Genetic Engineering Toward a 57-Codon Genome

Abstract

Scientific progress in fundamental biology has drastically transformed our ability to engineer biological systems, with diverse applications in medicine and industry, from insulin and artemisin production in prokaryotes to gene therapy in eukaryotes. In particular, DNA synthesis technologies and their recent plumetting cost have the potential to transform genome engineering by unlocking unlimited number of edits independent of the parent genome, paving the way for rational genome-wide changes. For example, a powerful strategy for enhancing genomes with functions not commonly found in nature is offered by recoding, the re-purposing of genetic codons. Indeed, since all living organisms share an identical genetic code composed of 64 genetic codons, changing this code allows exploration of new properties such as genetic isolation and possibilities for expansion of protein function. In that context, we set out to explore the feasibility of the construction of an entirely synthetic 57-codon genome in Escherichia coli. Construction of a 57-codon genome is a daunting task, due to the genome scale and to the unprecedented amount of modifications to perform. When I started my PhD, only the UAG stop codon had been successfully replaced genome-wide. To move forward, better knowledge of the consequences of synonymous codon replacements and improved genome design rules were required. Thus, I focused on understanding the consequences of synonymous codon replacements for two sense codons: AGA and AGG. Then, I tackled the validation and troubleshooting of a 57-codon genome in segments, our major effort toward the validation of the entire genome design in E. coli. Last, I developed safe genomically recoded genes to increase containment of recoded organisms, a major concern in the field of synthetic biology.