(70e) Synthetic Auxotrophy in a Spore-Forming Rhizobacterium Enables Tight Control of Germination

Synthetic biology has demonstrated immense promise in laboratory settings for the ability to control and predict activity of live cellular systems, yet engineered microbes have been slow to deploy given their efficacy in environmentally relevant settings and public concern about genetically modified organisms. Intrinsic biocontainment is one proposed solution for preventing the unintended proliferation of engineered microbes in the environment to help address concerns of biosecurity. Synthetic auxotrophy, the dependence of a microbe on a synthetic nutrient like a non-standard amino acid (nsAA), has been one of the most promising methods of biological containment with undetectable escape of an E. coli synthetic auxotroph, but there are many environments in which E. coli would not be a suitable host. We have adapted this technology to the natural rhizobacterium B. subtilis with the goal of developing an environmentally relevant microbe that is biocontained. We successfully generated synthetic auxotrophs dependent on two distinct nsAAs. For one synthetic auxotroph, we have demonstrated that the strain remains biocontained in minimal media conditions used for growth of tomato and Arabidopsis plants for up to a week. Further, the B. subtilis synthetic auxotroph can form spores that will only germinate in the presence of the synthetic nutrient. Lastly given the community goal to use naturally isolated microbes, we have demonstrated nsAA incorporation and synthetic auxotrophy in a non-domesticated B. subtilis strain UD1022.