Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions.
Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (alpha-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the beta-glucuronidase (gusA) and beta-galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.
Menne
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Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum, Germany.
Walz
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Kück
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D000164:Acremonium / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D002511:Cephalosporins / Q000096:biosynthesis; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D002511:Cephalosporins; D004271:DNA, Fungal; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D005966:Glucuronidase; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase