里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

doi:10.11949/j.issn.0438-1157.20151654

化工学报 ›› 2016, Vol. 67 ›› Issue (6): 2510-2518.DOI: 10.11949/j.issn.0438-1157.20151654

里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

钟立霞^1,2, 钱远超², 戴美学¹, 钟耀华²

1. 山东师范大学生命科学学院, 山东济南 250014;
2. 山东大学生命科学学院, 微生物技术国家重点实验室, 山东济南 250100

收稿日期:2015-11-03 修回日期:2015-12-26 出版日期:2016-06-05 发布日期:2016-06-05
通讯作者: 戴美学, 钟耀华
基金资助:
国家自然科学基金面上项目(31370135);山东省农业科技成果转化资金项目(2014No.45);山东大学基本科研业务费专项资金项目(2015CJ005)。

Improvement of uracil auxotrophic transformation system in Trichoderma reesei QM9414 and overexpression of β-glucosidase

ZHONG Lixia^1,2, QIAN Yuanchao², DAI Meixue¹, ZHONG Yaohua²

1. College of Life Sciences, Shandong Normal University, Jinan 250014, Shandong, China;
2. State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, Shandong, China

Received:2015-11-03 Revised:2015-12-26 Online:2016-06-05 Published:2016-06-05
Supported by:
supported by the National Natural Science Foundation of China (31370135), the Agricultural Science and Technology Achievement Transformation Fund of Shandong Province (2014No.45) and the Fundamental Research Funds of Shandong University (2015CJ005).

摘要/Abstract

摘要：

里氏木霉是广泛应用于纤维素酶生产的工业真菌,但高产突变株遗传操作困难,限制了菌种改良。首先利用定点整合策略敲除里氏木霉突变株QM9414的pyr4基因,成功构建尿嘧啶营养缺陷型菌株QP4,其遗传转化效率显著提高,而且产酶能力不受影响。进一步在QP4中过量表达β-葡萄糖苷酶(BGL)基因bgl1,经大量平板显色筛选获得2株BGL活力明显增强的工程菌QPB4和QPB5,其酶活分别提高10.01倍和8.26倍。利用发酵酶液对2种不同预处理的玉米芯底物进行水解糖化实验,结果显示以酸处理玉米芯为底物时QPB4和QPB5的葡萄糖得率比QP4分别提高60.98%和52.44%,而以脱木素处理玉米芯为底物时其葡萄糖得率分别提高80.01%和86.00%。研究表明改进里氏木霉高产突变株遗传转化体系可以显著促进菌株改良,提高糖化应用效果。

关键词: 纤维素酶, 里氏木霉高产突变株, 遗传转化, 水解糖化, 尿嘧啶营养缺陷型标记, 构建和表达, 生物技术, 生物工程

Abstract:

Trichoderma reesei is an industrial filamentous fungus widely used for cellulase production. However, the difficulty in genetic modification of the high-production mutants limits the strain improvement. Firstly, the pyr4 gene was successfully deleted by target integration strategy in T. reesei QM9414 to construct a uracil auxotroph strain QP4. Compared with those in the parental strain, not only the genetic transformation efficiency was improved, but also the cellulase production was not influenced in QP4. The β-glucosidase (BGL) encoding gene bgl1 was further overexpressed in the QP4 strain. The transformants QPB4 and QPB5 with increased BGL activity were obtained by rapid screening based on the esculin plate. The BGL activities in the culture supernatants of QPB4 and QPB5 exhibited 10.01- and 8.26- fold higher than that in QP4, respectively. The crude enzyme preparations were used to saccharify two types of differently pretreated corncob residues. The glucose yields from saccharification of acid-pretreated corncob by QPB4 and QPB5 were 60.98% and 52.44% higher than that by QP4, respectively, while using the delignined corncob as substrate, the glucose yields by QPB4 and QPB5 were improved 80.01% and 86.00%, respectively. These results suggested that improvement of the genetic manipulation system in T. reesei greatly facilitated the strain improvement and increased the saccharification efficiency.

Key words: cellulases, Trichoderma reesei high-production mutant, genetic transformation, saccharification, uracil auxotroph, construction and expression, biotechnology, biological engineering

中图分类号:

TQ812

钟立霞, 钱远超, 戴美学, 钟耀华. 里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达[J]. 化工学报, 2016, 67(6): 2510-2518.

ZHONG Lixia, QIAN Yuanchao, DAI Meixue, ZHONG Yaohua. Improvement of uracil auxotrophic transformation system in Trichoderma reesei QM9414 and overexpression of β-glucosidase[J]. CIESC Journal, 2016, 67(6): 2510-2518.

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里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

Improvement of uracil auxotrophic transformation system in Trichoderma reesei QM9414 and overexpression of β-glucosidase

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