Molecular breeding of white rot basidiomycete for efficient saccharification in biomass conversion process

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A versatile peroxidase MnP2, secreted by a white rot fungus Pleurotus ostreatus was characterized for its unique property of high molecular weight compounds oxidizing activity. A series of mutant MnP2 were produced by P. ostreatus transformants containing a recombinant mnp2 with site-directed base substitutions. Each mutant protein was purified to homogeneity and characterized with kinetics and reactivity for low- and high-molecular weight substrates, and spectroscopic methods. All variants assayed in this work showed substantially similar kinetic properties for Mn^<2+> and H_2O_2 to those of wild type MnP2. On the other hand, it was demonstrated that substitution of an exposing tryptophan residue (W170) to alanine lost oxidation activity for veratrylacohol (VA), Poly R-478 and Rnase A. Furthermore, substitution of the surrounding amino add residues affected oxidation of the high-molecular weight compounds but VA. From these results, it was clearly demonstrated that MnP2 oxidizes high-molecular weight compounds directly via long range electron transfer pathway from W170 to heme moiety. An extra modification of carbohydrate drain in a mutant MnP2 was also discussed. This is the first report of mutational analysis of fungal versatile peroxidase produced by a homologous expression system.For the development of a gene-targeting system in basidiomycete, a gene encoding for Ku70 protein which is involved in non-homologous end joining system was cloned and analyzed.