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Proteolytic analysis of Trichoderma reesei in celluase-inducing condition reveals a role for trichodermapepsin (TrAsP) in cellulase production

  • Bioenergy/Biofuels/Biochemicals - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Filamentous fungi produce a variety of proteases with significant biotechnological potential and show diverse substrate specificities. Proteolytic analysis of the industrial enzyme producer Trichoderma reesei has been sparse. Therefore, we determined the substrate specificity of T. reesei secretome and its main protease Trichodermapepsin (TrAsP) up to P1 position using FRETS-25Xaa-libraries. The role of TrAsP was analyzed using T. reesei QM9414 and the deletant QM∆trasp in Avicel. We observed higher activities of CMCase, Avicelase, and Xylanase in QM∆trasp compared to that of QM9414. Saccharification rate of cellulosic biomass also increased when using secretome of QM∆trasp but the effect was not significant due to the absence of difference in BGL activity compared to QM9414. Higher TrAsP was produced when monosaccharides were used as a carbon source compared to cellulase inducers such as Avicel and α-sophorose. These results elucidate the relationship between TrAsP and cellulase production in T. reesei and suggest a physiological role for TrAsP.

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References

  1. Ahmed ME (2018) Extraction and purification of protease from Aspergillus niger isolation. Pharm Pharmacol Int J 6:96–99. https://doi.org/10.15406/ppij.2018.06.00162

    Article  Google Scholar 

  2. Amore A, Giacobbe S, Faraco V (2013) Regulation of cellulase and hemicellulase gene expression in fungi. Curr Genom 14:230–249. https://doi.org/10.2174/1389202911314040002

    Article  CAS  Google Scholar 

  3. Braaksma M, Smilde AK, van der Werf MJ, Punt PJ (2009) The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger. Microbiology 155:3430–3439. https://doi.org/10.1099/mic.0.031062-0

    Article  CAS  PubMed  Google Scholar 

  4. de Castro RJS, Sato HH (2014) Protease from Aspergillus oryzae: biochemical characterization and application as a potential biocatalyst for production of protein hydrolysates with antioxidant activities. J Food Process 2014:1–11. https://doi.org/10.1155/2014/372352

    Article  Google Scholar 

  5. Delgado-Jarana J, Rincón AM, Benítez T (2002) Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization. Microbiology 148:1305–1315. https://doi.org/10.1099/00221287-148-5-1305

    Article  CAS  PubMed  Google Scholar 

  6. Eneyskaya EV, Kulminskaya AA, Shabalin KA et al (1999) Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases. Appl Microbiol Biotechnol 52:226–231. https://doi.org/10.1007/s002530051513

    Article  CAS  Google Scholar 

  7. Gente S, Billon-Grand G, Poussereau N, Févre M (2001) Ambient alkaline pH prevents maturation but not synthesis of ASPA, the aspartyl protease from Penicillium roqueforti. Curr Genet 38:323–328

    Article  CAS  PubMed  Google Scholar 

  8. Haab D, Hagspiel K, Szakmary K, Kubicek CP (1990) Formation of the extracellular proteases from Trichoderma reesei QM 9414 involved in cellulase degradation. J Biotechnol 16:187–198. https://doi.org/10.1016/0168-1656(90)90035-A

    Article  CAS  Google Scholar 

  9. Hagspiel K, Haab D, Kubicek C (1989) Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant. Appl Microbiol Biotechnol 32:61–67. https://doi.org/10.1007/BF00164824

    Article  CAS  Google Scholar 

  10. Hamin Neto YAA, da Rosa Garzon NG, Pedezzi R, Cabral H (2017) Specificity of peptidases secreted by filamentous fungi. Bioengineered 5979:1–8. https://doi.org/10.1080/21655979.2017.1373531

    Article  CAS  Google Scholar 

  11. Van Den Hombergh JPTW, Van De Vondervoort PJI, Fraissinet-Tachet L, Visser J (1997) Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 15:256–263. https://doi.org/10.1016/S0167-7799(97)01020-2

