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Deletion Analysis of GH7 Endoglucanase Gene (cel7B) Promoter Region in a Talaromyces cellulolyticus ligD-Disrupted Strain

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Abstract

Talaromyces cellulolyticus is expected to become an industrial cellulase producer. In this study, we performed deletion analysis of the promoter region of the GH7 endoglucanase gene (cel7B), which encodes one of the major cellulases, using a β-glucuronidase reporter system. To obtain strains that harbor each gene cassette at the same locus, we had to improve the homologous recombination frequency. Hence, the ligD gene, encoding DNA ligase IV, was disrupted by homologous recombination. After that, the introduced pyrF marker gene, encoding orotate phosphoribosyl transferase, was deleted by a marker recycling system. The resultant strain, YDLP, exhibits high homologous recombination frequency. These data suggest that this approach will drastically improve the genetic modification tools of T. cellulolyticus. We obtained 7 strains for reporter analysis using YDLP as the host strain. Reporter analysis revealed that the promoter region between −812 and −612 is important for expression of cel7B. These results imply a relationship between this region and novel transcriptional factors.

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References

  1. Yamanobe, T., Mitsuishi, Y., & Takasaki, Y. (1987). Isolation of a cellulolytic enzyme producing microorganism, culture conditions and some properties of the enzymes. Agricultural and Biological Chemistry, 51, 65–74.

    CAS  Google Scholar 

  2. Gusakov, A. V. (2011). Alternatives to Trichoderma reesei in biofuel production. Trends in Biotechnology, 29, 419–425.

    Article  CAS  Google Scholar 

  3. Yamanobe, T., Okuda, N., Oouchi, K., & Suzuki, K. (2003). Japanese patent, 2003–135052.

  4. Fang, X., Yano, S., Inoue, H., & Sawayama, S. (2009). Strain improvement of Acremonium cellulolyticus for cellulase production by mutation. Journal of Bioscience and Bioengineering, 107, 256–261.

    Article  CAS  Google Scholar 

  5. Fujii, T., Fang, X., Inoue, H., Murakami, K., & Sawayama, S. (2009). Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials. Biotechnology for Biofuels, 2, 24.

    Article  Google Scholar 

  6. Fujii, T., Inoue, H., & Ishikawa, K. (2013). Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus. AMB Express, 3, 73.

    Article  Google Scholar 

  7. Fujii, T., Inoue, H., & Ishikawa, K. (2014). Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). Bioscience, Biotechnology, and Biochemistry, 78, 1564–1567.

    Article  CAS  Google Scholar 

  8. Fujii, T., Inoue, H., & Ishikawa, K. (2015). Decreased cellulase and xylanase production in the fungus Talaromyces cellulolyticus by disruption of tacA and tctA genes, encoding putative zinc finger transcriptional factors. Applied Biochemistry and Biotechnology, 175, 3218–3229.

    Article  CAS  Google Scholar 

  9. Okuda, N., Fujii, T., Inoue, H., Ishikawa, K., & Hoshino, T. (2016). Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain. Bioscience, Biotechnology, and Biochemistry, 80, 2065–2068.

    Article  CAS  Google Scholar 

  10. Stricker, A. R., Mach, R. L., & de Graaff, L. H. (2008). Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei). Applied Microbiology and Biotechnology, 78, 211–220.

    Article  CAS  Google Scholar 

  11. Marui, J., Tanaka, A., Mimura, S., de Graaff, L. H., Visser, J., Kitamoto, N., Kato, M., Kobayashi, T., & Tsukagoshi, N. (2002). A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae. Fungal Genetics and Biology, 35, 157–169.

    Article  CAS  Google Scholar 

  12. Lockington, R. A., Rodbourn, L., Barnett, S., Carter, C. J., & Kelly, J. M. (2002). Regulation by carbon and nitrogen sources of a family of cellulases in Aspergillus nidulans. Fungal Genetics and Biology, 37, 190–196.

    Article  CAS  Google Scholar 

  13. Ilmén, M., Onnela, M. L., Klemsdal, S., Keränen, S., & Penttilä, M. (1996). Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei. Molecular & General Genetics, 253, 303–314.

    Google Scholar 

  14. Fujii, T., Iwata, K., Murakami, K., Yano, S., & Sawayama, S. (2012). Isolation of uracil auxotrophs of the fungus Acremonium cellulolyticus and the development of a transformation system with the pyrF gene. Bioscience, Biotechnology, and Biochemistry, 76, 245–249.

    Article  CAS  Google Scholar 

  15. Fujii, T., Koike, H., Sawayama, S., Yano, S., & Inoue, H. (2015). Draft genome sequence of Talaromyces cellulolyticus strain Y-94, a source of lignocellulosic biomass-degrading enzymes. Genome Announcements, 26, 3.

    Google Scholar 

  16. Kanna, M., Yano, S., Inoue, H., Fujii, T., & Sawayama, S. (2011). Enhancement of β-xylosidase productivity in cellulase producing fungus Acremonium cellulolyticus. AMB Express, 1, 15.

    Article  Google Scholar 

  17. Hayata, K., Asada, S., Fujii, T., Inoue, H., Ishikawa, K., & Sawayama, S. (2014). Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus. Applied Biochemistry and Biotechnology, 174, 1697–1704.

