Development of a synthetic cumate-inducible gene expression system for Bacillus
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.
KeywordsBacillus subtilis Bacillus megaterium Synthetic biology Promoter Gene expression Cumate
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.
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.
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