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Improvement of laccase activity by silencing PacC in Ganoderma lucidum

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Abstract

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5–5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC. Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

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References

  • Barda O, Maor U, Sadhasivam S, Bi Y, Zakin V, Prusky D, Sionov E (2020) The pH-responsive transcription factor PacC governs pathogenicity and ochratoxin a biosynthesis in Aspergillus carbonarius. Front Microbiol 11:210

    Article  PubMed  PubMed Central  Google Scholar 

  • Brijwani K, Rigdon A, Vadlani PV (2010) Fungal laccases: production, function, and applications in food processing. Enzyme Res 2010:1–10

    Article  Google Scholar 

  • Chen S, Ge W, Buswell JA (2004) Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: possible involvement in fruit body development. FEMS Microbiol Lett 230:171–176

    Article  CAS  PubMed  Google Scholar 

  • Chen S, Xu J, Liu C et al (2012) Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat Commun 3:913

    Article  PubMed  Google Scholar 

  • Courty PE, Hoegger PJ, Kilaru S, Kohler A, Buee M, Garbaye J, Martin F, Kües U (2009) Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor. New Phytol 182:736–750

    Article  CAS  PubMed  Google Scholar 

  • de Souza CG, Tychanowicz GK, de Souza DF, Peralta RM (2004) Production of laccase isoforms by Pleurotus pulmonarius in response to presence of phenolic and aromatic compounds. J Basic Microbiol 44:129–136

    Article  PubMed  Google Scholar 

  • Díez E, Alvaro J, Espeso EA, Rainbow L, Suárez T, Tilburn J, Arst HN Jr, Peñalva MA (2002) Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps. EMBO J 21:1350–2135

    Article  PubMed  PubMed Central  Google Scholar 

  • Elisashvili V, Kachlishvili E, Khardziani T, Agathos SN (2010) Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes. J Ind Microbiol Biotechnol 37:1091–1096

    Article  CAS  PubMed  Google Scholar 

  • Fang QH, Zhong JJ (2002) Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum. Biotechnol Prog 18:51–54

    Article  CAS  PubMed  Google Scholar 

  • Fang F, Zhang XL, Luo HH, Zhou JJ, Gong YH, Li WJ, Shi ZW, He Q, Wu Q, Li L, Jiang LL, Cai ZG, Oren-Shamir M, Zhang ZQ, Pang XQ (2015a) An intracellular laccase is responsible for epicatechin-mediated anthocyanin degradation in Litchi Fruit Pericarp. Plant Physiol 169:2391–2408

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fang ZM, Liu XM, Chen LY, Shen Y, Zhang XC, Fang W, Wang XT, Bao XM, **ao YZ (2015b) Identification of a laccase Glac15 from Ganoderma lucidum 77002 and its application in bioethanol production. Biotechnol Biofuels 8:54

    Article  PubMed  PubMed Central  Google Scholar 

  • Fonseca MI, Shimizu E, Zapata PD, Villalba LL (2010) Copper inducing effect on laccase production of white rot fungi native from Misiones (Argentina). Enzyme Microb Technol 46:534–539

    Article  CAS  PubMed  Google Scholar 

  • Galhaup C, Haltrich D (2001) Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper. Appl Microbiol Biotechnol 56:225–232

    Article  CAS  PubMed  Google Scholar 

  • Galhaup C, Goller S, Peterbauer CK, Strauss J, Haltrich D (2002) Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148:2159–2169

    Article  CAS  PubMed  Google Scholar 

  • Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, Sannia G (1999) Protein and gene structure of a blue laccase from Pleurotus ostreatus. Biochem J 34:655–663

    Article  Google Scholar 

  • Gunther H, Perner B, Gramss G (1998) Activities of phenol oxidizing enzymes of ectomycorrhizal fungi in axenic culture and in symbiosis with Scots pine (Pinus sylvestris L.). J Basic Microbiol 38:197–206

