## 4 Discussion  
Previous studies reported several ecotine biosynthesis clusters from different microorganisms, however only the genetically engineered strain of _Halomonas elongata_ or engineered _Escherichia coli_ with the gene clusters derived from this organism were successfully applied in current production and marketing at large scale. And the novel hyperproducers and heterologous production strains with improved characteristics, such as higher catalytic efficiency at low temperature, stronger substrate tolerance and at a certain pH, were suggested to be generated to meet the demand for more efficient production of ectoines in coming research [19; 30; 37]. In this study, we have firstly characterized the novel ectoine biosynthesis genes in _S. salarius_ 1A01339 isolated from the Indian Ocean. The bioinformmatical analyses showed that the three enzymes EctA, EctB and EctC shared the highest identities of 83.3%, 85.7% and 87.7% with reported proteins, respectively. As expected, EctA\({}_{S. salarius}\) had six strictly conserved amino acid residues (Asp55, Tyr60 Trp102, Gln103, His176, and Glu179) and belonged to the typical acetyltransferase family (PF00583). Likewise, EctB\({}_{S. salarius}\) was classified to aminotransferase class-III family (PF00202) containing the fingerprint residues of Lys394, Glu211, Glu384, Tyr17, Leu77, Arg299, Asn295 and Lys268. And EctC\({}_{S. salarius}\) was a typical dimer protein belonged to the cupin superfamily (PF06339) in which Trp23, Arg27, Thr42, Tyr54, Glu59, Tyr87 and His95 were completely conserved. These bioinformmatical analyses suggested that these novel ectoine biosynthetic proteins EctA\({}_{S. salarius}\), EctB\({}_{S. salarius}\) and EctC\({}_{S. salarius}\) most likely have similar activities and properties to the ectoine biosynthetic enzymes from _Halomonas elongata_ and _Chromolobacter salexigens_ and thus, have great potential for industrial production of ectoine. Figure 9: Time profiles of ectoine increasement during fed-batch fermentation. The 2 L-scale bioconversion was carried out in a 5 L-scale bioreactor using engineered strain BL-SsEt (20 OD/mL). Optimized reaction was performed under a flow feed of 200 mM sodium aspartate and the lower temperature of 25 \({}^{\circ}\)C for 24 h. Error bars represent standard deviations from triplicate biological replicates. In order to characterize the enzymatic properties of the \(\text{Et}\text{A}_{\text{S. salarius}}\), \(\text{Et}\text{B}_{\text{S. salarius}}\) and \(\text{Et}\text{C}_{\text{S. salarius}}\), and estimate their potentiality in manufacturing output, the engineered strain _Escherichia coli_ BL21(DE3) harboring \(\text{{ect}ABC}_{\text{S. salarius}}\) operon was cultured in flask and all three proteins corresponding to ectoine synthesis achieved successful expression under the control of _tac_ promoter. Afterwards, this transgenic _E. coli_ BL-SsEct was subjected to whole-cell catalysis reaction for biosynthesis of ectoines using aspartate as substrates. Compared with the reported heterologous productions by different engineered strains as shown in Table 1, _E. coli_ BL21-pDK-_ectABC\({}_{\text{S. salarius}}\)_ exhibited an excellent capability in ectoine biosynthesis and accumulated high extracellular ectoine concentrations (3.28 g/L) at the optimal condition in shaking flasks. Interestingly, this recombinant strain adapted a lower catalytic temperature with the optimum of 25 \({}^{\circ}\)C, and even maintained 80% bioconversion production at 20 \({}^{\circ}\)C, which was a clear difference from the optimal temperature (30-40 \({}^{\circ}\)C) for the bio-catalysis process utilizing recombinant strain harboring ectoine synthesis genes from other microorganisms (Table 2). This was in part because these enzymes were originated from _S. salarius_ 1A01339, which were isolated from the seawater and could produce evolved enzymes with desirable characteristics to survive in this environment.