高效转化法定向进化里氏木霉的纤维素酶  

Directed evolution of Trichoderma reesei cellulase by efficient transformation

作　　者：方浩[1,2,3] 邓禹[1,2] 毛银[1] 夏黎明[3] 

机构地区：[1]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]浙江大学化学工程与生物工程学院,浙江杭州310027

出　　处：《生物加工过程》2016年第1期1-7,共7页Chinese Journal of Bioprocess Engineering

基　　金：江苏省青年基金(BK20150130);中央高校基本科研业务费专项资金(JUSRP115A17)

摘　　要：优化了根癌农杆菌介导的里氏木霉的转化方法,得出转化的最适条件:共培养pH 5.3、共培养温度24℃、乙酰丁香酮浓度200μmol/L、根癌农杆菌OD660值0.8、里氏木霉孢子预萌发时间3 h。此时转化率为13 000个转化子(以107个里氏木霉孢子计),是未优化条件下的27倍以上。利用此高效转化法,结合强化纤维二糖水解酶基因II表达的操作,从324个转化子中筛选到了1株优良的里氏木霉转化子C10,发酵5 d后,纤维二糖水解酶活力达(122.44±5.91)U/m L,是初始菌株的5.4倍;滤纸酶活力达(28.92±2.45)IU/m L,是初始菌株的4.3倍。与初始菌株所产纤维素酶相比,转化子C10所产纤维素酶降解玉米秸秆和稻草的能力有明显的提高。本研究为里氏木霉的基因工程改造及其纤维素酶的定向进化提供了有价值的实验数据。The influencing factors of Agrobacterium tumefaciens-mediated transformation of Trichoderma reesei were optimized in this study and the optimal conditions were found to be co-culture at pH 5. 3,24 ℃,acetosyringone concentration 200 μmol / L,A. tumefaciens concentration OD660 value 0. 8,and pre-germination time of T. reesei spores for 3 h. Under these conditions,the transformation rate was 13 000 transformants per 107 spores,27 times higher than that before optimization. A well performed T. reesei transformant C10 was obtained among 324 transformants using the optimized transformation method and the genetic engineering strategy enhancing cellobiohydrolase II gene expression,which produced cellulase with（ 122. 44 ± 5. 91） U / m L cellobiohydrolase activity and（ 28. 92±2. 45） IU / m L filter paper activity,5. 4 and 4. 3 times higher than the original strain,respectively. Compared to the cellulase from the original strain,the cellulase from transformant C10 had an improved performance in the enzymatic hydrolysis of corn stover and rice straw. The result has significance to genetic engineering of T. reesei and directed evolution of T. reesei cellulase.

关 键 词：根癌农杆菌 转化 里氏木霉 优化 纤维二糖水解酶 纤维素酶 

分 类 号：Q812[生物学—生物工程]