Multistep Metabolic Engineering of <i>Escherichia coli</i> for High-Level Ectoine Production

Ectoine is an important natural macromolecule protector that helps extremophiles maintain cellular stability and function under high-salinity conditions. Recently, the development of microbial strains for high-level ectoine production has become an attractive research direction. In this study, we constructed an efficient plasmid-free ectoine-producing strain. We modified the 5'-untranslated region of the <i>ectABC</i> gene cluster from <i>Halomonas elongate</i> to fine-tune the expression of genes <i>ectA</i>, <i>ectB</i>, and <i>ectC</i>. Furthermore, we optimized the carbon flow across the MEP pathway, the TCA cycle, and the aspartic acid metabolic pathway. Subsequently, we blocked the production of byproducts from the aspartic acid metabolic pathway and dynamically regulated the TCA cycle to coordinate the balance between strain growth and production. The final strain was tested in a 5-L fermenter, which reached 118.5 g/L at 114 h of fermentation. The metabolic engineering strategies employed in this study can be used for the biosynthesis of other aspartate derivatives.