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Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

To investigate whether enzyme production can be enhanced in the Trichoderma reesei industrial hyperproducer strain RUT C30 by manipulation of cellulase regulation, the positive regulator Xyr1 was constitutively expressed under the control of the strong T. reesei pdc promoter, resulting in significantly enhanced cellulase activity in the transformant during growth on cellulose. In addition, constitutive expression of xyr1 combined with downregulation of the negative regulator encoding gene ace1 further increased cellulase and xylanase activities. Compared with RUT C30, the resulting transformant exhibited 103, 114, and 134 % greater total secreted protein levels, filter paper activity, and CMCase activity, respectively. Surprisingly, strong increases in xyr1 basal expression levels resulted in very high levels of CMCase activity during growth on glucose. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression, and suggest an attractive new single-step approach for increasing total cellulase productivity in T. reesei.

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References

  1. Aro N, Ilmén M, Saloheimo A, Penttilä M (2003) ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression. Appl Environ Microbiol 69(1):56–65

    Article  PubMed  CAS  Google Scholar 

  2. Aro N, Saloheimo A, Ilmén M, Penttilä M (2001) ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. J Biol Chem 276(26):24309–24314

    Article  PubMed  CAS  Google Scholar 

  3. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23(3):257–270

    Article  CAS  Google Scholar 

  4. Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18(5):355–383

    Article  PubMed  CAS  Google Scholar 

  5. Furukawa T, Shida Y, Kitagami N, Ota Y, Adachi M, Nakagawa S, Shimada R, Kato M, Kobayashi T, Okada H, Ogasawara W, Morikawa Y (2008) Identification of the cis-acting elements involved in regulation of xylanase III gene expression in Trichoderma reesei PC-3-7. Fungal Genet Biol 45(7):1094–1102

    Article  PubMed  CAS  Google Scholar 

  6. Ghose T (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268

    Article  CAS  Google Scholar 

  7. Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G (2006) Bio-ethanol: the fuel of tomorrow from the residues of today. Trends Biotechnol 24(12):549–556

    Article  PubMed  Google Scholar 

  8. Ilmén M, Saloheimo A, Onnela ML, Penttilä ME (1997) Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63(4):1298–1306

    PubMed  Google Scholar 

  9. Ilmén M, Thrane C, Penttilä M (1996) The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 251(4):451–460

    PubMed  Google Scholar 

  10. Kubicek CP, Mikus M, Schuster A, Schmoll M, Seiboth B (2009) Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina. Biotechnol Biofuels 2(19):1–14

    Google Scholar 

  11. Li J, Wang J, Wang S, Xing M, Yu S, Liu G (2012) Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters. Microb Cell Fact 11:84

    Google Scholar 

  12. Mach-Aigner AR, Pucher ME, Steiger MG, Bauer GE, Preis SJ, Mach RL (2008) Transcriptional regulation of xyr1, encoding the main regulator of the xylanolytic and cellulolytic enzyme system in Hypocrea jecorina. Appl Environ Microbiol 74(21):6554–6562

    Article  PubMed  CAS  Google Scholar 

  13. Mach RL, Zeilinger S (2003) Regulation of gene expression in industrial fungi: Trichoderma. Appl Microbiol Biotechnol 60(5):515–522

    PubMed  CAS  Google Scholar 

  14. Martinez D, Berka RM, Henrissat B et al (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 26(5):553–560

    Article  PubMed  CAS  Google Scholar 

  15. Nakari-Setälä T, Paloheimo M, Kallio J, Vehmaanperä J, Penttilä M, Saloheimo M (2009) Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production. Appl Environ Microbiol 75(14):4853–4860

    Article  PubMed  Google Scholar 

  16. Nemoto T, J-i Maruyama, Kitamoto K (2009) Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes. Biosci Biotechnol Biochem 73(11):2370–2373

    Article  PubMed  CAS  Google Scholar 

  17. Penttilä M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61(2):155–164

    Article  PubMed  Google Scholar 

  18. Peterson R, Nevalainen H (2012) Trichoderma reesei RUT-C30-thirty years of strain improvement. Microbiology 158:58–68

    Article  PubMed  CAS  Google Scholar 

  19. Portnoy T, Margeot A, Seidl-Seiboth V, Le Crom S, Ben Chaabane F, Linke R, Seiboth B, Kubicek CP (2011) Differential regulation of the cellulase transcription factors XYR1, ACE2 and ACE1 in high and low cellulase producing strains of Trichoderma reesei. Eukaryot Cell 10(2):262–271

