1887

Abstract

Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of for protein production.

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2009-10-01
2020-01-21
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References

  1. Bennett, J. W. & Lasure, L. L. ( 1991; ). Growth media. In More Gene Manipulations in Fungi, pp. 441–447. Edited by J. W. Bennett & L. L. Lasure. San Diego, CA: Academic Press.
  2. Berka, R. M., Ward, M., Wilson, L. J., Hayenga, K. J., Kodama, K. H., Carlomagno, L. P. & Thompson, S. A. ( 1990; ). Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86, 153–162.[CrossRef]
    [Google Scholar]
  3. Bos, C. J., Debets, A. J. M., Swart, K., Huybers, A., Kobus, G. & Slakhorst, S. M. ( 1988; ). Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14, 437–443.[CrossRef]
    [Google Scholar]
  4. Braaksma, M. & Punt, P. J. ( 2008; ). Aspergillus as a cell factory for protein production: controlling protease activity in fungal production. In The Aspergilli: Genomics, Medical Aspects, Biotechnology, and Research Methods, pp. 441–455. Edited by G. H. Goldman & S. A. Osmani. Boca Raton, FL: CRC Press.
  5. Buxton, F. P., Gwynne, D. I. & Davies, R. W. ( 1985; ). Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans. Gene 37, 207–214.[CrossRef]
    [Google Scholar]
  6. Cohen, B. L. ( 1972; ). Ammonium repression of extracellular protease in Aspergillus nidulans. J Gen Microbiol 71, 293–299.[CrossRef]
    [Google Scholar]
  7. Cohen, B. L. ( 1981; ). Regulation of protease production in Aspergillus. Trans Br Mycol Soc 76, 447–450.[CrossRef]
    [Google Scholar]
  8. de Vries, R. P., Burgers, K., van de Vondervoort, P. J., Frisvad, J. C., Samson, R. A. & Visser, J. ( 2004; ). A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production. Appl Environ Microbiol 70, 3954–3959.[CrossRef]
    [Google Scholar]
  9. Eilers, P. H. C. ( 2003; ). A perfect smoother. Anal Chem 75, 3631–3636.[CrossRef]
    [Google Scholar]
  10. Heerikhuisen, M., van den Hondel, C. A. M. J. J. & Punt, P. J. ( 2005; ). Aspergillus sojae. In Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems, pp. 191–214. Edited by G. Gelissen. Weinheim: Wiley.
  11. Holm, K. A. ( 1980; ). Automated colorimetric determination of acid proteinase activity in fermentation samples using a trinitrobenzenesulphonic acid reagent. Analyst 105, 18–24.[CrossRef]
    [Google Scholar]
  12. Iimura, Y., Gomi, K., Uzu, H. & Hara, S. ( 1987; ). Transformation of Aspergillus oryzae through plasmid-mediated complementation of the methionine-auxotrophic mutation. Agric Biol Chem 51, 323–328.[CrossRef]
    [Google Scholar]
  13. Jarai, G. & Buxton, F. ( 1994; ). Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger. Curr Genet 26, 238–244.[CrossRef]
    [Google Scholar]
  14. Katz, M. E., Flynn, P. K., vanKuyk, P. A. & Cheetham, B. F. ( 1996; ). Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 250, 715–724.
    [Google Scholar]
  15. Katz, M. E., Bernardo, S. M. & Cheetham, B. F. ( 2008; ). The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: Evidence for a role for CreA in the response to carbon starvation. Curr Genet 54, 47–55.[CrossRef]
    [Google Scholar]
  16. Kelly, J. M. & Hynes, M. J. ( 1985; ). Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4, 475–479.
    [Google Scholar]
  17. Kennedy, M. & Krouse, D. ( 1999; ). Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biotechnol 23, 456–475.[CrossRef]
    [Google Scholar]
  18. Li, Q., Harvey, L. M. & McNeil, B. ( 2008; ). The effects of bioprocess parameters on extracellular proteases in a recombinant Aspergillus niger B1-D. Appl Microbiol Biotechnol 78, 333–341.[CrossRef]
    [Google Scholar]
  19. Lin, Y., Means, G. E. & Feeney, R. E. ( 1969; ). The action of proteolytic enzymes on N,N-dimethyl proteins. Basis for a microassay for proteolytic enzymes. J Biol Chem 244, 789–793.
    [Google Scholar]
  20. Liu, F., Li, W., Ridgway, D., Gu, T. & Moo-Young, M. ( 1998; ). Inhibition of extracellular protease secretion by Aspergillus niger using cell immobilization. Biotechnol Lett 20, 539–542.[CrossRef]
    [Google Scholar]
  21. Lundstedt, T., Seifert, E., Abramo, L., Thelin, B., Nyström, Å., Pettersen, J. & Bergman, R. ( 1998; ). Experimental design and optimization. Chemom Intell Lab Syst 42, 3–40.[CrossRef]
    [Google Scholar]
  22. Mattern, I. E., van Noort, J. M., van den Berg, P., Archer, D. B., Roberts, I. N. & van den Hondel, C. A. ( 1992; ). Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet 234, 332–336.[CrossRef]
    [Google Scholar]
  23. Moralejo, F. J., Cardoza, R. E., Gutiérrez, S., Sisniega, H., Faus, I. & Martín, J. F. ( 2000; ). Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the pepA gene. Appl Microbiol Biotechnol 54, 772–777.[CrossRef]
    [Google Scholar]
  24. Moralejo, F. J., Cardoza, R. E., Gutiérrez, S., Lombraňa, M., Fierro, F. & Martín, J. F. ( 2002; ). Silencing of the aspergillopepsin B (pepB) gene of Aspergillus awamori by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production. Appl Environ Microbiol 68, 3550–3559.[CrossRef]
