Abstract:Ectoine is widely used in medicine, cosmetics and other fields. This study aimed to construct recombinant Escherichia coli strain to synthesize ectoine to solve the dependence of wild-type strain on high salt environment. Ectoine synthesis encoding gene cluster ectABC was cloned from Halomonas elongata ATCC 33173 and reconstructed in Escherichia coli. In order to confirm a suitable host, four commonly used E. coli strains, i.e., E. coli W3110, E. coli DH5α and E. coli (asp) and E. coli BL21(DE3) were compared, and E. coli BL21(DE3) was found to be the best host for ectoine synthesis(185.23 mg/L). Further analysis of the effect of expression systems with different copy numbers on ectoine synthesis was carried out, and the results showed that pRSFDuet-1 with high copy number generated the highest titer of ectoine(267.3 mg/L). On this basis, the ribosomal binding site optimization strategy was introduced to optimize the expression of three enzymes including EctA, EctB and EctC, producing ectoine to 521.24 mg/L. To increase the supply of precursors aspartate and aspartate semialdehyde, aspartate kinase, aspartate hemialdehyde dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate lyase were overexpressed. The results demonstrated that the single overexpression of aspartate kinase was beneficial to ectoine biosynthesis and the production was eventually enhanced to 551.24 mg/L. The present study provides a new strategy to construct ectoine-producing strain.
