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.
Highlights
Lignocellulose from plants is the largest organic renewable resource on earth, but it is the most difficult to degrade due to its complex structure
We found that could the laccase activity andits transcription levels be regulated by external pH, and PacC, as an important transcription factor that responded to ambient pH (Wu et al 2016), had a negative effect on laccase activity in G. lucidum
Amino acid sequence analysis by DNAMAN indicated that four copper-binding sites existed in the conserved domains, and each domain had conserved histidine residues (Fig. 1). These results indicated that 15 laccase genes were identified in G. lucidum genome and the proteins encoded by these genes could be classified as typical laccases in G. lucidum
Summary
Lignocellulose from plants is the largest organic renewable resource on earth, but it is the most difficult to degrade due to its complex structure. Fungi, especially white-rot basidiomycetes, could effectively degrade plant biomass (Kües 2015). A class of the most important fungal enzymes from white-rot basidiomycetes, is considered to be vital for the degradation of plant biomass (Brijwani et al 2010). Laccase was first found in the Rhusvernificera, and later was found to be widespread in plants, bacteria and insects, especially in white-rot basidiomycetes. Like most fungal extracellular secreted enzymes, laccase is a type of glycoprotein, that can use many phenolic compounds as electron donors to catalyze the reduction of O2 to H2O. Based on its broad range of substrates, fungal laccases have been used as the potential enzymes in industrial applications, including environmental pollutant degradation, baking industry and fruit juice stabilization (Mayer and Staples 2002; Brijwani et al 2010). Fungal laccases function in many physiological events, such as fruiting body formation in Laccaria bicolor and Volvariella volvacea (Courty et al 2009; Chen et al 2004), conidial pigment biosynthesis in Aspergillus fumigatus (Tsai et al 1999), and pathogenicity in Cryptococcus neoformans (Missal et al 2005)
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