Engineering a ‘Synthetic Auxotroph’ for Environmental Containment of Microorganisms

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Robin Prince, Nina DiPrimio, Gabriel Lopez, J. Christopher Anderson

University of California, United States

Synthetic biology is enabling new applications for cellular engineering, and with this comes a growing need to focus on biosafety for responsible containment of engineered organisms. We are taking a novel approach to address this concern by engineering an E. coli strain that is dependent on an inexpensive, synthetic small molecule for cell survival to create a ‘synthetic auxotroph’. Previous research has shown that indole-dependent allosteric control can be engineered into enzymes by mutating a buried tryptophan residue to glycine and rescuing protein function with indole. We utilized this strategy to identify sensitive sites in the protein core of homodimeric essential gene products to serve as a starting point for further engineering into a ligand-binding pocket. Candidates that displayed a disruption of dimerization upon tryptophan to glycine mutation and rescue in the presence of indole were subjected to saturation mutagenesis of neighboring residues to generate a ligand binding pocket library. Negative and positive selection systems based on the lambda repressor two-hybrid system were constructed and coupled with deep sequencing to identify library members which dimerize only in the presence of a synthetic small molecule of interest. With an essential protein that is dependent on a small molecule for proper protein folding and function, we can engineer a bacterial strain that is dependent on rescue with a synthetic chemical for survival.