(142c) Engineering Post-Translational Proofreading to Discriminate Non-Standard Amino Acids

One goal of engineers seeking to harness the advantages of biological processes is to expand the repertoire of biocompatible and bio-orthogonal chemistry. The use of non-standard amino acids (NSAAs) beyond the standard twenty amino acids is a salient example. NSAAs expand the structural and functional diversity of proteins ranging from catalysts to therapeutic agents. However, the low fidelity of existing engineered orthogonal translation systems (OTSs) hinders broader adoption of site-specific NSAA incorporation in protein engineering. Current methods to determine OTS fidelity require the low throughput and costly approaches of protein purification and mass spectrometry. To discriminate incorporation of desired NSAAs from incorporation of related standard amino acids into a target protein in vivo, we engineered the first example of a synthetic post-translational quality control system. We achieve Post-Translational Proofreading (PTP) by hijacking and modifying the N-end rule pathway of protein degradation for NSAAs. Under this scheme, desired NSAAs are targeted for incorporation at the N-terminus of a reporter protein, which is stabilized by correct incorporation and destabilized by misincorporation of undesired related standard amino acids. We show that PTP is compatible with a variety of useful reporter proteins. We further demonstrate that PTP is highly tunable by engineering variants of ClpS, the N-end rule discriminator protein, which distinguish incorporation of different types of NSAAs from one another. We illustrate the utility of PTP by incorporating this approach into a fluorescence activated cell sorting (FACS) pipeline for OTS evolution to achieve increased activity and selectivity for two different OTS families on desired NSAA substrates. Due to improved OTS fidelity resulting from this method, we improve the performance of Escherichia coli strains engineered for biocontainment.