Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2.
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.
Tsukihara
Takahisa
T
Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto, 611-0011, Japan.
Honda
Yoichi
Y
Watanabe
Takahito
T
Watanabe
Takashi
T
D001580:Benzopyrenes / Q000378:metabolism; D002261:Carboxin / Q000494:pharmacology; D003470:Culture Media; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005659:Fungicides, Industrial / Q000494:pharmacology; D005822:Genetic Vectors; D010070:Oxalates; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000187:drug effects; D020076:Pleurotus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D001580:Benzopyrenes; D003470:Culture Media; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D010070:Oxalates; D011994:Recombinant Proteins; D002261:Carboxin; D010544:Peroxidases; C051129:manganese peroxidase