    [Google Scholar]
  25. O'Donnell, D., Wang, L., Xu, J., Ridgway, D., Gu, T. & Moo-Young, M. ( 2001; ). Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity. Biochem Eng J 8, 187–193.[CrossRef]
    [Google Scholar]
  26. Papagianni, M. & Moo-Young, M. ( 2002; ). Protease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects. Process Biochem 37, 1271–1278.[CrossRef]
    [Google Scholar]
  27. Papagianni, M., Joshi, N. & Moo-Young, M. ( 2002; ). Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures. J Ind Microbiol Biotechnol 29, 259–263.[CrossRef]
    [Google Scholar]
  28. Pel, H. J., de Winde, J. H., Archer, D. B., Dyer, P. S., Hofmann, G., Schaap, P. J., Turner, G., de Vries, R. P., Albang, R. & other authors ( 2007; ). Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25, 221–231.[CrossRef]
    [Google Scholar]
  29. Punt, P. J., Schuren, F. H. J., Lehmbeck, J., Christensen, T., Hjort, C. & van den Hondel, C. A. M. J. J. ( 2008; ). Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes. Fungal Genet Biol 45, 1591–1599.[CrossRef]
    [Google Scholar]
  30. Sumner, J. B. & Somers, G. F. ( 1949; ). Dinitrosalicylic method for glucose. In Laboratory Experiments in Biological Chemistry, pp. 38–39. Edited by J. B. Sumner & G. F. Somers. New York: Academic Press.
  31. Unkles, S. E., Campbell, E. I., de Ruiter-Jacobs, Y. M. J. T., Broekhuijsen, M., Macro, J. A., Carrez, D., Contreras, R., van den Hondel, C. A. M. J. J. & Kinghorn, J. R. ( 1989; ). The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: a convenient and general selection system for filamentous fungal transformation. Mol Gen Genet 218, 99–104.[CrossRef]
    [Google Scholar]
  32. van den Hombergh, J. P. T. W., Jarai, G., Buxton, F. P. & Visser, J. ( 1994; ). Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger. Gene 151, 73–79.[CrossRef]
    [Google Scholar]
  33. van den Hombergh, J. P. T. W., van de Vondervoort, P. J. I., van der Heijden, N. C. B. A. & Visser, J. ( 1995; ). New protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target proteins; genetical and biochemical characterization of seven complementation groups. Curr Genet 28, 299–308.[CrossRef]
    [Google Scholar]
  34. van den Hombergh, J. P. T. W., Sollewijn-Gelpke, M. D., van de Vondervoort, P. J. I., Buxton, F. P. & Visser, J. ( 1997a; ). Disruption of three acid proteases in Aspergillus niger: effects on protease spectrum, intracellular proteolysis, and degradation of target proteins. Eur J Biochem 247, 605–613.[CrossRef]
    [Google Scholar]
  35. van den Hombergh, J. P., van de Vondervoort, P. J., Fraissinet-Tachet, L. & Visser, J. ( 1997b; ). Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 15, 256–263.[CrossRef]
    [Google Scholar]
  36. van Hartingsveldt, W., Mattern, I. E., van Zeijl, C. M. J., Pouwels, P. H. & van den Hondel, C. A. M. J. J. ( 1987; ). Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206, 71–75.[CrossRef]
    [Google Scholar]
  37. van Noort, J. M., van den Berg, P. & Mattern, I. E. ( 1991; ). Visualization of proteases within a complex sample following their selective retention on immobilized bacitracin, a peptide antibiotic. Anal Biochem 198, 385–390.[CrossRef]
    [Google Scholar]
  38. Wang, Y., Xue, W., Sims, A. H., Zhao, C., Wang, A., Tang, G., Qin, J. & Wang, H. ( 2008; ). Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression. Fungal Genet Biol 45, 17–27.[CrossRef]
    [Google Scholar]
  39. Weatherburn, M. W. ( 1967; ). Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39, 971–974.[CrossRef]
    [Google Scholar]
  40. Wiebe, M. G. ( 2003; ). Stable production of recombinant proteins in filamentous fungi – problems and improvements. Mycologist 17, 140–144.[CrossRef]
    [Google Scholar]
  41. Wiebe, M. G., Karandikar, A., Robson, G. D., Trinci, A. P. J., Candia, J.-L. F., Trappe, S., Wallis, G., Rinas, U., Derkx, P. M. F. & other authors ( 2001; ). Production of tissue plasminogen activator (t-PA) in Aspergillus niger. Biotechnol Bioeng 76, 164–174.[CrossRef]
    [Google Scholar]
  42. Xu, J. F., Wang, L. P., Ridgway, D., Gu, T. Y. & Moo-Young, M. ( 2000; ). Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth. Biotechnol Prog 16, 222–227.[CrossRef]
    [Google Scholar]
  43. Yoder, W. T. & Lembeck, J. ( 2004; ). Heterologous expression and protein secretion in filamentous fungi. In Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, pp. 201–219. Edited by J. S. Tkacz & L. Lange. New York: Kluwer Academic/Plenum Publishers.
  44. Zheng, X. F., Kobayashi, Y. & Takeuchi, M. ( 1998; ). Construction of a low-serine-type-carboxypeptidase-producing mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion. Appl Microbiol Biotechnol 49, 39–44.[CrossRef]
    [Google Scholar]
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vol. , part 10, pp. 3430-3439

Comprehensive ANOVA data for each of the six protease-related phenotypes are available hereas an Acrobat PDF file.



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