基因组重排技术提高白腐菌漆酶产量及降解木质素机理研究

Genome shuffling is a kind of typical comprehensive combination technical means, which is an extension of whole-genome metabolic engineering. In order to improve the laccase production of white-rot fungi Pleurotus ostreatus, the strains of P. ostreatus were transformed by genome shuffling in this study. The initial mutant population was generated by UV irradiation, ethyl methane sulfonate (EMS) and nitrosoguanidine (NTG) treatment of the spores. The mutants were subjected to recursive protoplast fusion. The efficient shuffled strains were selected after genome shuffling, which exhibited a higher production of laccase activity and shorter fermentation time. The randomly amplified polymorphic DNA (RAPD) system was established. The genetic similarity and clustering analysis between fusants and parent strains were carried out. Purification and characterization of laccase were studied. Degradation mechanism of lignin by laccase was studied.The successful implementation of this project will promote the development of biological industry during the period of “the 12th Five-year Plan” in China and solve the increasingly serious water pollution problems, which has a significant social benefits, theoretical significance and application value.

基因组重排技术是一种典型的全面组合技术手段，是全基因组代谢工程的延伸。为了提高白腐菌糙皮侧耳(Pleurotus ostreatus)的漆酶产量，本研究拟采用基因组重排技术改造糙皮侧耳菌株，以P. ostreatus B1为出发菌株，进行紫外线（UV）、甲基磺酸乙酯（EMS）、亚硝基胍（NTG）诱变及组合诱变处理，获得突变群体，从中筛选出漆酶产量高、遗传性状稳定的突变株作为正向突变文库，通过递推式原生质体融合，对突变子的全基因组进行随机重排，将优良表型重组在一起，构建出漆酶产量高、发酵时间短的高效工程菌株；建立随机扩增多态性DNA(RAPD)体系，对融合子和出发菌株进行遗传相似性和聚类分析；纯化漆酶并研究其酶学性质；研究漆酶降解木质素的降解机理。该项目的成功实施，将促进我国“十二五”期间生物产业的发展，对于解决我国日益严重的水污染问题有着显著的社会效益、理论意义和工程应用价值。

基因组重排技术是在传统诱变技术和细胞融合技术基础上发展而来的，是对整个微生物基因组重排，来选育具有优良性状菌株的新型育种技术。本项目提出对白腐菌糙皮侧耳(Pleurotus ostreatus)进行菌株改良，采用基因组重排技术来构建新型菌株，提高漆酶产量并进行降解木质素的研究。以P. ostreatus B1 为出发菌株，进行紫外线（UV）、甲基磺酸乙酯（EMS）、亚硝基胍（NTG）诱变及组合诱变处理，经过初筛和复筛，筛选出五株漆酶产量获得一定提高、遗传性状稳定的突变菌。研究P. ostreatus原生质体制备、再生和融合的方法和影响因素，确定选用第6d的菌丝，用1.5%纤维素酶+1.0%蜗牛酶在30℃酶解3h，用1.0M甘露醇为渗透压稳定剂进行原生质体的制备和再生。以诱变得到的突变株为亲本，采用3轮基因组重排技术选育高产漆酶菌株，考察其遗传稳定性，并对发酵条件进行优化。采用振荡培养方式，10%的接种量发酵，碳源为葡萄糖，氮源为蛋白胨，添加桔皮作为天然底物可提高7倍漆酶酶活。用硫酸铵盐析、超滤、离子交换色谱和DEAE-Sepharose等方法对漆酶进行分离和纯化，得到纯化漆酶，比原始粗酶液中漆酶比活力提高了5.1倍，漆酶总活力损失较小，得率为72%。确定漆酶最佳温度为45℃，最佳pH为3.0，Cu2+对漆酶酶活有促进作用，Mn2+和Fe3+对漆酶酶活有抑制作用。采用纯化漆酶和粗酶分别处理造纸废水，纯化漆酶降解率略高些，但纯化漆酶成本高，操作复杂，不适用于工业化大规模生产，而白腐菌粗酶液具有与纯化漆酶等同的脱色和降解效率，可降低废水处理成本。采用傅里叶红外光谱、气质联用等技术手段，研究木质素的降解机理。本项目采用新型细胞生物学手段，构建新型高效木质素分解菌，并开展相关研究，具有显著的社会效益、理论意义和突出的应用价值。