In Vitro Fermentation Evaluation of Engineered Sense and Respond Probiotics in Polymicrobial Communities.

Synthetic biology provides a means of engineering tailored functions into probiotic bacteria. Of particular interest is introducing microbial sense and response functions; however, techniques for testing in physiologically relevant environments, such as those for the intended use, are still lacking. Typically, engineered probiotics are developed and tested in monoculture or in simplified cocultures still within ideal environments. In vitro fermentation models using simplified microbial communities now allow us to simulate engineered organism behavior, specifically organism persistence and intended functionality, within more physiologically relevant, tailored microbial communities. Here, probiotic bacteria Escherichia coli Nissle and Lactobacillus plantarum engineered with sense and response functionalities were evaluated for the ability to persist and function without adverse impact on commensal bacteria within simplified polymicrobial communities with increasing metabolic competition that simulate gut microbe community dynamics. Probiotic abundance and plasmid stability, measured by viability qPCR, decreased for engineered E. coli Nissle relative to monocultures as metabolic competition increased; functional output was not affected. For engineered L. plantarum, abundance and plasmid stability were not adversely impacted; however, functional output was decreased universally as metabolic competition was introduced. For both organisms, adverse effects on select commensals were not evident. Testing engineered probiotics in more physiologically relevant in vitro test beds can provide critical knowledge for circuit design feedback and functional validation prior to the transition to more costly and time-consuming higher-fidelity testing in animal or human studies.