Rational design for fungal laccase production in the model host Aspergillus nidulans

Abstract

Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively. Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A. nidulans but also provide a general approach to applying regulatory methods to control gene expression.

This is a preview of subscription content, access via your institution.

References

  1. Ahuja, M., Chiang, Y.M., Chang, S.L., Praseuth, M.B., Entwistle, R., Sanchez, J.F., Lo, H.C., Yeh, H.H., Oakley, B.R., and Wang, C.C.C. (2012). Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. J Am Chem Soc 134, 8212–8221.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Aleksenko, A., and Clutterbuck, A.J. (1997). Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements. Fungal Genet Biol 21, 373–387.

    Article  CAS  PubMed  Google Scholar 

  3. Alves, A.M.C.R., Record, E., Lomascolo, A., Scholtmeijer, K., Asther, M., Wessels, J.G.H., and Wösten, H.A.B. (2004). Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus. Appl Environ Microbiol 70, 6379–6384.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Antošová, Z., and Sychrová, H. (2016). Yeast hosts for the production of recombinant laccases: a review. Mol Biotechnol 58, 93–116.

    Article  CAS  PubMed  Google Scholar 

  5. Baccile, J.A., Spraker, J.E., Le, H.H., Brandenburger, E., Gomez, C., Bok, J.W., Macheleidt, J., Brakhage, A.A., Hoffmeister, D., Keller, N.P., et al. (2016). Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus. Nat Chem Biol 12, 419–424.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Baldrian, P. (2006). Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30, 215–242.

    Article  CAS  PubMed  Google Scholar 

  7. Beauvais, A., Monod, M., Debeaupuis, J.P., Diaquin, M., Kobayashi, H., and Latgé, J.P. (1997). Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus. J Biol Chem 272, 6238–6244.

    Article  CAS  PubMed  Google Scholar 

  8. Benghazi, L., Record, E., Suárez, A., Gomez-Vidal, J.A., Martínez, J., and de la Rubia, T. (2014). Production of the Phanerochaete flavido-alba laccase in Aspergillus niger for synthetic dyes decolorization and biotransformation. World J Microbiol Biotechnol 30, 201–211.

    Article  CAS  PubMed  Google Scholar 

  9. Bok, J.W., and Keller, N.P. (2012). Fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis. Methods Mol Biol 944, 163–174.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Colao, M.C., Lupino, S., Garzillo, A.M., Buonocore, V., and Ruzzi, M. (2006). Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb Cell Fact 5, 31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gershenzon, N.I., and Ioshikhes, I.P. (2005). Promoter classifier: software package for promoter database analysis. Appl BioInf 4, 205–209.

    Article  CAS  Google Scholar 

  12. Hoopes, J.T., and Dean, J.F.D. (2004). Ferroxidase activity in a laccase-like multicopper oxidase from Liriodendron tulipifera. Plant Physiol Biochem 42, 27–33.

    Article  CAS  PubMed  Google Scholar 

  13. Iimura, Y., Sonoki, T., and Habe, H. (2018). Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae. Protein Express Purif 141, 39–43.

    Article  CAS  Google Scholar 

  14. Jin, F.J., Watanabe, T., Juvvadi, P.R., Maruyama, J., Arioka, M., and Kitamoto, K. (2007). Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae. Appl Microbiol Biotechnol 76, 1059–1068.

    Article  CAS  PubMed  Google Scholar 

  15. Kiiskinen, L.L., Kruus, K., Bailey, M., Ylösmäki, E., Siika-Aho, M., and Saloheimo, M. (2004). Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology 150, 3065–3074.

    Article  CAS  PubMed  Google Scholar 

  16. Kilaru, S., Hoegger, P.J., Majcherczyk, A., Burns, C., Shishido, K., Bailey, A., Foster, G.D., and Kües, U. (2006). Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Appl Microbiol Biotechnol 71, 200–210.

    Article  CAS  PubMed  Google Scholar 

  17. Kitamoto, N., Ono, N., and Yoshino-Yasuda, S. (2015). Construction of quintuple protease and double amylase gene deletant for heterologous protein production in Aspergillus oryzae KBN616. Food Sci Technol Res 21, 297–307.

    Article  CAS  Google Scholar 

  18. Kudanga, T., Nyanhongo, G.S., Guebitz, G.M., and Burton, S. (2011). Potential applications of laccase-mediated coupling and grafting reactions: a review. Enzyme Microbial Tech 48, 195–208.

