Expression of Fluorescence Reporters and Natural Products in Native Gut <i>Escherichia coli</i>

<i>Escherichia coli</i> 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 <i>E. coli</i> strains remains challenging. In this study, we employed the chassis-independent recombinase-assisted genome engineering (CRAGE) technique to genetically engineer the native gut strain <i>E. coli</i> EcAZ-1 and the probiotic strain <i>E. coli</i> Nissle 1917 (EcN). We successfully expressed a suite of heterologous genes, including the bioluminescent <i>lux</i> 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 <i>E. coli</i> strains as versatile platforms for synthetic biology, paving the way for innovative applications in biomedical research and therapeutic development.