Cre1 of the ascomyceteCarbon catabolite repression is a means by which cells manage priority use of easy and fast metabolizable carbon sources over more complex ones. In Saccharomyces cerevisiae, mutations in MIG1 or in respective binding sites in Mig1 regulated promoters partially relieve the yeast from carbon catabolite repression (1-4). To form an active repressor complex, Mig1 requires the co-repressors Ssn6 (Cyc8)-Tup1 (5-8). Mig1 function is regulated by glucose-dependent nuclear import/export, Mig1 being nuclear when glucose is present and being transported to the cytoplasm when glucose becomes limiting (9). Concomitantly, Mig1 has been shown to be phosphorylated under derepressing conditions (10, 11). Present genetic evidence suggests that both of these events are mediated by the Snf1 kinase complex: a mutation in MIG1 suppresses the snf1 mutant defects in SUC2 and GAL1 expression (3, 4), indicating that SNF1 negatively regulates repression by Mig1. Furthermore, Mig1 is permanently localized in the nucleus in snf1

The xylanase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of two specific xylanases, Xyn1 and Xyn2, which are simultaneously expressed during growth on xylan but respond differentially to low-molecular-weight inducers. Using in vivo footprinting analysis of xylan-induced and noninduced mycelia, we detected two adjacent nucleotide sequences (5-AGAA-3 on the noncoding strand and 5-GGGT AAATTGG-3, referred to as the xylanase-activating element [XAE], on the coding strand, respectively) to bind proteins. Among these, binding to the AGAA-box is only observed under noninduced conditions, whereas binding to XAE is constitutive. Electrophoretic mobility shift assay with heterologously expressed components of the H. jecorina Hap2/3/5 protein complex and the cellulase regulator Ace2 suggests that these two transactivators form the protein complex binding to XAE. H. jecorina transformants, containing correspondingly mutated versions of the xyn2 promoter fused to the Aspergillus niger goxA gene as a reporter, revealed that the elimination of protein binding to the AGAA-box resulted in a threefold increase in both basal and induced transcription, whereas elimination of Ace2 binding to its target in XAE completely eliminated transcription under both conditions. Destruction of the CCAAT-box by insertion of a point mutation prevents binding of the Hap2/3/5 complex in vitro and results in a slight increase in both basal and induced transcription. These data support a model of xyn2 regulation based on the interplay of Hap2/3/5, Ace2 and the AGAA-box binding repressor.

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Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

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