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Identification of a minimal cre1 promoter sequence promoting glucose-dependent gene expression in the β-lactam producer Acremonium chrysogenum

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Abstract

The promoter of the cre1 gene, encoding the glucose-dependent regulator CRE1 from the β-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.

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Acknowledgments

We thank Kerstin Kalkreuter (Bochum), Sylke Fricke (Jena), and Ingeborg Godehardt (Bochum) for excellent technical assistance, Prof. Eckhard Hofmann (Bochum) for the use of the JASCO spectrofluorometer, Dr. E. Friedlin and Dr. H. Kürnsteiner (Kundl) for fruitful discussions, and Prof. A. Brakhage (Hans-Knöll Institute, Jena) for institutional support. This work was funded by Sandoz GmbH (Austria, Kundl).

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Authors and Affiliations

  1. Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780, Bochum, Germany

    Danielle Janus & Ulrich Kück

  2. Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology (HKI) and Friedrich-Schiller-University, 07745, Jena, Germany

    Peter Hortschansky

Authors
  1. Danielle Janus
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  2. Peter Hortschansky
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Correspondence to Ulrich Kück.

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Communicated by K. Breunig.

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294_2007_164_MOESM1_ESM.doc

Supplementary Table 1: Data from spectrofluorometric measurements to determine the DsRed amount (ng DsRed/µg protein) in crude protein extracts from different low (lc) and multi copy (mc) transformants carrying promoter deletion derivatives as indicated. (DOC 38 kb)

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Janus, D., Hortschansky, P. & Kück, U. Identification of a minimal cre1 promoter sequence promoting glucose-dependent gene expression in the β-lactam producer Acremonium chrysogenum . Curr Genet 53, 35–48 (2008). https://doi.org/10.1007/s00294-007-0164-8

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