A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes
A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes
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用于研究里氏木霉基因功能的铜响应启动子替换系统：SAGA 基因特征的案例研究
DOI：
10.1007/s00253-016-8036-0
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发表时间：
2017
影响因子：
5
通讯作者：
Liu Weifeng
中科院分区：
文献类型：
--
作者：
Zheng Fanglin;Cao Yanli;Lv Xinxing;Wang Lei;Li Chunyan;Zhang Weixin;Chen Guanjun;Liu Weifeng
里氏木霉是纤维素酶和其他蛋白质工业生产的重要原料。里氏木霉纤维素酶产生的调节以及其他生理过程的分子机制仍然不够清楚。我们构建了一个基于PtCu1的启动子替换盒，它允许一步替换内源启动子，从而用铜来控制靶基因的表达。然后，我们发现组蛋白乙酰转移酶基因gcn5的铜抑制作用出现在gcn5缺失菌株上。使用相同的策略，我们进一步研究了另一个可能的SPT-Ada-Gcn5乙酰基转移酶(SAGA)复合亚单位编码基因ada2在里氏酵母中的功能。与gcn5的抑制作用类似，在ptcu1-ada2菌株中添加铜不仅显著降低了里氏木霉的营养生长和分生孢子，而且严重抑制了诱导的纤维素酶基因的表达。因此，开发的策略将潜在地有助于探索包括基因组中那些必需基因在内的大部分功能未知的里氏木霉基因的生物学功能，以扩大其非凡的生物技术潜力。
Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a Ptcu1-based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the Ptcu1-ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.