Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum: specific DNA-protein interactions and characterization of the transcription factor PACC

Abstract

An analysis of the pcbC promoter from the cephalosporin C-producing filamentous fungus Acremonium chrysogenum was performed using fungal transformants carrying reporter gene fusions. By investigating sequential deletion derivatives of the pcbC promoter region, a DNA fragment was identified, which is responsible for transcriptional activation of the pcbC gene. Sequence analysis of this fragment revealed a consensus binding site for the fungal transcription factor PACC. Gel-retardation experiments with crude extracts of A. chrysogenum confirmed the specific binding of a protein to the PACC binding site. The subsequent cloning of the pacC homolog from A. chrysogenum allowed the identification of an ORF of 621 amino acids encoded by four exons. The polypeptide shows about 35% sequence identity to other fungal PACC proteins. A PACC protein fragment synthesized in E. coli was used for in vitro binding assays, and specific binding of the zinc-finger transcription factor to its consensus binding sites in the promoter regions of four cephalosporin C biosynthesis genes could be demonstrated. The bi-directional promoters of the genes pcbAB-pcbC and cefEF-cefG contain two PACC binding sites each. The data obtained strongly suggest that, in A. chrysogenum, the zinc-finger transcription factor PACC is involved in the transcriptional regulation of the genes involved in cephalosporin C biosynthesis.