Sequential Plasmid Curing and Genome Editing in <i>Escherichia coli</i> Nissle 1917

Nissle 1917 (EcN) is a promising probiotic, but its native cryptic plasmids, pMUT1 and pMUT2, hinder exogenous plasmid transformation and impose a metabolic burden. Here, we established a workflow to sequentially cure these plasmids and perform genome editing using the pEcCas/pEcgRNA system. We first eliminated pMUT1 by targeting the sequence encoding an HTH domain protein. Subsequently, after removing the first pEcgRNA, a second pEcgRNA expressing the RelB antitoxin and targeting the pMUT2 replicase was introduced to eliminate pMUT2. A cryptic plasmid-free EcN strain was obtained after curing the pEcCas and pEcgRNA plasmids. Alternatively, a genome-targeting pEcgRNA can be introduced to modify the probiotic's genome. The cryptic plasmid-free strain exhibited improved transformability with plasmids of various replication origins and enabled efficient heterologous protein expression. This streamlined workflow facilitates iterative editing in EcN and can be adapted for curing endogenous plasmids in other bacteria.