    Article  CAS  Google Scholar 

  19. Larrondo, L.F., Avila, M., Salas, L., Cullen, D., and Vicuña, R. (2003). Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology 149, 1177–1182.

    Article  CAS  PubMed  Google Scholar 

  20. Liu, F., Yang, Y., and Zhou, Y. (2018). Polymerase delta in eukaryotes: how is it transiently exchanged with specialized DNA polymerases during translesion DNA synthesis? Curr Protein Pept Sci 19, 100–111.

    Article  CAS  PubMed  Google Scholar 

  21. Ma, Z., Li, W., Zhang, P., Lyu, H., Hu, Y., and Yin, W.B. (2018). Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans. Appl Microbiol Biotechnol 102, 297–304.

    Article  CAS  PubMed  Google Scholar 

  22. Majeau, J.A., Brar, S.K., and Tyagi, R.D. (2010). Laccases for removal of recalcitrant and emerging pollutants. Bioresource Tech 101, 2331–2350.

    Article  CAS  Google Scholar 

  23. Mander, G.J., Wang, H., Bodie, E., Wagner, J., Vienken, K., Vinuesa, C., Foster, C., Leeder, A.C., Allen, G., Hamill, V., et al. (2006). Use of laccase as a novel, versatile reporter system in filamentous fungi. Appl Environ Microbiol 72, 5020–5026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mayfield, M.B., Kishi, K., Alic, M., and Gold, M.H. (1994). Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium. Appl Environ Microbiol 60, 4303–4309.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Na, N., Huijun, T., Xinxin, S., and Jingfeng, N. (2017). Advance of heterologus expression study of eukaryote-origin laccases. Chin J Biotechnol 33, 565–577.

    Google Scholar 

  26. Nevalainen, K.M.H., Te’o, V.S.J., and Bergquist, P.L. (2005). Heterologous protein expression in filamentous fungi. Trends Biotech 23, 468–474.

    Article  CAS  Google Scholar 

  27. Nicolini, C., Bruzzese, D., Cambria, M.T., Bragazzi, N.L., and Pechkova, E. (2013). Recombinant laccase: I. Enzyme cloning and characterization. J Cell Biochem 114, 599–605.

    CAS  PubMed  Google Scholar 

  28. Otterbein, L., Record, E., Longhi, S., Asther, M., and Moukha, S. (2000). Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur J Biochem 267, 1619–1625.

    Article  CAS  PubMed  Google Scholar 

  29. Pezzella, C., Autore, F., Giardina, P., Piscitelli, A., Sannia, G., and Faraco, V. (2009). The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members. Curr Genet 55, 45–57.

    Article  CAS  PubMed  Google Scholar 

  30. Pezzella, C., Lettera, V., Piscitelli, A., Giardina, P., and Sannia, G. (2013). Transcriptional analysis of Pleurotus ostreatus laccase genes. Appl Microbiol Biotechnol 97, 705–717.

    Article  CAS  PubMed  Google Scholar 

  31. Record, E., Punt, P.J., Chamkha, M., Labat, M., van den Hondel, C.A.M.J. J., and Asther, M. (2002). Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem 269, 602–609.

    Article  CAS  PubMed  Google Scholar 

  32. Salony Garg, N., Baranwal, R., Chhabra, M., Mishra, S., Chaudhuri, T.K., and Bisaria, V.S. (2008). Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli. Biochim Biophys Acta 1784, 259–268.

    Article  CAS  PubMed  Google Scholar 

  33. Sigoillot, C., Record, E., Belle, V., Robert, J.L., Levasseur, A., Punt, P.J., van den Hondel, C.A.M.J.J., Fournel, A., Sigoillot, J.C., and Asther, M. (2004). Natural and recombinant fungal laccases for paper pulp bleaching. Appl Microbiol Biotechnol 64, 346–352.

    Article  CAS  PubMed  Google Scholar 

  34. Soden, D.M., and Dobson, A.D.W. (2003). The use of amplified flanking region-PCR in the isolation of laccase promoter sequences from the edible fungus Pleurotus sajor-caju. J Appl Microbiol 95, 553–562.