    Article  PubMed  CAS  Google Scholar 

  20. Pucher ME, Steiger MG, Mach RL, Mach-Aigner AR (2011) A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation. Catal Today 167(1):122–128

    Article  CAS  Google Scholar 

  21. Rauscher R, Würleitner E, Wacenovsky C, Aro N, Stricker AR, Zeilinger S, Kubicek CP, Penttilä M, Mach RL (2006) Transcriptional regulation of xyn1, encoding xylanase I. Hypocrea jecorina. Eukaryot Cell 5(3):447–456

    Article  PubMed  CAS  Google Scholar 

  22. Saloheimo A, Aro N, Ilmén M, Penttilä M (2000) Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei. J Biol Chem 275(8):5817–5825

    Article  PubMed  CAS  Google Scholar 

  23. Seiboth B, Hartl L, Pail M, Fekete E, Karaffa L, Kubicek CP (2004) The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose. Mol Microbiol 51(4):1015–1025

    Article  PubMed  CAS  Google Scholar 

  24. Seiboth B, Karimi RA, Phatale PA, Linke R, Hartl L, Sauer DG, Smith KM, Baker SE, Freitag M, Kubicek CP (2012) The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei. Mol Microbiol 84(6):1150–1164

    Article  PubMed  CAS  Google Scholar 

  25. Steiger MG, Mach RL, Mach-Aigner AR (2010) An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei). J Biotechnol 145(1):30–37

    Article  PubMed  CAS  Google Scholar 

  26. Strauss J, Mach RL, Zeilinger S, Hartler G, Stöffler G, Wolschek M, Kubicek CP (1995) Cre1, the carbon catabolite repressor protein from Trichoderma reesei. FEBS Lett 376(1–2):103–107

    Article  PubMed  CAS  Google Scholar 

  27. Stricker AR, Grosstessner-Hain K, Würleitner E, Mach RL (2006) Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and d-xylose metabolism in Hypocrea jecorina. Eukaryot Cell 5(12):2128–2137

    Article  PubMed  CAS  Google Scholar 

  28. Stricker AR, Trefflinger P, Aro N, Penttilä M, Mach RL (2008) Role of Ace2 (Activator of Cellulases 2) within the xyn2 transcriptosome of Hypocrea jecorina. Fungal Genet Biol 45(4):436–445

    Article  PubMed  CAS  Google Scholar 

  29. Wang S-W, Xing M, Liu G, Yu S-W, Wang J, Tian S-L (2012) Improving cellulase production in Trichoderma koningii through RNA interference on ace1 gene expression. J Microbiol Biotechnol 22(8):1133–1140

    Article  PubMed  CAS  Google Scholar 

  30. Wilson DB (2009) Cellulases and biofuels. Curr Opin Biotechnol 20(3):295–299

    Article  PubMed  CAS  Google Scholar 

  31. Zeilinger S, Schmoll M, Pail M, Mach RL, Kubicek CP (2003) Nucleosome transactions on the Hypocrea jecorina (Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction. Mol Genet Genomics 270(1):46–55

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research was supported in part by the National Natural Science Foundation of China (No. 31070044), the Shenzhen Municipal Science and Technology Basic Research Program (JC201005280559A), and Shenzhen Municipal Science and Technology key projects of Basic Research Program (JC201005250041A, JCYJ20120613115323982).

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The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, 518060, China

    Shaowen Wang, Gang Liu, Juan Wang & Miao Xing

  2. School of Life Sciences, Northeast Normal University, Changchun, 130024, China

    Shaowen Wang & Baiqu Huang

  3. Department of Marine Biology, Shenzhen Key Laboratory of Marine Biotechnology and Ecology, Shenzhen, 518060, China

    Jianteng Yu

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  1. Shaowen Wang
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  2. Gang Liu
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  3. Juan Wang
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  4. Jianteng Yu
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  5. Baiqu Huang
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  6. Miao Xing
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Correspondence to Gang Liu or Miao Xing.

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Wang, S., Liu, G., Wang, J. et al. Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes. J Ind Microbiol Biotechnol 40, 633–641 (2013). https://doi.org/10.1007/s10295-013-1253-y

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  • DOI: https://doi.org/10.1007/s10295-013-1253-y

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