    Article  Google Scholar 

  • Hoegger PJ, Navarro-González M, Kilaru S, Hoffmann M, Westbrook ED, Kües U (2004) The laccase gene family in Coprinopsis cinerea (Coprinus cinereus). Curr Genet 45:9–18

    Article  CAS  PubMed  Google Scholar 

  • Huang W, Shang YF, Chen PL, Gao Q, Wang CS (2015) MrPacC regulates sporulation, insect cuticle penetration and immune evasion in Metarhizium robertsii. Environ Microbiol 17:994–1008

    Article  CAS  PubMed  Google Scholar 

  • Janusz G, Rogalski J, Szczodrak J (2007) Increased production of laccase by Cerrena unicolor in submerged liquid cultures. World J Microbiol Biotechnol 23:1459–1464

    Article  CAS  Google Scholar 

  • Jiao X, Li G, Wang Y, Nie F, Cheng X, Abdullah M, Lin Y, Cai Y (2018) Systematic analysis of the Pleurotus ostreatus laccase gene (PoLac) family and functional characterization of PoLac2 involved in the degradation of cotton-straw lignin. Molecules 23:880

    Article  PubMed Central  Google Scholar 

  • Keyser P, Kirk TK, Zeikus J (1978) Ligninolytic enzyme system of Phanaerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J Bacteriol 135:790–797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kilaru S, Hoegger PJ, Kües U (2006) The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies. Curr Genet 50:45–60

    Article  CAS  PubMed  Google Scholar 

  • Kim HM, Park MK, Yun JW (2006) Culture pH affects exopolysaccharide production in submerged mycelial culture of Ganoderma lucidum. Appl Biochem Biotechnol 134:249–262

    Article  CAS  PubMed  Google Scholar 

  • Ko EM, Leem YE, Choi HT (2001) Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum. Appl Microbiol Biotechnol 57:98–102

    Article  CAS  PubMed  Google Scholar 

  • Kües U (2015) Fungal enzymes for environmental management. Curr Opin Biotechnol 33:268–278

    Article  PubMed  Google Scholar 

  • Kurose T, Saito Y, Kimata K, Nakagawa Y, Yano A, Ito K, Kawarasaki Y (2014) Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein. J Biosci Bioeng 117:659–663

    Article  CAS  PubMed  Google Scholar 

  • Levin L, Melignani E, Ramos AM (2010) Effect of nitrogen sources and vitamins on ligninolytic enzyme production by some white-rot fungi. Dye decolorization by selected culture filtrates. Bioresour Technol 101:4554–4563

    Article  CAS  PubMed  Google Scholar 

  • Litvintseva AP, Henson JM (2002) Cloning, characterization, and transcription of three laccase genes from gaeumannomyces graminis var. tritici, the take-all fungus. Appl Environ Microbiol 68:1305–1311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu YH, Huang L, Guo W, Jia LB, Fu Y, Gui S, Lu FP (2017) Cloning, expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization. Process Biochem 53:125–134

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Mayer AM, Staples RC (2002) Laccase: new functions for old enzyme. Phytochemistry 60:551–565

    Article  CAS  PubMed  Google Scholar 

  • Missal TA, Moran JM, Corbett JA, Lodge JK (2005) Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1. Eukaryo Cell 4:202–208

    Article  Google Scholar 

  • Miyara I, Shafran H, Kramer Haimovich H, Rollins J, Sherman A, Prusky D (2008) Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits. Mol Plant Pathol 9:281–291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nobile CJ, Nobile CJ, Solis N, Myers CL, Fray AJ, Deneault JS, Nantel A, Mitichell AP, Filler S (2008) Candida albicans transcription factor Rim101 mediates pathogenic interaction through cell wall functions. Cell Microbiol 10:2180–2196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Palmieri G, Giardina P, Bianco C, Fontanella B, Sannia G (2000) Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl Environ Microbiol 66:920–924

