Cloning And Expression Of Lignin - Degrading Enzyme Related Genes In Ganoderma Lucidum

In nature, white-rot fungi were with the best ability to secrete extracellularligninolytic enzymes, and to degrade lignocellulosic biomass. The genus ofGanoderma was a large group of white-rot fungi. Therefore, the study of taxonomy ofthe genus Ganoderma, establishment of an effective method for screening theGanoderma strain with the best secreting ability of ligninolytic enzymes and cloningand analysis of the related genes will make an active role in screening for best strainsfrom white-rot fungi and application of these fungi on environmental protection.In the present study, we firstly analyzed genetic diversity among twentyGanoderma strains based on the gene sequences of fungal immunomodulatoryproteins (FIPs), the ITS1-5.8S-ITS2of nuclear ribosomal DNA genes and themitochondrial small subunit rDNA (mt SSU rDNA) genes. Secondly, we set up aneffective method for screening the Ganoderma strain with the best secreting abilityof laccase enzymes. Meanwhile, the genomic laccase sequences were clonedsuccessfully by the method of exon spicing from Ganoderma strains, and expressedthe gene in Pichia pastoris. Thirdly, we had cloned a gene encoding manganeseperoxidase (MnP) by the method of rapid amplification of cDNA ends (RACE) fromone Ganoderma strain and expressed the gene in Pichia pastoris.The results showed as following.①Among the selected marker genes,different gene had the different ability in the identification of Ganoderma;②In theexperiments of screening the Ganoderma strain with the best secreting ability oflaccase enzymes from four different Ganoderma strains, the best one was the G.lucidum00679. The full-length cDNA sequences of G. lucidum00679and G. sinensis50817laccase were1566bp and1563bp, encoded521and520amino acidsrespectively, and belonged to the copper oxidase super family. Further, the GlLacgene fragment of G. lucidum00679was subcloned to pPICZαA with double enzymedigestion and connection, constructing eukaryotic co-expression plasmidpPICZαA-GlLac. The recombinant plasmid was successfully transfected into a native yeast strain X-33by electrotrans formation.③The MnP gene was successfullyamplified from G. lucidum00679, which was1337bp, encoded363amino acids. Theencoded protein included a catalytic site of lignin oxidase, and was similar to plantperoxidase superfamily. Further, the GlMnP gene fragment was subcloned to pAO815with double enzyme digestion and connection, constructing eukaryotic co-expressionplasmid pAO815-GlMnP. The recombinant plasmid was transfected into a native yeaststrain SMD1168by electrotrans formation. The purified protein was showed to bebioactive.The conclusion of this study was presented as follows. Among the selectedmarker genes, the V4-V6regions sequences of mt SSU rRNA gene could distinguishdifferent strain within the genus Ganoderma. The cDNA sequence of laccase encoded520amino acids was cloned successfully from Ganoderma strain00679; the laccasebelonged to the copper oxidase superfamily. While the cDNA sequences of MnPencoded363amino acids was also cloned from the same strain, the MnP belonged tothe plant peroxidase superfamily. Both the Lac and MnP genes were successfullyexpressed in P. pastoris respectively. This study lays a foundation for furtherdegradation of lignocellulosic biomass and bioremediation of the environmentpollution.