Recombinant Production of Ectoine from Virgibacillus salarius in Escherichia coli Using BASIC Assembly.

Ectoine, a widely recognized osmoprotectant noted for its capacity to stabilize biomolecules and diverse biotechnological applications, has attracted considerable research interest. Whole-genome sequencing of Virgibacillus salarius, isolated from the northern Java Sea, revealed a secondary metabolite gene cluster responsible for ectoine biosynthesis. Under 5% (w/v) NaCl,V. salarius produced ectoine at 2.73 × 10-3 g/L during the midexponential growth phase. In this study, the ectoine biosynthetic gene cluster fromV. salarius was reconstructed in Escherichia coli BL21-(DE3), and two expression strategies were evaluated: operon-level expression using a single ribosome binding site (RBS) and gene-specific expression using individually assigned RBS sequences. The results showed that operon-level expression outperformed individual RBS optimization, resulting in higher ectoine production. Furthermore, induction using 5 g/L lactose in this construct enabled the recombinant E. coli BL21-(DE3) to produce ectoine at 1.560 ± 0.013 g/L, representing a 133-fold increase compared to the wild-type V. salarius. FT-IR analysis confirmed the functional groups characteristic of ectoine, and 1H NMR spectroscopy further validated the identity against standard ectoine. In this study, the ectoine biosynthetic gene cluster fromV. salarius was identified and reconstructed for heterologous expression inE. coli. This approach provides a complementary platform for further optimization and biotechnological applications.