Using a DNA-mediated transformation technique, a molecular breeding approach to isolate Pleurotus ostreatus strains with enhanced productivity of its versatile peroxidase MnP2 was conducted. A recombinant mnp2 construct under the control of P. ostreatus sdi1 expression signals was introduced into the wild-type P. ostreatus strain by cotransformation with a carboxin-resistant marker plasmid. A total of 32 transformants containing the recombinant mnp2 sequence were isolated in a screening with specific amplification by PCR. Productivity of MnP2 in the recombinants was evaluated by the decolorization ability of Poly R-478 on agar plates in the absence of Mn2+. Recombinant P. ostreatus strains with elevated manganese peroxidase (MnP) productivity were successfully isolated. One of the recombinants, TM2-10, was demonstrated to secrete recombinant MnP2 predominantly on a synthetic medium containing 15 mM ammonium oxalate, which was confirmed by reverse transcription PCR (RT-PCR) and isozyme profile analysis using anion-exchange chromatography. The benzo[a]pyrene-removing activity by fungal treatment was also analyzed using the isolated recombinant strains.

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Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn 2؉ and H 2 O 2 were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.Pleurotus ostreatus, known as the oyster mushroom, is a white rot basidiomycete that degrades plant cell wall lignin effectively (33). It secrets a series of isozymes of extracellular oxidizing enzymes belonging to manganese peroxidase (MnP)

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl 2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase ( hph ) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene ( gpd ) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

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