Metabolic Engineering of Escherichia\ncoli for Efficient Production of Ectoine

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