    Article  PubMed  Google Scholar 

  12. Kamitori S, Ohtaki A, Ino H, Takeuchi M (2003) Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9 Å resolution. J Mol Biol 326:1503–1511. https://doi.org/10.1016/S0022-2836(03)00078-0

    Article  CAS  PubMed  Google Scholar 

  13. Karaffa L, Fekete E, Gamauf C et al (2006) D-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates. Microbiology 152:1507–1514. https://doi.org/10.1099/mic.0.28719-0

    Article  CAS  PubMed  Google Scholar 

  14. Kawai T, Nakazawa H, Ida N et al (2012) Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts. J Ind Microbiol Biotechnol 39:1741–1749. https://doi.org/10.1007/s10295-012-1195-9

    Article  CAS  PubMed  Google Scholar 

  15. Kawamori M, Morikawa Y, Shinsha Y et al (1985) Preparation of mutants resistant to catabolite repression of Trichoderma reesei. Agric Biol Chem 49:2875–2879. https://doi.org/10.1080/00021369.1985.10867203

    Article  CAS  Google Scholar 

  16. Kubicek-Pranz EM, Gsur A, Hayn M, Kubicek CP (1991) Characterization of commercial Trichoderma reesei cellulase preparations by denaturing electrophoresis (SDS-PAGE) and immunostaining using monoclonal antibodies. Biotechnol Appl Biochem 14:317–323

    CAS  PubMed  Google Scholar 

  17. Landowski CP, Huuskonen A, Wahl R et al (2015) Enabling low cost biopharmaceuticals: a systematic approach to delete proteases from a well-known protein production host Trichoderma reesei. PLoS One 10:1–28. https://doi.org/10.1371/journal.pone.0134723

    Article  CAS  Google Scholar 

  18. Li Q, Yi L, Marek P, Iverson BL (2013) Commercial proteases: present and future. FEBS Lett 587:1155–1163. https://doi.org/10.1016/j.febslet.2012.12.019

    Article  CAS  PubMed  Google Scholar 

  19. Mach-Aigner AR, Pucher ME, Steiger MG et al (2008) Transcriptional regulation of xyr1, encoding the main regulator of the xylanolytic and cellulolytic enzyme system in Hypocrea jecorina. Appl Env Microbiol 74:6554–6562. https://doi.org/10.1128/AEM.01143-08

    Article  CAS  Google Scholar 

  20. Mandels M, Reese ET (1957) Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol 73:269–278. https://doi.org/10.1002/path.1700730133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mandels M, Parrish FW, Reese ET (1962) Sophorose as an inducer of cellulase in Trichoderma viride. J Bacteriol 83:400–408

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Mäntylä M, Paloheimo MSP (1998) Industrial mutants and recombinant strains of Trichoderma reesei. In: Christian P, Harman GE, Ondik KL, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor and Francis, London, pp 291–309

    Google Scholar 

  23. Margolles-Clark E, Hayes CK, Harman GE, Penttilä M (1996) Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei. Appl Env Microbiol 62:2145–2151

    CAS  Google Scholar 

  24. Mojzita D, Herold S, Metz B et al (2012) l- xylo -3-Hexulose reductase is the missing link in the oxidoreductive pathway for d-galactose catabolism in filamentous fungi. J Biol Chem 287:26010–26018. https://doi.org/10.1074/jbc.M112.372755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Moralejo FJ, Cardoza RE, Gutierrez S et al (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. https://doi.org/10.1128/AEM.68.7.3550-3559.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moreno-Mateos MA, Delgado-Jarana J, Codón AC, Benítez T (2007) pH and Pac1 control development and antifungal activity in Trichoderma harzianum. Fungal Genet Biol 44:1355–1367. https://doi.org/10.1016/j.fgb.2007.07.012

    Article  CAS  PubMed  Google Scholar 

  27. Muthukrishnan S, Mukilarasi K (2016) Industrial important protease screening and optimization from micro-fungal isolates of Ayyanar Falls Forest Samples, Rajapalalyam. World Appl Sci J 34:343–347. https://doi.org/10.5829/idosi.wasj.2016.34.3.23113

