Escherichia coli is a widely studied model organism and an integral component of the human gut microbiome, offering significant potential for bacteria-based therapeutic applications. Despite this promise, engineering native E. coli strains remains challenging. In this study, we employed the chassis-independent recombinase-assisted genome engineering (CRAGE) technique to genetically engineer the native gut strain E. coli EcAZ-1 and the probiotic strain E. coli Nissle 1917 (EcN). We successfully expressed a suite of heterologous genes, including the bioluminescent lux operon, green fluorescent protein (GFP), and oxygen-independent fluorescent protein IFP2.0, in both strains. Optimization of IFP2.0 fluorescence was achieved under both aerobic and anaerobic conditions by coexpressing a heme oxygenase gene and/or supplementing the chromophore biliverdin or hemin. Additionally, we engineered these strains to biosynthesize the bioactive compounds naringenin and mycosporine-like amino acids. This work highlights the potential of native E. coli strains as versatile platforms for synthetic biology, paving the way for innovative applications in biomedical research and therapeutic development.
Keywords: E. coli Nissle 1917; fluorescent proteins; hypoxic conditions; native E. coli; natural products; synthetic biology.