Dataset Information

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

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 (?38) and sigma 70 (?70) 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 ?38 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.

SUBMITTER: Stiller LM

PROVIDER: S-EPMC5924526 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Stiller Lisa M LM Galinski Erwin A EA Witt Elisabeth M H J EMHJ

Genes 20180328 4

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 i ...[more]

PMID: 29597294

Similar Datasets

Project description:Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions.

Project description:BACKGROUND:As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. RESULTS:The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L-1 NaCl in a 500-mL flask, the double mutant of Y2/?ectD/?doeA synthesized 3.13 g L-1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L-1), and also exceeds the production of Y2/?ectD (2.21 g L-1). The remarkably enhanced accumulation of ectoine by Y2/?ectD/?doeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/?ectD/?doeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L-1 or 500 mg (g DCW) -1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L-1 NaCl stress, a maximum 10.5 g L-1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)-1 and a yield of 0.21 g g-1 monosodium glutamate. CONCLUSION:The remarkably enhanced production of ectoine by Y2/?ectD/?doeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets