[Engineered probiotic Escherichia coli Nissle 1917 for controlled delivery of lycopene]

Escherichia coli Nissle 1917(EcN), a non-pathogenic probiotic strain isolated from the human gut, exhibits intrinsic intestinal colonization, tumor-targeting capability, and proven biosafety, which make it an ideal live delivery system. This study engineered EcN to achieve dual functionality in intestinal colonization and controlled release of active compounds. Starting with wild-type EcN, this study used lycopene biosynthesis as a proof-of-concept. First, lycopene biosynthetic genes under arabinose-inducible promoters were integrated into the EcN genome via CRISPR/Cas9, which generated the base strain EcN-C1. Subsequent overexpression of rate-limiting genes in the endogenous methylerythritol phosphate(MEP) pathway: including isopentenyl diphosphate isomerase(idi), 1-deoxy-D-xylulose-5-phosphate synthase(dxs), and farnesyl diphosphate synthase(ispA): enhanced lycopene production by 4.28-fold. Further integration of a heterologous mevalonate(MVA) pathway augmented precursor supply, resulting in a further 2.58-fold increase in yield, elevating titer to(15.24±0.87)mg·L~(-1)(11.04-fold over initial strain). Dose-and time-dependent arabinose induction enabled precise control of lycopene release across engineered strains: EcN-C1 to EcN-C4 exhibited titers spanning 0.02-1.38, 0.22-2.81, 0.67-5.90, and 1.24-15.24 mg·L~(-1), respectively. This system achieved an 800-fold dynamic range(0.02-15.24 mg·L~(-1)), demonstrating fine-tuned control over compound delivery. This work lays an important foundation for the development of novel delivery systems based on probiotics for active compound release.