Metabolic engineering of the probiotic Escherichia coli Nissle 1917 for lacto-N-triose II production

Abstract

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. In recent years, the development of metabolic engineering has promoted the application of EcN in synthetic biology. Lacto-N-triose II (LNT II), as an important component in breast milk, has unique benefits for infant health. This study introduces a metabolic engineering method to produce LNT II in EcN. Firstly, construct promoters with different expression intensities to optimize transcriptional regulation levels and further fine-tune plasmid copy numbers to optimize gene expression levels. The highest titer of LNT II was detected when the lgtA gene was expressed under the tac promoter and pETDuet-1 vector. Secondly, the gene editing efficiency of the EcN strain was improved by knocking out the endA gene (encoding the endonuclease I). Then, a series of engineered strains were constructed by knocking out the wecB gene and cryptic plasmid. It was found that the deletion of the wecB gene increased the titer of LNT II, but the elimination of the cryptic plasmid had no effect. Finally, we validated the production performance of the engineering strain E13 in a 3-L bioreactor with batch feeding culture. The LNT II titer reached 2040 mg/L, which was 6.6 times higher than that of shake flask cultivation and has potential for industrial application.