Massively-Parallel Laboratory Evolution of Bacteriophage T7 Reveals Differential Evolutionary Utility of Expanded Genetic Codes

The development of the first synthetic organisms with truly reassigned codons has spurred research projects to determine the potential evolutionary utility of genetically-incorporated nonstandard amino acids in proteins and whole organisms. Initial results have been promising, and have included enzymes with greater activity on novel substrates as well as organisms with improved fitness due to nsAA incorporation in laboratory evolution experiments. We sought a more rigorous quantification of the evolutionary utility of a variety of expanded genetic codes in the context of an evolving biological system. To this end, we evolved 96 independent lines of bacteriophage T7 each on Escherichia coli hosts with six different expanded genetic codes, for a total of 576 independently evolved populations. Each independently-evolved population was deep-sequenced, and the frequency of nsAA incorporation in bacteriophage proteins were compared to the 20 canonical amino acids and each other. The nsAAs were found to be incorporated throughout the proteome of the bacteriophage, including instances of strong signals of positive selection. We also developed a substitution matrix for each nsAA to determine which adjacent canonical amino acids it most readily substituted for in the bacteriophage proteome. Finally, bacteriophage protein variants found to incorporate nsAAs at high frequency, such as T7 RNA polymerase, were assayed to assess the impact of the nsAA on protein function. Our results offer several lessons for the productive use of expanded genetic codes in complex evolving biological systems and biotechnology applications.