title: Marker recycling via 5-fluoroorotic acid and 5-fluorocytosine counter-selection in the white-rot agaricomycete Pleurotus ostreatus.
abstract: Of all of the natural polymers, lignin, an aromatic heteropolymer in plant secondary cell walls, is the most resistant to biological degradation. White-rot fungi are the only known organisms that can depolymerize or modify wood lignin. Investigating the mechanisms underlying lignin biodegradation by white-rot fungi would contribute to the ecofriendly utilization of woody biomass as renewable resources in the future. Efficient gene disruption, which is generally very challenging in the white-rot fungi, was established in Pleurotus ostreatus (the oyster mushroom). Some of the genes encoding manganese peroxidases, enzymes that are considered to be involved in lignin biodegradation, were disrupted separately, and the phenotype of each single-gene disruptant was analysed. However, it remains difficult to generate multi-gene disruptants in this fungus. Here we developed a new genetic transformation marker in P. ostreatus and demonstrated two marker recycling methods that use counter-selection to generate a multigene disruptant. This study will enable future genetic studies of white-rot fungi, and it will increase our understanding of the complicated mechanisms, which involve various enzymes, including lignin-degrading enzymes, underlying lignin biodegradation by these fungi.
authors: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan. Electronic address: tnakazaw@kais.kyoto-u.ac.jp.
authors: Takehito
authors: T
authors: Nakazawa
authors: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors: Masami
authors: M
authors: Tsuzuki
authors: Environmental Science Graduate School, The University of Shiga Prefecture, Hikone, Shiga, 522-8533, Japan.
authors: Toshikazu
authors: T
authors: Irie
authors: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors: Masahiro
authors: M
authors: Sakamoto
authors: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors: Yoichi
authors: Y
authors: Honda
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism*; D005437:Flucytosine / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods; D005819:Genetic Markers; D005827:Genetics, Microbial / Q000379:methods; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics; D020076:Pleurotus / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D005819:Genetic Markers; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D005437:Flucytosine; D010544:Peroxidases; C051129:manganese peroxidase
keywords: Basidiomycete; Mushroom; fcy1; pyrG; wood decay