title: DNA affinity capturing identifies new regulators of the heterologously expressed novobiocin gene cluster in Streptomyces coelicolor M512.
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.
authors.affiliation: Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.|German Centre for Infection Research (DZIF), Partner site Tübingen, Tübingen, Germany.
authors.forename: Paulina
authors.initials: P
authors.lastname: Bekiesch
authors.affiliation: Proteome Center Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany.
authors.forename: Mirita
authors.initials: M
authors.lastname: Franz-Wachtel
authors.affiliation: Microbiology/Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kulik
authors.affiliation: IBG-1 Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, 52425, Jülich, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Brocker
authors.affiliation: Microbiology/ Department of Organismic Interactions, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Forchhammer
authors.affiliation: Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.|German Centre for Infection Research (DZIF), Partner site Tübingen, Tübingen, Germany.
authors.forename: Bertolt
authors.initials: B
authors.lastname: Gust
authors.affiliation: Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany. kristian.apel@uni-tuebingen.de.|German Centre for Infection Research (DZIF), Partner site Tübingen, Tübingen, Germany. kristian.apel@uni-tuebingen.de.
authors.forename: Alexander Kristian
authors.initials: AK
authors.lastname: Apel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D009675:Novobiocin / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C071941:HrdB protein, Streptomyces; D009675:Novobiocin; D005982:Glutathione Transferase
keywords: DNA affinity capturing assay; Heterologous expression; Novobiocin; Regulation; Streptomyces; Surface plasmon resonance