The study on synthesis of ectoine by metabolic engineering of Escherichia coli

• 1、School of Bioengineering,Jiangnan University ,Wuxi,Jiangsu 214000

Abstract：Halophilic bacteria are highly efficient microorganisms for ectoine synthesis in industry, but the high salt concentration can cause equipment corrosion, which is not conducive to large-scale production. Escherichia coli is an important industrial host bacterium with a clear genetic background and simple nutritional and equipment requirements. In this study, we constructed a heterosynthetic pathway in E. coli for efficient ectoine synthesis. We investigated the effects of gene cluster sequence, plasmid copy number, and key gene ecThe study on synthesis of ectoine by metabolic engineering of Escherichia colitB copy number on ectoine synthesis in E. coli.The results showed that when the recombinant plasmid was constructed using the ectABC gene cluster sequence and pRSFDuet-1 high-copy plasmid as vectors, the yield of ectoine was highest (0.44 g/L). Furthermore, by knocking out diaminopimaric acid decarboxylase on the competing branch, we significantly increased ectoine production (0.541 g/L). Subsequently, overexpression of aspartate semi-aldehyde dehydrogenase, aspartate aminotransferase and aspartate aminolyase were performed to investigate the influence of key gene copy number; the results showed that having three copies of aspA coding genes in the genome had optimal effect. Additionally, overexpressing phosphoenolpyruvate carboxylase coding genes ppc or pyc from Corynebacterium glutamicum enhanced metabolic flow of oxaloacetic acid leading to 4.48 g/L concentration of ectoine in culture medium. Finally,combining all above mentioned operations resulted into construction of high-yield engineered strain. After 72 h batch fermentation in 5 L fermenter ,ectoine yield reached up to 30.4 g/L with glucose conversion rate at 0.194g/g.This study provides good foundation for industrial scale production not only limited to ectoine but also other aspartic acid derivatives.

Keywords： Ectoine Escherichia coli；Metabolic engineering