Identification of a minimal cre1 promoter sequence promoting glucose-dependent gene expression in the beta-lactam producer Acremonium chrysogenum.
The promoter of the cre1 gene, encoding the glucose-dependent regulator CRE1 from the beta-lactam producer Acremonium chrysogenum, carries 15 putative CRE1 binding sites (BS1 to BS15). For a detailed analysis, we fused cre1 promoter deletion derivatives with the DsRed reporter gene to perform a comparative gene expression analysis. Plate assays, Northern hybridizations, and spectrofluorometric measurements of DsRed identified the minimal D4 promoter sequence that promoted glucose-dependent expression. Truncated recombinant CRE1 interacted with D4 in electromobility shift analysis and these binding studies were further extended with two oligonucleotides, carrying putative CRE1 binding sites BS14 and BS15. Surface plasmon resonance analysis was performed using BS14 and BS15, along with four derivatives containing 2 or 4 bp substitutions within BS14 and BS15, respectively. Substitutions within BS14 abolished the high affinity interaction with CRE1, while mutations in BS15 only marginally diminished the affinity with CRE1. In vivo analysis of a modified D4 sequence with substitutions in the two binding sites confirmed the in vitro binding results and still promoted glucose-dependent gene expression. Our results will contribute to the construction of versatile expression vectors carrying a minimal cre1 promoter sequence that still confers glucose-dependent induction of gene expression.
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780 Bochum, Germany. danielle.janus@rub.de
Danielle
D
Janus
Peter
P
Hortschansky
Ulrich
U
Kück
D000164:Acremonium / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D017384:Sequence Deletion; D047090:beta-Lactams / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D047090:beta-Lactams; D005947:Glucose