ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei
ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei
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DOI：
10.1074/jbc.m003624200
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发表时间：
2001-06-29
影响因子：
4.8
通讯作者：
Penttilä, M
中科院分区：
文献类型：
--
作者：
Aro, N;Saloheimo, A;Penttilä, M
利用一种基于酵母分离转录激活因子的新方法，克隆了与丝状真菌里氏木霉（Trichoderma reesei）纤维素酶启动子cbh1结合的调节因子。这导致分离纤维素酶激活剂ace2编码的蛋白质属于锌双核簇蛋白类，只在真菌中发现。ACEII的dna结合域在大肠杆菌中以谷胱甘肽s -转移酶融合蛋白的形式表达，并且ACEII在体外被证明与cbh1启动子中的5'-GGCTAATAA位点结合。该位点还包含黑曲霉木聚糖酶激活剂XlnR的结合序列。GGC三联体突变使ACEII结合消失。通过分析ace2缺失对高酶解T. reesei菌株ALKO2221的影响，研究了ACEII的功能。缺失ace2基因导致编码主要纤维素酶-纤维素生物水解酶I和II以及内切葡聚糖酶I和II的mrna诱导动力学降低，当真菌在含有Solka escape纤维素的培养基上生长时，纤维素酶活性降低30-70%。编码木聚糖酶的基因表达量也受到影响。在纤维素诱导培养中，Ace2缺失导致xyn2表达降低。纤维素酶的诱导。磷酸化不受ace2缺失的影响。
A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2 encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bind in vitro to the 5'-GGCTAATAA site present in the cbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects of ace2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30-70% reduced cellulase activity when the fungus was grown on medium containing Solka flee cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by so. phorose was not affected by ace2 deletion.