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Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis
Open AccessArticle

Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment

Institute of Microbiology and Biotechnology, Rheinische Friedrich-Wilhelms University Bonn, 53115 Bonn, Germany
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Genes 2018, 9(4), 184; https://doi.org/10.3390/genes9040184
Received: 31 January 2018 / Revised: 19 March 2018 / Accepted: 21 March 2018 / Published: 28 March 2018
(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)
It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strain Halomonas elongata accumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (promA). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ38) and sigma 70 (σ70) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of a promA-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression—even in a deletion construct lacking the σ38 promoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Both sec and tat leader sequences from H. elongata proved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength. View Full-Text
Keywords: Halomonas; compatible solutes; heterologous expression; salt-induced promoter; ribosome binding site; RBS Calculator; 16S rRNA, GFP; mCherry; ectoine Halomonas; compatible solutes; heterologous expression; salt-induced promoter; ribosome binding site; RBS Calculator; 16S rRNA, GFP; mCherry; ectoine
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Stiller, L.M.; Galinski, E.A.; Witt, E.M.H.J. Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment. Genes 2018, 9, 184.

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