Escherichia coli Nissle 1917 (EcN) is a promising chassis strain in synthetic biology, but its application is limited by inefficient genetic manipulation. This study established an efficient genetic manipulation system for EcN via electroporation optimization and recombinase-mediated cassette exchange (RMCE). Systematic screening revealed that using SB medium for cultivation and sterile deionized water as the wash buffer significantly improved EcN electroporation efficiency. Further optimization of electroporation conditions enhanced RMCE efficiency. We constructed the recombinant strain EcN-lox by inserting the loxP-hyg-lox5171 cassette into the EcN genome (replacing the colibactin-synthesizing gene clbB ), which served as a stable landing pad for site-specific integration of exogenous genes via RMCE. Compared with direct electroporation, this RMCE system exhibited superior efficiency in integrating large exogenous DNA fragments, successfully mediating the integration of 17 kb and 29 kb gene cluster segments, while direct electroporation failed to stably maintain large plasmids in wild-type EcN. Finally, the RMCE system was applied to integrate a 10-kb artificial astaxanthin biosynthetic operon into EcN, achieving successful heterologous astaxanthin production. The highest yield (0.627 mg g −1 DCW) was obtained when the recombinant strain was cultured in LB medium at 37 °C for 24 h in shake flasks. Collectively, the optimized electroporation protocol and RMCE-mediated genome integration system developed in this study provide valuable tools for EcN genetic engineering, facilitating its applications in heterologous production of valuable natural products and other synthetic biology fields.
Jiang et al. (Thu,) studied this question.