Metabolic Engineering of <i>Escherichia coli</i> for Efficient Production of Ectoine

Ectoine is a valuable compatible solute with extensive applications in bioengineering, cosmetics, medicine, and the food industry. While certain halophilic bacteria can naturally produce ectoine, as a model organism for biomanufacturing, <i>Escherichia coli</i> offers significant advantages to be engineered for potentially high-level ectoine production. However, complex metabolic flux distributions and byproduct formation present bottlenecks that limit ectoine production in <i>E. coli</i>. In this study, we aimed to enhance ectoine production in <i>E. coli</i> BL21(DE3) through systematic metabolic engineering strategies. We investigated the effects of the <i>ectABC</i> gene cluster sequence, plasmid copy number, and key gene copy number on ectoine synthesis. Using the original <i>ectABC</i> sequence with the high-copy-number plasmid pRSFDuet-1 resulted in the highest level of ectoine production. Knocking out genes encoding homoserine dehydrogenase and diaminopimelate decarboxylase reduced competing pathways, further increasing ectoine yield. Overexpression of aspartate semialdehyde dehydrogenase, aspartate kinase I (<i>thrA</i>*), aspartate aminotransferase, and aspartate ammonia-lyase (<i>aspA</i>) was performed, and optimal gene copy numbers were determined. When the copy numbers of <i>thrA</i>* and <i>aspA</i> were both three, ectoine synthesis improved, reaching 1.91 g/L. Enhancing the oxaloacetate pool by overexpressing phosphoenolpyruvate carboxylase (<i>ppc</i>) or introducing pyruvate carboxylase (<i>pyc</i>) from <i>Corynebacterium glutamicum</i> further increased ectoine production to 4.99 g/L. Balancing NADPH and ATP levels through cofactor engineering contributed to additional production improvements. Combining these strain engineering strategies, we ultimately constructed strain C24, which produced 35.33 g/L ectoine in a 5 L fermenter with a glucose conversion rate of 0.21 g/g. These results demonstrate that targeted metabolic engineering can significantly enhance ectoine production in <i>E. coli</i>, providing a foundation for industrial-scale production.