A regulator based “semi-targeted” approach to activate silent biosynthetic gene clusters

Erik Mingyar; Lucas Mühling; Andreas Kulik; Anika Winkler; Daniel Wibberg; Jörn Kalinowski; Kai Blin; Tilmann Weber; Wolfgang Wohlleben; Evi Stegmann

doi:10.3390/ijms22147567

A regulator based “semi-targeted” approach to activate silent biosynthetic gene clusters

Erik Mingyar, Lucas Mühling, Andreas Kulik, Anika Winkler, Daniel Wibberg, Jörn Kalinowski, Kai Blin, Tilmann Weber, Wolfgang Wohlleben, Evi Stegmann^*

^*Corresponding author for this work

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Abstract

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Original language	English
Article number	7567
Journal	International Journal of Molecular Sciences
Volume	22
Issue number	14
Number of pages	14
ISSN	1661-6596
DOIs	https://doi.org/10.3390/ijms22147567
Publication status	Published - 2 Jul 2021

Bibliographical note

Funding Information:
Acknowledgments: The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation.

Funding Information:
Funding: This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021).

Funding Information:
This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021). The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation.

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

Regulation
Secondary metabolites
Silent biosynthetic gene cluster
Streptomyces

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title = "A regulator based “semi-targeted” approach to activate silent biosynthetic gene clusters",

abstract = "By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the T{\"u}bingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. T{\"U}17, Streptomyces sp. T{\"U}10 and Streptomyces sp. T{\"U}102. Introduction of the CSRs-plasmid into strain S. sp. T{\"U}17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. T{\"U}102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. T{\"U}10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. T{\"U}102 and aur1P and pntR in of S. sp. T{\"U}10.",

keywords = "Regulation, Secondary metabolites, Silent biosynthetic gene cluster, Streptomyces",

author = "Erik Mingyar and Lucas M{\"u}hling and Andreas Kulik and Anika Winkler and Daniel Wibberg and J{\"o}rn Kalinowski and Kai Blin and Tilmann Weber and Wolfgang Wohlleben and Evi Stegmann",

note = "Funding Information: Acknowledgments: The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation. Funding Information: Funding: This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021). Funding Information: This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021). The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation. Publisher Copyright: {\textcopyright} 2021 by the authors. Licensee MDPI, Basel, Switzerland.",

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AU - Mingyar, Erik

AU - Mühling, Lucas

AU - Kulik, Andreas

AU - Winkler, Anika

AU - Wibberg, Daniel

AU - Kalinowski, Jörn

AU - Blin, Kai

AU - Weber, Tilmann

AU - Wohlleben, Wolfgang

AU - Stegmann, Evi

N1 - Funding Information: Acknowledgments: The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation. Funding Information: Funding: This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021). Funding Information: This research was funded by German Center for Infection Research (DZIF), grant number 9.818 (01.01.2017). T.W. would like to acknowledge funding from the Novo Nordisk Foundation, grant numbers NNF16OC0021746 (01.03.2017) and NNF20CC0035580 (1.1.2021). The authors thank J. Wiese (Helmholz-Center for Ocean Research Kiel) for providing mayamycin. E.S. and W.W. were supported by the DZIF, the position of E.M. was funded by DZIF. The position of T.W. and K.B. were funded by the Nordisk Foundation. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

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N2 - By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

AB - By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

KW - Regulation

KW - Secondary metabolites

KW - Silent biosynthetic gene cluster

KW - Streptomyces

U2 - 10.3390/ijms22147567

DO - 10.3390/ijms22147567

M3 - Journal article

C2 - 34299187

AN - SCOPUS:85110498578

SN - 1661-6596

VL - 22

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

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M1 - 7567

ER -

A regulator based “semi-targeted” approach to activate silent biosynthetic gene clusters

Abstract

Bibliographical note

Keywords

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