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Applied Microbiology and Biotechnology

, Volume 103, Issue 1, pp 303–313 | Cite as

Development of a synthetic cumate-inducible gene expression system for Bacillus

  • Seung-Oh Seo
  • Claudia Schmidt-Dannert
Applied genetics and molecular biotechnology

Abstract

A novel inducible gene expression system using p-isopropyl benzoate (cumate) as an inducer was developed for the industrial production hosts, Bacillus subtilis and Bacillus megaterium. Cumate is non-toxic to the host, inexpensive, and carbon source-independent inducer which provides an economical option for large-scale production of valuable proteins and chemicals from Bacillus strains. The synthetic cumate-inducible system was constructed by combining the strong constitutive Bacillus promoter Pveg with regulatory elements of the Pseudomonas putida, CymR repressor, and its operator sequence CuO. The designed expression cassette containing a sfGFP reporter under the cumate-inducible promoter was assembled into a Bacillus-E. coli shuttle and gene expression investigated in the two Bacillus strains. Characterization of gene expression levels, expression kinetics, and dose-response to cumate inducer concentration confirmed high-level, but tightly controlled GFP reporter expression in tunable, cumate concentration-dependent manner. Unexpectedly, this expression system works equally well for Escherichia coli, resulting in a platform that can be used both in gram-positive and gram-negative expression host. Its tight regulation and controllable expression makes this system useful for metabolic engineering, synthetic biology studies as well industrial protein production.

Keywords

Bacillus subtilis Bacillus megaterium Synthetic biology Promoter Gene expression Cumate 

Notes

Acknowledgments

The authors thank Dr. Maureen Quin for helpful discussion about the project, and Dr. Antony Dean and Dr. Xiao Yi at the University of Minnesota for flow cytometer use.

Funding information

This work was supported by funding from the Defense Advanced Research Projects Agency (DARPA) #HR0011-17-2-0038.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

253_2018_9485_MOESM1_ESM.pdf (1 mb)
ESM 1 (PDF 1074 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biochemistry, Molecular Biology and BiophysicsUniversity of MinnesotaSt. PaulUSA

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