The impact of synthetically expanded genetic codes on evolvability

Synthetic biology allows researchers to address previously unanswerable biological questions through the wholesale reengineering of living systems. Even one of the most fundamental properties of biology – the canonical genetic code for the translation of genetic information into proteins – may now be altered to include nonstandard amino acids (nsAAs) because synthetic incorporation systems for nsAAs now match native host machinery in terms of efficiency and fidelity. However, the evolutionary stability of these systems and the potential for them to promote the evolvability of the organisms in which they are deployed remains mostly unexplored. In chapter one of this dissertation, I explore how Escherichia coli strains that incorporate a 21st nsAA at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. I found a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance, and I interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways. I consider the implications of these results for the evolution of alternative genetic codes, in particular their support for the codon capture model of genetic code evolution. In chapters 2 and 3, I employ bacteriophage T7, which utilizes the translation apparatus of its E. coli host, as a model organism for whole-genome evolution with an expanded genetic code. I show that phages evolved on an E. coli host that incorporates 3-iodotyrosine became dependent on an alternative genetic code as they adapted to higher fitness. In particular, phages with a 3-iodotyrosine substitution in the type II holin protein outcompete phages with canonical amino acids at this position. Inspired by this surprising outcome, I performed massively parallel evolution experiments in T7 with six expanded genetic codes, finding over a thousand substitutions to nsAAs in these experiments, many of which displayed genomic signatures of positive selection. Two of the nsAAs accumulated in genomes at a rate comparable to a mock genetic code expansion with the canonical amino acid tyrosine. Together these results show that expanded genetic codes alter functional sequence space and create new possibilities for adaptive mutations, thereby improving evolvability in these organisms.