A glucose-derepressed promoter for expression of heterologous products in the filamentous fungus Aspergillus nidulans.
We describe a putative binding sequence (GCGGGGC) for the glucose-responsive repressor protein CreA at two positions upstream of the transcription start site of the alcohol dehydrogenase I (alcA) gene of Aspergillus nidulans. To positively identify the putative binding sites as CreA-specific, the GCGGGGC blocks were mutated at five internal nucleotide positions to GTACTAC and reintroduced into the wild type alcA promoter driving expression of the endogenous alcohol dehydrogenase I gene. This CreA-binding site variant was then transformed into an AlcR constitutive A. nidulans host strain (T2625) and growth was monitored in the presence of the non-metabolized glucose analogue, 2-deoxyglucose. Positive transformants were selected by their ability to grow using ethanol as a carbon source in the presence of 2-deoxyglucose. Similar CreA binding site variant alcA promoters should permit the alcA-driven expression of heterologous genes in A. nidulans in the presence of glucose, the preferred carbon source for biomass accumulation and provides a model for controlling carbon-catabolite regulated expression in other expression systems.
Hintz
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Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.
Lagosky
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PA
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans; D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D005947:Glucose / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D012641:Selection, Genetic; D014170:Transformation, Genetic
D005656:Fungal Proteins; D012097:Repressor Proteins; C071237:CreA protein, Aspergillus nidulans; D005947:Glucose