Heterologous Expression In Aspergillus Nidulans Of Three Manganese Peroxidase Genes From Lenzites Gibbosa

The white rot fungi have a remarkable ability to degrade lignin. These enzymes mainly include manganese peroxidases,lignin peroxidases, and laccases. MnP have an attractive in biotechnological applications. It has a specile ability to degrade extracellular lignin and polycyclic aromatic hydrocarbons. Lenzites gibbosa is a white rot fungi which have strong decomposition of lignin in Northeast China Forest. In recent years, its cultural characteristics from Lenzites gibbosa, and its manganese peroxidase gene cloning and expression have been studied. Eukaryotic expression system is feasible and effective method to express protein. But the studies show that the manganese peroxidases of Lenzites gibbosa have lower expression in Pichia pastoris. Filamentous fungi genetic transformation system is established. Aspergillus nidulans have rapid growth, expression of large, high extracellular secretion, the exogenous protein having a natural activity. Aspergillus nidulans transformation method has been established and has been greatly developed. To explore a new way to increase the production of enzymes, we can study manganese peroxidase of white rot fungi transformation in Filamentous fungi. It has important significance and theoretical value for further building strains. This study constructed recombinant plasmids. By protoplast transformation method, the3MnP isozyme gene of Lenzites gibbosa was transformed into Aspergillus nidulans. Results this study obtained3MnP isozyme gene engineering strain of Aspergillus nidulans.The main research conclusions of the present study are as follows:The study firstly explored the methods how the conidiophores of auxotrophic stain TN02A7of Aspergillus nidulans were transformed to protoplasts by different cell wall lyases, results showed that lywallzyme,cellulase and snailase mixed together by1:1:1could effectively make the spores to become protoplasts.By RT-PCR, the complete manganese peroxidase (MnP) isozyme CDS gene Lg-mnpl, Lg-mnp2and Lg-mnp1were cloned from Lenzites gibbosa and heterologous expression in Aspergillus nidulans. Firstly, the vector plasmid pMDTM18-T/Lg-mnpl, pMDTM18-T/Lg-mnp2and pMDTM18-T/Lg-mnp3were constructed. Then, the recombinant plasmid pLB01/Lg-mnpl, pLB01/Lg-mnp2and pLB01/Lg-mnp3were constructed by ligating pMDTM18-T/Lg-mnpl, pMDTM18-T/Lg-mnp2, pMDTM18-T/Lg-mnp3and T4ligase integrated expression plasmid pLB01both digested by restriction enzymes, respectively. Then, by CaCl2PEG protoplast transformation method, Lg-mnpl, Lg-mnp2and Lg-mnp3were transferred to auxotrophic stain TN02A7of Aspergillus nidulans. The new tranformant stain TN02A7-Lg-mnpl, TN02A7-Lg-mnp2and TN02A7-Lg-mnp3, Auxotrophic stain TN02A7and Lenzites gibbosa CB1, were cultured in the same shaking medium containing lignin and detected the MnP activity in liquid MMPGRT, result showed that TN02A7-Lg-mnpl, TN02A7-Lg-mnp2and TN02A7-Lg-mnp3could produce MnP activity, the MnP high activity was up to26.7U·L-1,17U·L-1and12U·L-1on96h, but their MnP activity are less than Lenzites gibbosa CB1. TN02A7could not produce MnP activity at any time. In solution with heme, the MnP activity is roughly the same. The biomass of recombinant strains was increased over inducible time. This results show that the3MnP isozyme gene of Lenzites gibbosa was transformed into Aspergillus nidulans.