Efficient Synthesis of Ectoine in <i>Escherichia coli</i> via Multistep Metabolic Engineering Modification

expression to maintain cofactor equilibrium. Similarly, we fortified the glucose-to-oxaloacetate synthetic pathway through a series of metabolic modifications, achieving a yield of 5.94 g/L in shake flask fermentation. Finally, under controlled batch glucose feeding, strain E20 produced 88.1 g/L of ectoine over a 60 h fermentation period, and a glucose conversion rate of 0.26 g/g. This study employed metabolic engineering strategies to enhance the accumulation of oxaloacetate and utilized 5'-UTR engineering to finely regulate GltA expression, thereby balancing cell growth, These strategies, including increasing aspartate accumulation, enhancing the catalytic efficiency of the key heterologous enzyme LysCpa in the aspartate to aspartate phosphate pathway, and boosting glutamate as an amino donor to enhance the synthesis of aspartate from oxaloacetic acid, can be applied to the synthesis of other amino acids in the aspartate family.