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

白腐菌平菇（Pleurotus ostreatus）分泌的多功能过氧化物酶MnP2具有独特的高分子量化合物氧化活性。通过对P. ostreatus进行转化，产生了一系列的MnP2突变体，这些突变体含有一个具有定点碱基置换的重组MnP2。每个突变蛋白被纯化到均匀性，并对低分子量和高分子量底物的动力学和反应性以及光谱方法进行了表征。在本研究中，所有变异的Mn^<2+>和H_2O_2的动力学性质与野生型MnP2基本相似。另一方面，研究表明，将暴露的色氨酸残基（W170）替换为丙氨酸会使veratrylacohol （VA）、Poly R-478和Rnase a的氧化活性降低。此外，替换周围的氨基添加残基会影响VA以外的高分子量化合物的氧化。从这些结果可以清楚地证明，MnP2通过从W170到血红素片段的远程电子转移途径直接氧化高分子量化合物。还讨论了突变体MnP2中碳水化合物排放的额外修饰。本文首次报道了真菌同源表达系统产生的多功能过氧化物酶的突变分析。为建立担子菌基因靶向系统，克隆并分析了一个参与非同源末端连接系统的Ku70蛋白编码基因。

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.