Characterizing Pseudomonas Putida to Engineer a Novel Synthetic Auxotroph

• Defne Elbeyli, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware

Microbes are being engineered to address issues in both environmental and public health sectors. However, these engineered organisms require a biocontainment strategy in order to be deployed outside of a laboratory setting to prevent proliferation in unintended regions. Synthetic auxotrophy is an effective form of biocontainment that relies on redesigning an essential gene to depend on a nonstandard amino acid (nsAA) to maintain vital cellular activities. The Kunjapur lab has successfully created multiple synthetic auxotrophs using multiplex automated genome engineering (MAGE) to target and redesign various essential genes. There are several synthetic auxotrophs that exist in Escherichia coli that are dependent on different nsAAs and involve various genome modifications, but engineered E. Coli is not applicable in many sectors due to public perception and its inability to thrive in many environmental niches. The more relevant Pseudomonas putida, however, has the potential to be a workhorse for industrial biotechnology ranging in applications from biocontainment to bioremediation to biosensing with its hardiness under physicochemical stress, natural environmental niche, and HV1 certification from the FDA. Relying on the Kunjapur lab’s extensive experience with engineering synthetic auxotrophs, we must first characterize P. Putida’s growth dynamics so we can determine if tools used previously to engineer E. Coli synthetic auxotrophs, specifically MAGE, will be viable in P. Putida. We need to additionally investigate the behavior of P. Putida in the presence of our chosen nsAA and then rely on the lab’s previous knowledge of creating E. Coli synthetic auxotrophs to translate that machinery into P. Putida.