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White-rot fungi, especially Trametes strains, are the primary source of industrial laccases in bioenergy and bioremediation. Trametes strains express members of the laccase gene family with different physicochemical properties and expression patterns. However, the literature on the expression pattern of the laccase gene family in Trametes trogii S0301 and the response mechanism to Cu(2+), a key laccase inducer, in white-rot fungal strains is scarce. In the present study, we found that Cu(2+) could induce the mRNAs and proteins of the two alternative splicing variants of heat shock transcription factor 2 (TtHSF2). Furthermore, the overexpression of alternative splicing variants TtHSF2α and TtHSF2β-I in the homokaryotic T. trogii S0301 strain showed opposite effects on the extracellular total laccase activity, with maximum laccase activities of approximately 0.6 and 3.0 U ml(−1), respectively, on day 8, which are 0.4 and 2.3 times that of the wild-type strain. Similarly, TtHSF2α and TtHSF2β-I play opposite roles in the oxidation tolerance to H(2)O(2). In addition, the direct binding of TtHSF2α to the promoter regions of the representative laccase isoenzymes (TtLac1 and TtLac13) and protein-protein interactions between TtHSF2α and TtHSF2β-I were detected. Our results demonstrate the crucial roles of TtHSF2 and its alternative splicing variants in response to Cu(2+). We believe that these findings will deepen our understanding of alternative splicing of heat shock transcription factors (HSFs) and their regulatory mechanism of the laccase gene family in white-rot fungi. IMPORTANCE The members of laccase gene family in Trametes strains are the primary source of industrial laccase and have gained widespread attention. Increasing the yield and enzymatic properties of laccase through various methods has always been a topic worthy of attention, and there is no report on the regulation of laccase expression through HSF transcription factor engineering. Here, we found that two alternative splicing variants of TtHSF2 functioned oppositely in regulating the expression of laccase genes, and copper can induce the expression of almost all members of the laccase gene family. Most importantly, our study suggested that TtHSF2 and its alternative splicing variants are vital for copper-induced production of laccases in T. trogii S0301.

Background Trametes trogii is a member of the white-rot fungi family, which has a unique ability to break down recalcitrant lignin polymers to CO2 and water, and they have enormous potential to biodegrade a wide range of toxic environmental pollutants. Because of its industrial potential, the identification of lignin-degrading enzyme systems in Trametes is an important area of research. Development and utilization of industrial value genes are suffering due to deficiency knowledge of genome available for their manipulation. Results In the present study, Homokaryotic strains of T. trogii S0301 were screened and sequencing by PacBio Sequel II platform. The final draft genome is ~ 39.88 Mb, with a contig N50 size of 2.4 Mb, this was the first genome sequencing and assembly of T. trogii species. Further analyses predicted 14,508 protein-coding genes. Results showed that T. trogii S0301 contains 602 genes encoding CAZymes, include 211 glycoside hydrolase and 117 lignin-degrading family genes, nine laccases related genes. Small subunit ribosomal RNA gene (18S rRNA) sequencing confirms its phylogenetic position. Moreover, T. trogii S0301 has the largest number of cytochromes P450 (CYPs) superfamily genes compare to other fungi. All these results are consistent with enzymatic assays and transcriptome analysis results. We also analyzed other genome characteristics in the T. trogii S0301genome. Conclusion Here, we present a nearly complete genome for T. trogii S0301, which will help elucidate the biosynthetic pathways of the lignin-degrading enzyme, advancing the discovery, characterization, and modification of novel enzymes from this genus. This genome sequence will provide a valuable reference for the investigation of lignin degradation in the Trametes genus.

Using response surface methodology, the maximum laccase activity of 122.9 U ml −1 was obtained in Trametes trogii S0301. The major isoform of the laccase secreted in the optimized medium was purified by 4-fold to a specific activity of 352.1 U mg −1 protein. The laccase (a molecular mass of 56 kDa), acted optimally at pH 3.0 and exhibited an optimum temperature of 45 °C using ABTS as substrate, with the half-life at 60 °C and 75 °C for 3 h and 10 min, respectively. The purified laccase was highly resistant to Co 2+ , Cu 2+ , Zn 2+ and Mn 2+ (100 mM). In addition, the purified laccase was effective to decolorize malachite green, bromophenol blue, crystal violet and acid red without the addition of redox mediators. Peptide-mass fingerprinting analysis by MALDI-TOF MS showed the purified laccase of T. trogii S0301 was a typical laccase isoform, which shared 99.3% identity with a laccase from Coriolopsis gallica . Further, the full-length DNA of the laccase was cloned based on the highly conservative copper-binding domains using degenerate PCR and TAIL-PCR, and the deduced amino acid sequence of the matured protein matched exactly with the peptides of the purified laccase.