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

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.

The hydroxyectoine gene cluster of the non-halophilic acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic α-Proteobacterium which thrives in acidic, metal-rich environments (e.g. acid mine drainage). Recently, an ectABCDask gene cluster for biosynthesis of the compatible solutes ectoine and hydroxyectoine was detected in the genome sequence of A. cryptum JF-5. We were able to demonstrate that the type strain A. cryptum DSM 2389(T) is capable of synthesizing the compatible solute hydroxyectoine in response to moderate osmotic stress caused by sodium chloride and aluminium sulphate, respectively. Furthermore, we used the A. cryptum JF-5 sequence to amplify the ectABCDask gene cluster from strain DSM 2389(T) and achieved heterologous expression of the gene cluster in Escherichia coli. Hence, we could for the first time prove metabolic functionality of the genes responsible for hydroxyectoine biosynthesis in the acidophile A. cryptum. In addition, we present information on specific enzyme activity of A. cryptum DSM 2389(T) ectoine synthase (EctC) in vitro. In contrast to EctCs from halophilic microorganisms, the A. cryptum enzyme exhibits a higher isoelectric point, thus a lower acidity, and has maximum specific activity in the absence of sodium chloride.

Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC.

A new cyclic amino acid was detected in a deletion mutant of the moderately halophilic bacterium Halomonas elongata deficient in ectoine synthesis. Using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques, the substance was identified as 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). We were able to demonstrate that ADPC is the product of a side reaction of lone ectoine synthase (EC 4.2.1.108), which forms ADPC by cyclic condensation of glutamine. This reaction was shown to be reversible. Subsequently, a number of ectoine derivatives, in particular 4,5-dihydro-2-methylimidazole-4-carboxylate (DHMICA) and homoectoine, were also shown to be cleaved by ectoine synthase, which is classified as a hydro-lyase. This study thus reports for the first time that ectoine synthase accepts more than one substrate and is a reversible enzyme able to catalyze both the intramolecular condensation into and the hydrolytic cleavage of cyclic amino acid derivatives. As ADPC supports growth of bacteria under salt stress conditions and stabilizes enzymes against freeze-thaw denaturation, it displays typical properties of compatible solutes. As ADPC has not yet been described as a natural compound, it is presented here as the first man-made compatible solute created through genetic engineering.