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DNA affinity capturing identifies new regulators of the heterologously expressed novobiocin gene cluster in Streptomyces coelicolor M512

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Abstract

Understanding the regulation of a heterologously expressed gene cluster in a host organism is crucial for activation of silent gene clusters or overproduction of the corresponding natural product. In this study, Streptomyces coelicolor M512(nov-BG1) containing the novobiocin biosynthetic gene cluster from Streptomyces niveus NCIMB 11891 was chosen as a model. An improved DNA affinity capturing assay (DACA), combined with semi-quantitative mass spectrometry, was used to identify proteins binding to the promoter regions of the novobiocin gene cluster. Altogether, 2475 proteins were identified in DACA studies with the promoter regions of the pathway-specific regulators novE (PnovE) and novG (PnovG), of the biosynthetic genes novH-W (PnovH) and of the vegetative σ-factor hrdB (PhrdB) as a negative control. A restrictive classification for specific binding reduced this number to 17 proteins. Twelve of them were captured by PnovH, among them, NovG, two were captured by PnovE, and three by PnovG. Unexpectedly some well-known regulatory proteins, such as the global regulators NdgR, AdpA, SlbR, and WhiA were captured in similar intensities by all four tested promoter regions. Of the 17 promoter-specific proteins, three were studied in more detail by deletion mutagenesis and by overexpression. Two of them, BxlRSc and BxlR2Sc, could be identified as positive regulators of novobiocin production in S. coelicolor M512. Deletion of a third gene, sco0460, resulted in reduced novobiocin production, while overexpression had no effect. Furthermore, binding of BxlRSc to PnovH and to its own promoter region was confirmed via surface plasmon resonance spectroscopy.

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Acknowledgments

We are grateful to Lutz Heide for his valuable advice and support, to Boris Maček for his help and advice with the mass-spectrometric protein analysis, and to Ksenia Fedorova for her help with the Biacore SPR System. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 766).

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Authors and Affiliations

  1. Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany

    Paulina Bekiesch, Bertolt Gust & Alexander Kristian Apel

  2. German Centre for Infection Research (DZIF), Partner site Tübingen, Tübingen, Germany

    Paulina Bekiesch, Bertolt Gust & Alexander Kristian Apel

  3. Proteome Center Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany

    Mirita Franz-Wachtel

  4. Microbiology/Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany

    Andreas Kulik

  5. IBG-1 Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, 52425, Jülich, Germany

    Melanie Brocker

  6. Microbiology/ Department of Organismic Interactions, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany

    Karl Forchhammer

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  1. Paulina Bekiesch
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Correspondence to Alexander Kristian Apel.

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This study was funded by the Deutsche Forschungsgemeinschaft (grant number SFB 766) and the German Centre for Infection Research (DZIF) (grant number TTU 9.802).

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Paulina Bekiesch declares that she has no conflict of interest.

Mirita Franz-Wachtel declares that she has no conflict of interest.

Andreas Kulik declares that he has no conflict of interest.

Melanie Brocker declares that she has no conflict of interest.

Karl Forchhammer declares that he has no conflict of interest.

Bertolt Gust declares that he has no conflict of interest.

Alexander Kristian Apel declares that he has no conflict of interest.

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Bekiesch, P., Franz-Wachtel, M., Kulik, A. et al. DNA affinity capturing identifies new regulators of the heterologously expressed novobiocin gene cluster in Streptomyces coelicolor M512. Appl Microbiol Biotechnol 100, 4495–4509 (2016). https://doi.org/10.1007/s00253-016-7306-1

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