Affiliation:
1. Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, D-44780 Bochum, Germany
2. Sandoz GmbH, A-6250 Kundl, Austria
Abstract
ABSTRACT
Winged helix transcription factors, including members of the forkhead and the RFX subclasses, are characteristic for the eukaryotic domains in animals and fungi but seem to be missing in plants. In this study, in vitro and in vivo approaches were used to determine the functional role of the RFX transcription factor CPCR1 from the filamentous fungus
Acremonium chrysogenum
in cephalosporin C biosynthesis. Gel retardation analyses were applied to identify new binding sites of the transcription factor in an intergenic promoter region of cephalosporin C biosynthesis genes. Here, we illustrate that CPCR1 recognizes and binds at least two sequences in the intergenic region between the
pcbAB
and
pcbC
genes. The in vivo relevance of the two sequences for gene activation was demonstrated by using
pcbC
promoter-
lacZ
fusions in
A. chrysogenum
. The deletion of both CPCR1 binding sites resulted in an extensive reduction of reporter gene activity in transgenic strains (to 12% of the activity level of the control). Furthermore,
Acremonium
transformants with multiple copies of the
cpcR1
gene and knockout strains support the idea of CPCR1 being a regulator of cephalosporin C biosynthesis gene expression. Significant differences in
pcbC
gene transcript levels were obtained with the knockout transformants. More-than-twofold increases in the
pcbC
transcript level at 24 and 36 h of cultivation were followed by a reduction to approximately 80% from 48 to 96 h in the knockout strain. The overall levels of the production of cephalosporin C were identical in transformed and nontransformed strains; however, the knockout strains showed a striking reduction in the level of the biosynthesis of intermediate penicillin N to less than 20% of that of the recipient strain. We were able to show that the complementation of the
cpcR1
gene in the knockout strains reverses
pcbC
transcript and penicillin N amounts to levels comparable to those in the control. These results clearly indicate the involvement of CPCR1 in the regulation of cephalosporin C biosynthesis. However, the complexity of the data points to a well-controlled or even functional redundant network of transcription factors, with CPCR1 being only one player within this process.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Reference44 articles.
1. Abe, Y., C. Ono, M. Hosobuchi, and H. Yoshikawa. 2002. Functional analysis of mlcR, a regulatory gene for ML-236B (compactin) biosynthesis in Penicillium citrinum.Mol. Genet. Genomics268:352-361.
2. Ahn, J. H., and J. D. Walton. 1998. Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats. Mol. Gen. Genet.260:462-469.
3. Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi
4. Brakhage A. A. and M. L. Caruso. 2004. Biotechnical genetics of antibiotic biosynthesis. In U. Kück (ed.) The Mycota II 2nd ed. Springer Press Berlin Germany.
5. Durand, B., C. Vandaele, D. Spencer, S. Pantalcci, and P. Couble. 2000. Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene246:285-293.
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