Abstract
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.
Highlights
The gamma proteobacterium Halomonas elongata DSM 2581T was first described by Vreeland et al, 1980 [1]
To develop an expression system that is suitable for H. elongata, a broad host range vector was chosen as a backbone and one of the hosts own promoters was selected
The promoter region upstream of the ectoine biosynthetic gene cluster seemed a good choice as it is active under standard growth conditions and can be further regulated by increasing or decreasing the salinity of the medium
Summary
The gamma proteobacterium Halomonas elongata DSM 2581T (family: Halomonadaceae) was first described by Vreeland et al, 1980 [1] It belongs to a group of versatile halophiles with a broad range of halotolerance and a high degree of adaptability to changing salinity [2,3]. Its prime compatible solutes responsible for osmotic equilibrium are the tetrahydropyrimidine derivatives L-ectoine and S,S-5-hydroxyectoine. Their biosynthesis and degradation has been investigated in depth [4,5,6,7] and members of the Halomonadaceae have since become a popular object for research on osmotic adaptation [8,9,10]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call