    Article  CAS  PubMed  Google Scholar 

  35. Soukup, A.A., Fischer, G.J., Luo, J., and Keller, N.P. (2017). The Aspergillus nidulans Pbp1 homolog is required for normal sexual development and secondary metabolism. Fungal Genet Biol 100, 13–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Staaden, S., Milcu, A., Rohlfs, M., and Scheu, S. (2010). Fungal toxins affect the fitness and stable isotope fractionation of Collembola. Soil Biol Biochem 42, 1766–1773.

    Article  CAS  Google Scholar 

  37. Van Dijk, J.W., and Wang, C.C. (2016). Heterologous expression of fungal secondary metabolite pathways in the Aspergillus nidulans host system. Methods Enzymol 575, 127–142.

    Article  CAS  PubMed  Google Scholar 

  38. van den Hombergh, J.P.T.W., van de Vondervoort, P.J.I., Fraissinet-Tachet, L., and Visser, J. (1997). Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotech 15, 256–263.

    Article  Google Scholar 

  39. Wang, Y., Xue, W., Sims, A.H., Zhao, C., Wang, A., Tang, G., Qin, J., and 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.

    Article  CAS  PubMed  Google Scholar 

  40. Yang, J., Ng, T.B., Lin, J., and Ye, X. (2015). A novel laccase from basidiomycete Cerrena sp.: cloning, heterologous expression, and characterization. Int J Biol Macromol 77, 344–349.

    Article  CAS  PubMed  Google Scholar 

  41. Yaropolov, A.I., Skorobogat’ko, O.V., Vartanov, S.S., and Varfolomeyev, S. D. (1994). Laccase—properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49, 257–280.

    Article  CAS  Google Scholar 

  42. Yin, W.B., Amaike, S., Wohlbach, D.J., Gasch, A.P., Chiang, Y.M., Wang, C.C.C., Bok, J.W., Rohlfs, M., and Keller, N.P. (2012). An Aspergillus nidulans bZIP response pathway hardwired for defensive secondary metabolism operates through aflR. Mol Microbiol 83, 1024–1034.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yin, W.B., Chooi, Y.H., Smith, A.R., Cacho, R.A., Hu, Y., White, T.C., and Tang, Y. (2013a). Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth Biol 2, 629–634.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yin, W.B., Reinke, A.W., Szilágyi, M., Emri, T., Chiang, Y.M., Keating, A. E., Pócsi, I., Wang, C.C.C., and Keller, N.P. (2013b). bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans. Microbiology 159, 77–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Yoon, J., Maruyama, J., and Kitamoto, K. (2011). Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89, 747–759.

    Article  CAS  PubMed  Google Scholar 

  46. Yu, J.H., Hamari, Z., Han, K.H., Seo, J.A., Reyes-Domínguez, Y., and Scazzocchio, C. (2004). Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41, 973–981.

    Article  CAS  PubMed  Google Scholar 

  47. Zhang, J., Qu, Y., Xiao, P., Wang, X., Wang, T., and He, F. (2012). Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2. J Biosci Bioeng 113, 697–703.

    Article  CAS  PubMed  Google Scholar 

  48. Zhang, P., Wang, X., Fan, A., Zheng, Y., Liu, X., Wang, S., Zou, H., Oakley, B.R., Keller, N.P., and Yin, W.B. (2017). A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici. Mol Microbiol 105, 469–483.

    Article  CAS  PubMed  Google Scholar 

  49. Zhang, S.Y., Zhao, C.B., Guo, L., and Li, W. (2015). Decolorization of Congo red with laccase from Pycnoporus sanguineus mk528 under mediator conditions. J Biol 32, 30–36.

    CAS  Google Scholar 

Download references

Acknowledgements

We thanked Dr. Wei Xue for his help to clone the promoter and terminator sequence of pslcc gene in Pycnoporus sanguineus mk528. This work was supported by Beijing Natural Science Foundation (5152018), the National Natural Science Foundation of China (31470178) and Wen-Bing Yin is a scholar of “the 100 Talents Project” of CAS.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Wen-Bing Yin.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, W., Yu, J., Li, Z. et al. Rational design for fungal laccase production in the model host Aspergillus nidulans. Sci. China Life Sci. 62, 84–94 (2019). https://doi.org/10.1007/s11427-017-9304-8

Download citation

Keywords

  • laccase
  • heterologous expression
  • promoter
  • Aspergillus nidulans