Escherichia coli Nissle 1917 (EcN) is an endotoxin-free probiotic in the treatment of gastrointestinal diseases. However, two cryptic plasmids are stable in EcN causing metabolic burden, which limits its application in genetic engineering. In this study, two different strategies were employed to enhance the applicability of EcN. First, CRISPR/Cas9-based plasmid curing was designed with two sgRNA targeting sites for each cryptic plasmid. A plasmid-free EcN called EcNP, was successfully and efficiently constructed within one week. EcNP could overexpress sfGFP with high intensity under constitutive promoter J23100. The plasmid copy number (PCN) in EcNP corresponds to the type of replication origin of the plasmid which is in the order of pUC > pMB1 > p15A, consistent with other E. coli. Second, two stable expression vectors based on the cryptic plasmid, pMT1 and pMT2, were developed and showed a high-level expression of sfGFP and RFP, respectively. The stability of pMT1 and pMT2 were superior to other plasmids with 100 % maintenance over 8 passages for every 12 h. Finally, we demonstrated the expression of gadB gene in the stable vector pMT1, and expressed in EcNP, with 17.9 g/L of gamma-aminobutyric acid (GABA) production in an antibiotic-free system. Thus, EcNP and the plasmid pMT1 paved the way to efficiently engineer E. coli Nissle as live therapeutics.
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