    Article  CAS  Google Scholar 

  18. Tani, S., Tsuji, A., Kunitake, E., Sumitani, J., & Kawaguchi, T. (2013). Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus aculeatus. AMB Express, 3(1), 4.

    Article  Google Scholar 

  19. Jiang, B., Zhang, R., Feng, D., Wang, F., Liu, K., Jiang, Y., Niu, K., Yuan, Q., Wang, M., Wang, H., Zhang, Y., & Fang, X. (2016). A tet-on and Cre-loxP based genetic engineering system for convenient recycling of selection markers in Penicillium oxalicum. Frontiers in Microbiology, 7, 485.

    Google Scholar 

  20. Bugeja, H. E., Boyce, K. J., Weerasinghe, H., Beard, S., Jeziorowski, A., Pasricha, S., Payne, M., Schreider, L., & Andrianopoulos, A. (2012). Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei. Fungal Genetics and Biology, 49, 772–778.

    Article  CAS  Google Scholar 

  21. Mizutani, O., Kudo, Y., Saito, A., Matsuura, T., Inoue, H., Abe, K., & Gomi, K. (2008). A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genetics and Biology, 45, 878–889.

    Article  CAS  Google Scholar 

  22. Inoue, H., Fujii, T., Yoshimi, M., Taylor 2nd, L. E., Decker, S. R., Kishishita, S., Nakabayashi, M., & Ishikawa, K. (2013). Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. Journal of Industrial Microbiology & Biotechnology, 40, 823–830.

    Article  CAS  Google Scholar 

  23. Jefferson, R. A. (1987). Plant reporter genes: the GUS gene fusion system. Plant Mol. Biol. Rep., 5, 387–405.

    Article  CAS  Google Scholar 

  24. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin-phenol reagent. The Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  25. Noguchi, Y., Tanaka, H., Kanamaru, K., Kato, M., & Kobayashi, T. (2011). Xylose triggers reversible phosphorylation of XlnR, the fungal transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus oryzae. Bioscience, Biotechnology, and Biochemistry, 75, 953–959.

    Article  CAS  Google Scholar 

  26. Cubero, B., & Scazzocchio, C. (1994). Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. The EMBO Journal, 13, 407–415.

    CAS  Google Scholar 

  27. Kato, M., Aoyama, A., Naruse, F., Tateyama, Y., Hayashi, K., Miyazaki, M., Papagiannopoulos, P., Davis, M. A., Hynes, M. J., Kobayashi, T., & Tsukagoshi, N. (1998). The Aspergillus nidulans CCAAT-binding factor AnCP/AnCF is a heteromeric protein analogous to the HAP complex of Saccharomyces cerevisiae. Molecular & General Genetics, 257, 404–411.

    Article  CAS  Google Scholar 

  28. Arst, H. N. J. (1968). Genetic analysis of the first steps of sulphate metabolism in Aspergillus nidulans. Nature, 219, 268–270.

    Article  CAS  Google Scholar 

  29. Daboussi, M., Djeballi, A., Gerlinger, C., Blaiseau, P., Bouvier, I., Cassan, M., Lebrun, M., Parisot, D., & Brygoo, Y. (1989). Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Current Genetics, 15, 453–456.

    Article  CAS  Google Scholar 

  30. Stricker, A., Grosstessner-Hain, K., Würleitner, E., & Mach, R. (2006). Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryotic Cell, 5, 2128–2137.

    Article  CAS  Google Scholar 

  31. van Peij, N., Gielkens, M., de Vries, R., Visser, J., & de Graaff, L. (1998). Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger. Applied and Environmental Microbiology, 64, 3615–3619.

    Google Scholar 

  32. Brakhage, A. A., Andrianopoulos, A., Kato, M., Steidl, S., Davis, M. A., Tsukagoshi, N., & Hynes, M. J. (1999). HAP-like CCAAT-binding complexes in filamentous fungi: implications for biotechnology. Fungal Genetics and Biology, 27, 243–252.

    Article  CAS  Google Scholar 

  33. 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. The Journal of Biological Chemistry, 275, 5817–5825.

    Article  CAS  Google Scholar 

  34. Li, D., Sirakova, T., Rogers, L., Ettinger, W. F., & Kolattukudy, P. E. (2002). Regulation of constitutively expressed and induced cutinase genes by different zinc finger transcription factors in Fusarium solani f. sp. pisi (Nectria haematococca). The Journal of Biological Chemistry, 277, 7905–7912.

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Dr. Katsuji Murakami, Dr. Masahiro Watanabe, Dr. Hironaga Akita, and Dr. Saori Kamachi of AIST for the helpful discussions. This study was supported by MEXT/JSPS KAKENHI Grant Number 26850058, Japan.

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Authors and Affiliations

  1. Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan

    Tatsuya Fujii, Hiroyuki Inoue & Tamotsu Hoshino

  2. Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan

    Kazuhiko Ishikawa

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  1. Tatsuya Fujii
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  2. Hiroyuki Inoue
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  4. Tamotsu Hoshino
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Correspondence to Tatsuya Fujii.

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Fujii, T., Inoue, H., Ishikawa, K. et al. Deletion Analysis of GH7 Endoglucanase Gene (cel7B) Promoter Region in a Talaromyces cellulolyticus ligD-Disrupted Strain. Appl Biochem Biotechnol 183, 1516–1525 (2017). https://doi.org/10.1007/s12010-017-2519-z

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  • DOI: https://doi.org/10.1007/s12010-017-2519-z

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