FX174 Attenuation by Whole Genome Codon Deoptimization

James T. Van Leuven; Martina M. Ederer; Katelyn Burleigh; LuAnn Scott; Randall A. Hughes; Vlad Codrea; Andrew D. Ellington; Holly Wichman; Craig Miller

Synonymous mutations that disrupt important genome compositional features can have substantial phenotypic effects (95) (96) (97) . For example, of 48 cefotaxime resistance mutations in TEM-1 β -lactamase that were identified from a randomly mutagenized pool of variants, 10 were silent (98) . One mutation increased nitrocefin hydrolysis by 7.5-fold, probably by increasing the amount of functional TEM-1 (99) . Our work on ΦX174 also shows that synonymous mutations can have massive fitness effects, as the virus can be completely attenuated by recoding with non-preferred, synonymous codons. Although we introduced many synonymous mutations in each recoded ΦX174 strain, the largest observed fitness impact of any single deoptimized fragment contained only 29 synonymous codon changes. These 29 synonymous changes resulted in a 50% decrease in fitness, which is a decrease of about 10 doublings per hour or about a 1000-fold change in the number of offspring. Aside from using codons that ensure the use of preferred tRNAs, organisms must also balance codon usage with codon pair biases, di-nucleotide preferences, mRNA structural motifs, restriction enzyme site avoidances, etc.