Efficient Synthesis of Ectoine in Escherichia coli via Multistep Metabolic Engineering Modification.

Ectoine is a highly valuable amino acid derivative with multiple functionalities, which finds extensive applications in cosmetics, pharmaceuticals, and life sciences. Developing microbial strains capable of high-level ectoine production has emerged as a prominent research focus in recent years. Here, we employed a systematic metabolic engineering strategy to transform wild-type Escherichia coli into a high-yield ectoine-producing strain. First, we constructed a heterologous ectoine synthesis pathway in E. coli W3110. By knocking out the bifunctional enzymes ThrA and MetL, as well as LysA to block byproduct formation and overexpressing an optimized LysC as a substitute, we enhanced the supply of the direct precursor. We also investigated the impact of the copy number on ectoine synthesis. Subsequently, we modified the 5' untranslated region of citrate dehydrogenase gltA to fine-tune its expression, balancing cellular growth with product synthesis. To augment glutamate amino donor availability, we heterologously overexpressed Bacillus subtilis gltAB to enhance glutamate supply, while boosting pntAB 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.