Generalization of Genetic Code Expansion

Abstract

The standard genetic code directs the assembly of the 20 standard amino acids into proteins and defines function in biology. Through the central dogma, DNA is transcribed into RNA which is translated by the well-understood machinery of the ribosome and accompanying tRNA, using the genetic code to create the proteins that accomplish most tasks in life. The field of genetic code expansion has focused on incorporating synthetic ‘non-standard amino acids’ (nsAAs) with novel chemical structures into the genetic code. This is done by engineering an aminoacyl-tRNA synthetase to conjugate an externally provided nsAA onto an engineered tRNA in vivo such that it will proceed to the ribosome for standard translation, being incorporated into a growing polypeptide chain. Once incorporation has been achieved, nsAAs allow for site-specific encoding of a defined chemical function, without the limitations of the standard genetic code or the requirement of complex protein engineering. With over 150 nsAAs demonstrated in the literature, a broad array of functions are available for experiment and application. However, the contexts in which they can be used are limited. In this thesis, I investigate ways to broaden the applications of existing genetic code expansion tools. I begin with a description of a post-translational proofreading tool capable of distinguishing between proteins successfully charged with a ‘correct’ nsAA and proteins with an ‘incorrect’ nsAA or standard amino acid. We repurposed a natural protein degradation pathway, the N-end rule, to degrade proteins that were not properly charged with the target nsAA. This system could be tuned by engineering an adaptor protein to change the desired nsAA profile, allowing different versions of post-translational proofreading to check for distinct nsAAs. Finally, we demonstrated that this tool improved the purity of desired product for promiscuous genetic code expansion systems and facilitated the directed evolution of more specific genetic code expansion systems. Next, I explore genetic code expansion beyond the optimal conditions of strains specifically engineered to enhance nsAA incorporation. My coauthors and I investigate the use of peptides derived from honeybee antimicrobial molecules which could transiently inhibit competition with genetic code expansion. These peptides allow improved nsAA incorporation into various biotechnologically relevant E. coli strains as well as facilitate the expansion of the Agrobacterium tumefaciens genetic code for the first time. Finally, I apply the tools of genetic code expansion to the bacteria Bacillus subtilis and demonstrate that nearly any nsAA used in E. coli can be applied in B. subtilis using identical synthetases. I explain that nsAA incorporation into native stop codons is much more common than in E. coli, suggesting differences in translational termination between the two organisms. I also utilize nsAAs for translational titration and photocrosslinking in B. subtilis, showing that these tools can be easily utilized for novel kinds of experiments. Together, these tools will help expand the scope of genetic code expansion beyond specifically engineered strains and nsAAs.