus niger is correctly processed and folded. Biotechnology 8:741–745Google Scholar
  2. Barbesgaard P (1977) Industrial enzymes produced by members of the genus Aspergillus. In: Smith JE, Pateman JA (eds) Genetics and physiology of Aspergillus. Brit Mycol Soc Symp Series No 1, Academic Press, London pp 391–404Google Scholar
  3. Bennett JW (1985) Molds, manufacturing and molecular genetics. In: WE Timberlake (ed) Molecular genetics of filamentous fungi Alan R Liss, New York, pp 345–366Google Scholar
  4. Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and deletion of the aspergillopepsin A gene from Aspergillus awamori. Gene 86:153–162Google Scholar
  5. Bos CJ, Debets AIM, Swart K, Huybers A, Kobus G, Slakhorst SM (1988) Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14:437–443Google Scholar
  6. Cohen BL (1977) The proteases of Aspergilli. In: Smith JE and Pateman JA (eds) Genetics and physiology of Aspergillus. Academic Press, London, pp 281–292Google Scholar
  7. Debets AIM, Holub EF, Swart K, van den Broek HWJ, Bos CJ (1990) An electrophoretic karyotype of Aspergillus niger. Mol Gen Genet 224:264–268Google Scholar
  8. Pontecorvo G, Roper JA, Hemmons LM, MacDonald KD, Bufton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238Google Scholar
  9. Timberlake WE (1991) Cloning and analysis of fungal genes. In: Bennett JW, Lasure LL (eds) More gene manipulations in filamentous fungi. Academic Press, New York, pp 51–85Google Scholar
  10. Thompson SA (1991) Fungal aspartic proteinases. In: Leong SA, Berka RM (eds) Molecular industrial mycology: Systems and applications in filamentous fungi. Marcel Dekker, New York, pp 107–128Google Scholar
  11. Van den Hondel CAMJJ, Punt PJ, van Gorcom RFM (1991) Heterologous gene expression in filamentous fungi. In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, London, pp 396–428Google Scholar
  12. Van Hartingsveldt W, Mattern IE, Van Zeijl CMJ, Pouwels PH, Van den Hondel, CAMJJ (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mot Gen Genet 206:71–75Google Scholar
  13. Van Noort JM, van den Berg P, Mattern IE (1991) Visualization of proteases within a complex sample following their selective retention on immobilized bacitracin, a peptide antibiotic. Analyt Biochem 198:385–390Google Scholar
  14. Ward M (1991) Chymosin production in Aspergillus. In: Leong SA, Berka RM (eds) Molecular industrial mycology: Systems and applications in filamentous fungi. Marcel Dekker, New York, pp 83–105Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Ineke E. Mattern
    • 1
  • Johannes M. van Noort
    • 1
  • Paul van den Berg
    • 1
  • David B. Archer
    • 2
  • Ian N. Roberts
    • 2
  • Cees A. M. J. J. van den Hondel
    • 1
  1. 1.TNO Medical Biological LaboratoryRijswijkThe Netherlands
  2. 2.AFRC Institute of Food ResearchColneyUK

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