The Preliminary Sduty On The Genetic Operation Of Phanerochaete Chrysosporium And Expression Of Manganese Peroxidase Gene In The Trichoderma Reesei

The white-rot basidiomycete Phanerochaete chrysosporium has oxidation and can degrade ligin with no substrate specificity, used as the type strain in the ligin degradation. P. chrysosporium also can degrade toxic aromatic pollutants and has potential on the bioremediation. There are more studies on characterization of P. chrysosporium on the ligin degradation and cellulose metabolism. But studies on the regulation and expression of these genes have many difficulties. Transformation of this fungus would be a major step toward use of this organism for industrial bioprocessing. Currently, auxotrophic marker strains of P. chrysosporium are used in the transformation. But transformation of the wild type strain of p. chrysosporium has been made little progress.In the paper, we describe the transformation of protoplast of the wild type strain ME-446and the haploid strain RP-78of P. chrysosporium. The optimum conditions of protoplast formation of P. chrysosporium are, as following, useing germinated spores to form protoplast and0.5mol/L MgSO4as osmotic stabilizer,30mg/ml Vinoflow FCE to digest spores, lμg/ml pyrithiamine cannot surpress the protoplasts. The minimum inhibitory concentration of Hygromycin B on protoplasts is670μg/ml and Basta on protoplasts of ME-446is4μg/ml, on protoplasts of RP-78is5μg/ml.With the Double-joint PCR, we construct PgpdA-Bar-Tmnp1gene expression cassette in which the PgpdA is from P. chrysosporium. With PCR, we construct hph gene expression cassette in which Ptrpc is from Aspergilliis nidulans and bar gene expression cassette in which PgpdA is also from A.nidulans. Then three expression cassettes are transformated into the protoplasts from P. chrysosporium. Transformants with hph gene expression cassette have not been obtained. We obtain three ME-446transformants with bar gene expression cassette. PCR analysis of these ME-446transformants have a success, but southern blot analysis failure. We suggest that the wild type strain ME-446is dioploid which maybe easy to drop the bar gene expression cassette. Manganese peroxidase (MnP) is one of P. chrysosporium extracellular enzymes involved in ligin degradation. MnP can degrade many substrates, which play an important role in many areas. But MnP from P. chrysosporium is not conducive to industrial applications. Heterologous expression of MnP from P. chrysosporium is a major step toward use of the MnP. Currently, MnPl has been successfully expressed in the Aspergillus niger and the Aspergillus oryzae, but MnP2has been given little attention to. Trichoderma reesei has been recognized as the industrial workhorse to produce cellulose several years.In the paper, we have successfully cloned the MnP2cDNA from the wild type strain ME-446of P. chrysosporium. With Double-joint PCR, we constaict mup2-pyrG gene expression cassette which is used to transform protoplasts from uracil auxotrophic strain Atku70of T. reesei. We have obtained nine T. reesei transformants which have intergaration of the nuip2-pyrG gene expression cassette at cbhl gene locus. The probability of this intergaration is60%. We have successfully got one T. reesei transformant K13-1with active MnP2. The maximum enzyme activity of MnP2from K13-1is1.35U/L.We have successfully got the optimum conditions of protoplast formation of P. chrysosporium. We have also studied the influence of Hygromycin B, pyrithiamine and Basta resistance gene on the transformation of P. chrysosporium. It is convenient to complete the transformation of P. chrysosporium. The T. reesei transformant with active MnP2has obtained, which provide a new example for studying the MnP2and improving the enzyme system of T. reesei.