Abstract
Ectoine, a heterocyclic amino acid produced by various bacteria, was widely used in the fields of cosmetics and medicine. In this study, a novel ectoine synthesis cluster from marine bacterium Salinicola salarius 1A01339 was firstly introduced into Escherichia coli BL21(DE3) for heterologous production of ectoine. The bioinformatic analysis proved the function of these ectoine synthesis enzymes, and showed the highest identities of 83.3–87.7% with enzymes from other microorganisms. Using the whole-cell biocatalytic method, 3.28 g/L ectoine was synthesized and excreted into the medium with the substrate of 200 mM sodium aspartate at 25 °C, pH 6.5 in flask-level. Further bioconversion was performed in the fermentor system at the high cell density of 20 OD/mL, and the concentration of extracellular ectoine was increased to 22.5 g/L in 24 h (equivalent to the specific productivity of 0.94 g/L·h), achieving over 6 times of production compared with that in flasks. Significantly, the recombinant strain demonstrated a lower catalytic temperature with the optimum of 25 °C, and a stronger tolerance to the substrate aspartate of 300 mM. These results might provide a compelling case for ectoine synthesis as well as potential applications in large-scale industrial production.
Highlights
Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimdine-carboxylic acid), a heterocyclic amino acid derived from aspartate, is one of the widely used compatible solutes which could be functional as a protective extremolyte under environmental stresses, e.g., extreme temperature, high osmolarity and dryness
The sequencing data showed that ectABCS.salarius was 2416 bp in length and consisted of ectAS. salarius (588 bp), ectBS. salarius (1,266 bp), and ectCS. salarius (393 bp), encoding three proteins with 195 amino acids, 421 amino acids and 130 amino acids, respectively
Previous studies reported several ectoine biosynthesis clusters from different miPreviouscroorganisms, studies reported severalonly ectoine clusters from different micro- elongata or the biosynthesis genetically engineered strain of Halomonas organisms, only the genetically engineered strain of Halomonas elongata or engi- were sucengineered Escherichia coli with the gene clusters derived from this organism neered Escherichia coli with the gene clusters derived from this organism were successfully cessfully applied in current production and marketing at large scale
Summary
One of the ways is through inorganic salts in the cytosol, which could counterbalance the high extracellular salt concentrations [1,2]. Another method is to synthesize small organic molecules called compatible solutes (including sugars, polyols, methylamines, betaines, amino acids and their derivatives) as osmotic counterweights, such as Halorhodospim halochloris [3], Halomonas elongata [4] and Marinococcus halophilus [5,6]. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimdine-carboxylic acid), a heterocyclic amino acid derived from aspartate, is one of the widely used compatible solutes which could be functional as a protective extremolyte under environmental stresses, e.g., extreme temperature, high osmolarity and dryness. Besides being an osmotic protector, ectoine was found to act as skin protector against aging and cell damage and medicine to treat diseases such as Alzheimer’s Disease, atopic dermatitis, colitis, allergic rhinitis, and lung inflammation [7,8,9]
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