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J. Biol. Chem., Vol. 276, Issue 26, 24309-24314, June 29, 2001
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From VTT Biotechnology, Tietotie 2, FIN-02044
VTT, Espoo, Finland
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF220671.
ACEII, a Novel Transcriptional Activator Involved in Regulation
of Cellulase and Xylanase Genes of Trichoderma reesei*
,
*
This work was supported by the Foundation for Biotechnical
and Industrial Fermentation Research, Helsinki Graduate School in
Biotechnology and Molecular Biology, Roal Oy, and the National Technology Agency (Tekes).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: VTT
Biotechnology, Tietotie 2, P.O. Box 1500, FIN-02044 VTT, Espoo,
Finland. Tel.: 358-9-4561; Fax: 358-9-4552103; E-mail:
nina.aro@vtt.fi.
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