    Article  CAS  Google Scholar 

  28. Nascimento AS, Krauchenco S, Golubev AM et al (2008) Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A. J Mol Biol 382:763–778. https://doi.org/10.1016/j.jmb.2008.07.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nevalainen KMH, Te’o VSJ, Bergquist PL (2005) Heterologous protein expression in filamentous fungi. Trends Biotechnol 23:468–474. https://doi.org/10.1016/j.tibtech.2005.06.002

    Article  CAS  PubMed  Google Scholar 

  30. Peciulyte A, Anasontzis GE, Karlström K et al (2014) Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates. Fungal Genet Biol 72:64–72. https://doi.org/10.1016/j.fgb.2014.07.011

    Article  CAS  PubMed  Google Scholar 

  31. Pitts JE, Crawford MD, Nugent PG et al (1995) The three-dimensional X-ray crystal structure of the aspartic proteinase native to Trichoderma reesei complexed with a renin inhibitor CP-80794. Springer, Boston, pp 543–547

    Google Scholar 

  32. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Rauscher R, Wurleitner E, Wacenovsky C et al (2006) Transcriptional regulation of xyn1, encoding xylanase I, in Hypocrea jecorina. Eukaryot Cell 5:447–456. https://doi.org/10.1128/EC.5.3.447-456.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Siala R, Sellami-Kamoun A, Hajji M et al (2009) Extracellular acid protease from Aspergillus niger I1: purification and characterization. Afr J Biotechnol 8:4582–4589. https://doi.org/10.5897/AJB09.063

    Article  CAS  Google Scholar 

  35. da Silva RR, de Oliveira LCG, Juliano MA et al (2017) Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium. Food Chem 225:45–54. https://doi.org/10.1016/j.foodchem.2017.01.009

    Article  CAS  PubMed  Google Scholar 

  36. da Silva RR, Souto TB, de Oliveira TB et al (2016) Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei. J Ind Microbiol Biotechnol 43:1059–1069. https://doi.org/10.1007/s10295-016-1780-4

    Article  CAS  PubMed  Google Scholar 

  37. de Souza PM, de Assis Bittencourt ML, Caprara CC et al (2015) A biotechnology perspective of fungal proteases. Braz J Microbiol 46:337–346. https://doi.org/10.1590/S1517-838246220140359

    Article  PubMed  PubMed Central  Google Scholar 

  38. Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 139:761–769

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Tanskul S, Oda K, Oyama H et al (2003) Substrate specificity of alkaline serine proteinase isolated from photosynthetic bacterium, Rubrivivax gelatinosus KDDS1. Biochem Biophys Res Commun 309:547–551

    Article  CAS  PubMed  Google Scholar 

  40. Yu L-X, Gray BN, Rutzke CJ et al (2007) Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco. J Biotechnol 131:362–369. https://doi.org/10.1016/J.JBIOTEC.2007.07.942

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was partially supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO) Project (P16009). Furthermore, our sincere thanks go to Nobuyuki Homma; Nagaoka University of Technology, for contributing the substrate specificity and kinetic analysis.

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  1. Department of Bioengineering, Nagaoka University of Technology, 1603-1, Kamitomioka, Nagaoka, 940-2188, Japan

    Nayani Dhanushka Daranagama, Koki Shioya, Masahiro Yuki, Haruna Sato, Yuki Ohtaki, Yoshiyuki Suzuki, Yosuke Shida & Wataru Ogasawara

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  1. Nayani Dhanushka Daranagama
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Daranagama, N.D., Shioya, K., Yuki, M. et al. Proteolytic analysis of Trichoderma reesei in celluase-inducing condition reveals a role for trichodermapepsin (TrAsP) in cellulase production. J Ind Microbiol Biotechnol 46, 831–842 (2019). https://doi.org/10.1007/s10295-019-02155-9

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