Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2

Abstract

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.

Enhanced saccharification of corn straw pretreated by alkali combining crude ligninolytic enzymes

“…5 Pretreatment by white-rot fungi has been intensively investigated, 6 however, most wild white-rot fungi are dikaryonic, which are unstable in ligninolytic enzyme production and lignin degradation. 7 Monokaryons are relatively stable in the lignin degradation process, but its application was limited by long pretreatment time and low lignin degradation capacity. 8,9 Hence, pretreatment with ligninolytic enzyme might be an efficient strategy towards reducing treatment time.…”

Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4 , as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

“…Honda and coworkers (13,14) developed a protoplast-based polyethylene glycol and calcium salt (PEG-CaCl 2 )-mediated method for transformation and recombinant gene expression in P. ostreatus, based on the homologous drug-resistant marker cassette Cbx r , which confers dominant resistance to the systemic fungicide carboxin. This system has since been used for the homologous expression of recombinant Mn 2ϩ -dependent peroxidase (mnp3) and versatile-peroxidase (VP) (mnp2) genes (15,39). However, homologous recombination (HR) has been shown to occur only rarely, making gene knockout studies difficult (13)(14)(15)(16)31).…”

Phenanthrene Removal from Soil by a Strain of Aspergillus niger Producing Manganese Peroxidase of Phanerochaete chrysosporium

“…The results also confirmed that MnP production occurred in the transformants by introducing the mnp1 gene, since this activity was not detected in the wild-type strain. Differences observed between transformants are often explained by a differential integration of the heterologous gene within the fungal genome [33,55]. After 48 h, all transformant strains showed MnP activity in liquid medium.…”

“…It is therefore hypothesized that heterologous expression of genes codifying MnP and LiP in non-ligninolytic fungi will complement the degradation pathway of cytochrome P450 to obtain complete mineralization of the hydrocarbon without leaving more toxic intermediary compounds which accumulate in the soil (figure 1). Some studies on the homologous expression of peroxidases in ligninolytic fungi in submerged culture, have shown that a transformed Pleurotus ostreatus was better at removing recalcitrant pollutant than the wild-type strain [33]. Other researchers reported the development of a homologous expression system for MnP and LiP in P. chrysosporium.…”