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Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria

  • Applied genetics and molecular biotechnology
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Abstract

Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-d-thiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (β-glucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyper-inducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.

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Acknowledgements

This work was supported by the European Commission under the 7th Framework Program through the “Collaborative Project” action, “STREPSYNTH” grant No. 613877, and through the European Research Council (ERC) starting grant EXPLOGEN No. 281623 to AL.

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Authors and Affiliations

  1. Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University Campus, Building C2 3, 66123, Saarbrücken, Germany

    Liliya Horbal & Andriy Luzhetskyy

  2. Ivan Franko National University of Lviv, Hrushevskogo 4, 79005, Lviv, Ukraine

    Liliya Horbal & Victor Fedorenko

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  1. Liliya Horbal
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Correspondence to Andriy Luzhetskyy.

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Horbal, L., Fedorenko, V. & Luzhetskyy, A. Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria. Appl Microbiol Biotechnol 98, 8641–8655 (2014). https://doi.org/10.1007/s00253-014-5918-x

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  • DOI: https://doi.org/10.1007/s00253-014-5918-x

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