Transcriptional Regulation And Functional Analysis Of Laccase Gene From White Rot Fungi

White rot fungi can efficiently degrade different natural lignocellulose and synthetic chemicals non-specifically depend on a series of ligninolytic enzymes. Laccase, a group of copper-containing polyphenol oxidases, is an important ligninolytic enzyme responsible for the lignin degradation by white rot fungi. Due to its wide range of substrates and unique biodegradability, laccase has great application values and potentialities in lignin degradation and the substrate acquisition for biomass energy, xenobiotics degradation, dye decolorization, delignified in paper pulping as well as food processing. Therefore, acquisition of laccase gene and studying its biological functions and expression regulation are very important for enriching the theory and technique of molecular biology of white rot fungi. Based on the importance of laccase, this dissertation focuses on the study of molecular biology of laccase from white rot fungi. The main results are described as following:By using the degenerate PCR, thermal asymmetric interlaced PCR (TAIL-PCR) and RT-PCR, the laccase gene lac48424-1and its corresponding full-length cDNA were cloned and characterized from Trametes sp.48424which had the high yield of laccase and strong ability for decolorizing different dyes. The5’-flanking sequence upstream of start codon was obtained by Self-Formed Adaptor PCR (SEFA-PCR). Many putative cis-acting responsive elements which may be involved in the transcriptional regulation were predicted in the promoter region of Iac48424-1. The functionality of lac48424-1gene encoding active laccase was verified by expressing this gene in the yeast Pichia pastoris successfully.The recombinant laccase rLac48424-1was purified from the yeast transformant in which the laccase gene lac48424-1was expressed successfully. The enzymology property of rLac48424-1and its ability to degrade different dyes and simulated dye effluents were studied. The results indicated that rLac48424-1had excellent enzymology property. Compared with some other laccases reported previously, rLac48424-1showed better thermal stability and pH stability. rLac48424-1also exhibited strong tolerance to different organic solvents such as acetonitrile, acetone, methanol and ethanol. The recombinant laccase rLac48424-1had stronger capacity for decolorizing different dyes with different structure and simulated dye effluents compared with some other known laccases. The mediator syringaldehyde could improve the ability to degrade the more recalcitrant dyes by rLac48424-1.The biological function and mechanism of laccase involved in the defense against the oxidative stress were deeply studied by the heterologous expression of laccase gene in Pichia pastoris. It was found that the expression of laccase gene from white rot fungus in Pichia pastoris could significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. Further research suggested that the transcription of the laccase gene in Pichia pastoris could be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system. The expression of the laccase gene in Pichia pastoris could enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system, including the intracellular glutathione content and the enzymatic activity of glutathione peroxidase, glutathione reductase, and y-glutamylcysteine synthetase.A series of inducers were designed according to the different cis-acting responsive elements in the promoter region of lac48424-1, such as MRE (metal-responsive element) and XRE (xenobiotic-responsive element). The regulation of transcription of laccase gene lac48424-1in Trametes sp.48424by these different inducers and environment factors was studied. The results suggested that different metal ions, many kinds of aromatic compounds, different lignins could stimulate the transcription of laccase gene lac48424-1. The cis-acting elements in the promoter region of laccase gene may be involved in the regulation of laccase gene transcription by different factors.In conclusion, in this study, we not only expressed the laccase gene from white rot fungi in the yeast Pichia pastoris successfully and verified the correct function of laccase gene encoding the active laccase protein, but also found that the heterologous expression of laccase gene in Pichia pastoris could enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-dependent antioxidative system. The prediction of cis-acting responsive elements in the promoter region of laccase gene and the results suggesting that the transcription of lac48424-1gene could be regulated by different factors will lay a solid foundation for revealing the regulatory mechanism of laccase expression in the further research.