Improvement of wood-rotting activity of basidiomycetes using cell fusion techniques

降解木质素的担子菌在木质纤维素的各种商业加工方案中具有潜在的应用价值。然后，本研究旨在获得将细胞融合方法应用于木质素降解担子菌，特别是黄孢原毛平革菌，以获得木质素降解率较高的菌株的基本结果。黄孢原毛平革菌在0.6M硫酸镁或0.8M甘露醇中与诺维菌素234(1%)和纤维素酶Onozuka RS(1%)的混合物在37ﾟC下温和摇动3h，获得了黄孢原生菌原生质体。原生质体呈球形，直径3-10微米。原生质体再生率约为5.0%。常规的聚乙二醇法和电融合法均适用于黄孢原生菌的原生质体融合。用聚乙二醇或电融合器处理黄孢原生菌的两个营养缺陷型突变株的原生质体导致…融合在最小的培养皿上通过互补测定更多的原生质体。获得的融合子通过一些传代数的继代培养，保持了互补性(在最小培养基板上生长的能力)，并保持了相对稳定。还获得了营养缺陷型突变株和无过氧化物酶突变株之间的融合子，可用荧光化合物异硫氰酸荧光素(FITC)和罗丹明B异硫氰酸酯(RITC)标记原生质体。FITC和RITC标记的原生质体在荧光显微镜下可相互区分。用FITC或RITC分别标记黄孢原毛霉和杂色云芝、平菇的原生质体，用聚乙二醇法进行融合。黄孢原毛霉和埃利氏鸡腿霉的融合子也是通过电融合产生的。在亲本菌株所处的两种培养条件下，融合子均能产生在木质素降解过程中起重要作用的酚氧化酶(或过氧化物酶)，表明融合子具有较强的木质素降解能力。较少

The lignin-degrading basidiomycetes have potential application in a variety of schemes for the commercial processing of lignocellulose. Then, this research was intended to obtain the basic results for applying the cell fusion method to lignin-degrading basidiomycetes, especially Phanerochaete chrysosporium, for producing strains with superior capacities of lignin degradation.Protoplasts of P. chrysosporium were obtained by treating mycelia in 0.6 M MgSO_4 or 0.8 M mannitol with the mixture of Novozyme 234 (1%) and Cellulose ONOZUKA RS (1%) at 37ﾟC for 3 h with gentle shaking. The protoplasts showed the spherical feature with a diameter of 3-10 mum. The frequency of protoplast regeneration was calculated as about 5.0%. Both of the usual polyethylene glycol (PEG) treatment method and electrofusion method were available for the protoplast fusion of P.chrysosporium. Treatment of the protoplasts from two of the auxotrophic mutants of P. chrysosporium with PEG or electrofuser led to fusion o … More f protoplasts as measured by complementation on a minimal medium plate. The fusants obtained maintained the complementarity (the ability to grow on a minimal medium plate) with relatively stability through subcultures of some passage numbers. The fusants between auxotrophic mutants and peroxidase less mutants were also obtained.The protoplasts could be labeled with the fluorescent compounds, fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). The labeled protoplasts with FITC and RITC were distinguishable to each other under the fluorescence microscope. The separately labeled protoplasts with FITC or RITC of P. chrysosporium and of Coriolus versicolor or Pleurotus ostreatus were observed to fuse by PEG treatment. The fusants between P. chrysosporium and Coprinus aff ellisii were also produced by electrofusion. The fusants produced phenol oxidases (or peroxidases), which are known to play important roles in lignin degradation, under the both culture conditions in which each parental strain did, indicating that these fusants might show the superior capacities of lignin dergadation. Less