Molecular Cloning Of CDNA Gene Encoding A Manganese Peroxidase From Pseudotrametes Gibbosa And Heterologous Expression Of This Gene In Pichia Pastoris

Manganese peroxidase(MnP;EC1.11.1.13),a common lignin-degrading peroxidase,is a kind of heme glycoprotein enzyme which depend on the presence of H₂O₂.MnP occurs widely in white rot fungi.So far,the studies on MnP are mainly focused on its enzymatic characteristics in China,only limited researches are carried out on the molecular biology of MnP.MnP shows great potential in biological applications,due to its excellent capacity in degradation of lignin,polycyclic aromatic hydrocarbons(PAHs) and other xenobiotics.The investigation of the gene structure and heterologous expression of MnP would make a further understanding of the degradation mechanism and the regulation of mnp gene expression,which will have important significance in the construction of fine engineering strain.Pseudotrametes gibbosa is a white rot fungus belonging to the family of Polyporaceae.This fungus is commonly found in the forest of northeastern China.The cultural characteristics and production of laccase of Pseudotrametes gibbosa has been studied.However,research on MnP, the other main lignin-modifying enzyme of this fungus,has not been reported.In this paper, therefore,the activities of the lignin-modifying enzymes of Pseudotrametes gibbosa were determined,and the cDNA of its MnP was cloned and sequence analysed.The mnp gene was heterologously expressed in Pichia pastoris,and the recombinant MnP was successfully produced in an active form.The main research conclusions of the present work are listed as follows:1.The activities of manganese peroxidase,laccase and lignin peroxidase of Pseudotrametes gibbosa and Pleurotus djamor cultured in 4 different media were determined. Laccase activity but not MnP activity was found in the media not supplemented with Mn²⁺.In the medium supplemented with Mn²⁺ and enzyme substrates such as wood and 2,6-DMP,both MnP and laccase activities were detected in the culture supematant.Lignin peroxidase activity was not found in the supernatant of all tested media.The result demonstrated that the lignindegrading enzyme system of Pseudotrametes gibbosa and Pleurotus djamor contain both MnP and laccase,but lignin peroxidase was not produced by the two fungi.2.In comparison with the enzyme activities of 4 culture supernatant,the activities of MnP and laccase was remarkably enhanced by the addition of wood and 2,6-DMP into the medium, indicating that the production of the two lignin-degrading enzymes were inducible by suitable substrates.The two substrates had different effects on enzymatic activities,wood showed a better effect on the improvement of MnP and laccase activity than 2,6-DMP.Pseudotrametes gibbosa produced more laccase and MnP than Pleurotus djamor,the highest MnP and laccase activity of Pseudotrametes gibbosa were 165 and 445 U/L,respectively. 3.The cDNA of MnP was cloned from mycelium of Pseudotrametes gibbosa by degenerate PCR,RT-PCR and RACE,and was named as Pg-MnP.The gene contained a full open reading frame(ORF) of 1095 bp in length.The start codon was ATC,and the stop codon was TAA.The cDNA encoded a putative polypeptide of 364 amino acids.The length of 5' and 3' untranslating region was 51 and 160 nucleotides,respectively.The whole length cDNA of Pg-MnP had the highest homology with the MnP gene of Trametes versicolor(86%homology), and a homology of 68-80%was found with the MnP gene of Phlebia radiata,Spongipellis sp, Ceriporiopsis sp and Pleurotus ostreatus.A high sequence homology of 67%was also found with the gene sequence of lignin peroxidase from T.versicolor.4.The G+C content of Pg-MnP was 62.65%,and the A+T content was 37.35%.Among the amino acids composition,the highest content was Ala(12.05%),followed by Gly and Thr (8.22%),and no Tyr was found in the amino aicds of Pg-MnP.The predicted molecular mass of Pg-MnP was 38.24 kDa,and the pI was 4.49.There were 45 amino acid residues with negative charge(Asp+Glu) and 20 amino acid residues with positive charge(Arg+Lys).The putative protein contained 5311 atoms with a molecular formula of C₁₆₉₀H₂₆₁₉N₄₅₅O₅₃₆S₁₁.The protein was an unstable protein with a instability index of 49.13.The aliphatic index was 80.22 and the total average hydrophobicity is 0.010.5.The most probable splice site of signal peptide of Pg-MnP was predicted to be located between the 21st and 22nd amino acid,which was recognized as ANG-AA.The protein contained two hydrophobic regions which were located between the 30th～50th amino acid and 129th～147th amino acid.The transmembrane region was also predicted to be located in these regions.The secondary structure was mainly composed of random coil and a helix,with a percentage of 51.92%and 36.81%,respectively.There was only few 13 strands in the secondary structure.The result of subcellular location of Pg-MnP showed that this protein was mainly located extracellularly and in peroxisomes,seperating membrane and tubule of endoplasmic reticulum.The tertiary structure of Pg-MnP was predicted using SWISS-MODEL online and the structure was exported by Chimera.The functional sites of this protein was simulated using Cn3D,and the combiantional sites of Fe heme,calcium atom and manganese ions as well as the location of His residues were also predicted.6.The full CDS gene fragment of Pg-MnP was cloned by RT-PCR to be used for heterologous expression in Pichia pastoris.The objective gene of Pg-MnP was digested with restriction enzymes and ligated with the expression plasmid pPICZB.The recombinant plasmid pPICZB/MnP was transformed into P.pastoris by electroporation.The recombinant Pichia colonies was screened using the PCR method.The result demonstrated that the recombinant pPICZB/MnP had been successfully integrated into the genome of P.Pastoris.The heterologous MnP was produced under the induction of methanol.The biomass of P.pastoris was increased in 96 h after cultivation under inducible conditions.The highest MnP activity in the culture supernatant reached 7 U/L after 72-h incubation.The addition of heme into the culture medium had no effect on the productin of MnP activity.