Engineered orthogonal and obligate bacterial commensalism mediated by a non-standard amino acid

Microorganisms can be genetically engineered for intrinsic biological containment based on synthetic chemical provision. However, reliance on an exogenous chemical limits the contexts where a contained microorganism could survive. Here we design an orthogonal obligate commensalism in Escherichia coli that autonomously creates environments permissive for survival of a partner microbe. We engineer one E. coli strain (the producer) to biosynthesize a non-standard amino acid (nsAA) from simple carbon sources through heterologous expression. We engineer a second E. coli strain (the utilizer) to rely on the same nsAA for growth as a synthetic auxotroph, with a 14-day escape rate of 2.8 × 10^-9 escapees per colony-forming unit. Co-culture experiments show utilizer dependence on the producer, with no escape detected during co-inoculation of ~10⁷ colony-forming units of utilizer and a non-producer E. coli strain. Dependence is maintained within a simplified synthetic maize root-associated community. This work provides ecological insights and presents a potential biocontainment strategy independent of an exogenous chemical.