Aro N, Saloheimo A, Ilmén M, Penttilä M
VTT Biotechnology, Tietotie 2, FIN-02044 VTT, Espoo, Finland.
J Biol Chem. 2001 Jun 29;276(26):24309-14. doi: 10.1074/jbc.M003624200. Epub 2001 Apr 13.
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 floc 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 sophorose was not affected by ace2 deletion.
一种基于酵母的分离转录激活因子的新方法被应用于克隆与丝状真菌里氏木霉（枝状腐质霉）纤维素酶启动子cbh1结合的调控因子。这导致分离出纤维素酶激活因子ace2，其编码一种属于仅在真菌中发现的锌双核簇蛋白类的蛋白质。ACEII的DNA结合结构域在大肠杆菌中作为谷胱甘肽S - 转移酶融合蛋白表达，并且ACEII在体外被证明与cbh1启动子中存在的5'-GGCTAATAA位点结合。该位点还包含黑曲霉木聚糖酶激活因子XlnR的推测结合序列。GGC三联体的突变消除了ACEII的结合。通过分析纤维素酶高产里氏木霉菌株ALKO2221中ace2缺失的影响来研究ACEII的功能。ace2基因的缺失导致编码主要纤维素酶纤维二糖水解酶I和II以及内切葡聚糖酶I和II的mRNA的诱导动力学降低，并且当真菌在含有索尔卡纤维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的表达水平也受到影响。ace2缺失导致在纤维素诱导培养中xyn2表达降低。纤维二糖对纤维素酶的诱导不受ace2缺失的影响。
