An easy engineerable bacterial chassis opens a spectrum of biomedical applications such as antigen or drug delivery. However, most cell-walled bacteria are readily recognized and cleared by the immune system and encode various effector proteins potentially interacting with the target species. Mycoplasma feriruminatoris, a cell-wall-less minimal organism with excellent growth attributes, was genetically engineered to stably and almost seamlessly incorporate foreign DNA fragments into the chromosome in a single step using an orthogonal recombinase system. Using this mechanism, we characterized and developed promoters and proved their functionality, including a kill-switch based on a temperature-sensitive promoter that drives expression of a toxin from a toxin-antitoxin system. Additionally, we designed a streptavidin-based functional dynamic display system at the surface of the bacterium. The engineered strains have great potential such as to deliver vaccine antigens to different livestock species among other biomedical applications.