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peñalva MA, Arst HN Jr (2004) Recent advances in the characterization of ambient pH regulation of gene expression in filamentous fungi and yeasts. Annu Rev Microbiol 58:425–451

    Article  PubMed  Google Scholar 

  • Peñalva MA, Tilburn J, Bignell E, Arst HN Jr (2008) Ambient pH gene regulation in fungi: making connections. Trends Microbiol 16:291–300

    Article  PubMed  Google Scholar 

  • Piscitelli A, Giardina P, Lettera V, Pezzella C, Sannia G, Faraco V (2011) Induction and transcriptional regulation of laccases in fungi. Curr Genomics 12:104–112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schneider P, Caspersen MB, Mondorf K, Halkier T, Skov LK, Østergaard PR, Brown KM, Brown SH, Xu F (1999) Characterization of a Coprinus cinereus laccase. Enzyme Microb Technol 25:502–508

    Article  CAS  Google Scholar 

  • Sitarz AK, Mikkelsen JD, Hojrup P, Meyer AS (2013) Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation. Enzyme Microb Technol 53:378–385

    Article  CAS  PubMed  Google Scholar 

  • Tsai HF, Wheeler MH, Chang YC, Kwon-Chung KJ (1999) A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus. J Bacteriol 181:6469–6477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang W, Liu F, Jiang Y, Wu G, Guo L, Chen R, Chen B, Lu Y, Dai Y, **e B (2015) The multigene family of fungal laccases and their expression in the white rot basidiomycete Flammulina velutipes. Gene 563:142–149

    Article  CAS  PubMed  Google Scholar 

  • Wu FL, Zhang G, Ren A, Dang ZH, Shi L, Jiang AL, Zhao MW (2016) The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynthesis in Ganoderma lucidum. Mycologia 108:1104–1113

    CAS  PubMed  Google Scholar 

  • **ao YZ, Hong YZ, Li JF, Hang J, Tong PG, Fang W, Zhou CZ (2006) Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl Microbiol Biotechnol 71:493–501

    Article  CAS  PubMed  Google Scholar 

  • Zhang TY, Sun XP, Xu Q, Candelas LG, Li HY (2013) The pH signaling transcription factor PacC is required for full virulence in Penicillium digitatum. Appl Microbiol Biotechnol 97:9087–9098

    Article  CAS  PubMed  Google Scholar 

  • Zhou XW, Cong WR, Su KQ, Zhang YM (2013) Ligninolytic enzymes from Ganoderma spp: current status and potential applications. Crit Rev Microbiol 39:416–426

    Article  CAS  PubMed  Google Scholar 

  • Zhu J, Ying SH, Feng MG (2016) The Pal pathway required for ambient pH adaptation regulates growth, conidiation, and osmotolerance of Beauveria bassiana in a pH-dependent manner. Appl Microbiol Biotechnol 100:4423–4433

    Article  CAS  PubMed  Google Scholar 

  • Zhu J, Wu F, Yue S, Chen C, Song S, Wang H, Zhao M (2019) Functions of reactive oxygen species in apoptosis and ganoderic acid biosynthesis in Ganoderma lucidum. FEMS Microbiol Lett 366:fnaa015

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was financially supported by the Natural Science Foundation of Jiangsu Province (Project No. BK20180535), Fundamental Research Funds for the Central Universities (Project No. KJQN202058 and KYYZ202002) and Postdoctoral Research Foundation of China (Project No. 2017M611835).

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  1. Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nan**g Agricultural University, Nan**g, 210095, Jiangsu, P. R. China

    **g Zhu, Shuqi Song, Lindan Lian, Liang Shi, Ang Ren & Mingwen Zhao

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Zhu, J., Song, S., Lian, L. et al. Improvement of laccase activity by silencing PacC in Ganoderma lucidum. World J Microbiol Biotechnol 38, 32 (2022). https://doi.org/10.1007/s11274-021-03216-x

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