Engineering Orthogonal Translation Systems

The translation apparatus is the cell’s factory for protein synthesis, stitching amino acid substrates into sequence-defined polymers (proteins) from a defined template. With protein elongation rates of up to 20 amino acids per second and an accuracy of > 99.9%, prokaryotic translation is faster and more accurate than many enzymatic conversions that require only a single biocatalyst and a single reactant. This extraordinary synthetic capability, combined with the ability to produce long polymers sustained by adapter molecules for genetic encodability, has driven extensive efforts to harness the translation apparatus for novel functions. Pioneering efforts have demonstrated that it is possible to genetically encode more than the 20 natural amino acids and that this expansion can be a powerful tool. For example, the site-specific introduction of non-standard amino acids (nsAAs) into proteins has generated novel biological insights and applications that would be difficult, if not impossible, to achieve by other means. Despite establishment of ~100 orthogonal translation systems (OTSs) for nsAA incorporation and a rapidly growing number of cases showing their utility, building designer OTSs with high activity and specificity remains a significant challenge. Common challenges include poor efficiency of engineered OTS components, compatible ribosomes and elongation factors, and constraints arising from the fact that microbial growth and adaptation objectives are often opposed to the engineer’s process objectives. In this presentation, I will discuss our efforts to address these challenges by engineering synthetic ribosomes and using cell-free systems for the synthesis of proteins containing multiple nsAAs. Our work is enabling a deeper understanding of why nature’s designs work the way they do and opening new frontiers for harnessing a dramatically expanded genetic code for manufacturing novel therapeutics, synthesizing genetically-encoded materials, and elucidating fundamental biological insights.