Engineering the Escherichia coli Nissle strain for monitoring the bacterial cell distribution and therapeutic protein expression within the intestinal tract of animal models.

The probiotic Escherichia coli Nissle 1917 (EcN) has been developed as a therapeutic carrier capable of enabling in vivo production of functional proteins. To optimize these processes, precise selection of promoters and monitoring of heterologous protein expression are important. Here, we designed a hypoxia-induced expression system in EcN by integrating a bicistronic cassette under the control of the P_vhb promoter. This construct enabled simultaneous transcription of oxdC (encoding oxalate decarboxylase, OxdC) and mCherry (a fluorescent reporter gene), achieving co-expression of both therapeutic and reporter proteins under hypoxic conditions. We confirmed that the P_vhb promoter efficiently initiated oxdC and mCherry co-expression under both in vitro hypoxic culture conditions and in vivo hypoxic environments within the intestinal tracts of animal models. Crucially, this system establishes mCherry as a noninvasive indicator for dual monitoring of probiotic localization and therapeutic protein expression within animal intestinal tracts. This work provides valuable insights for designing novel engineered bacteria for disease treatment.