Translation in a Box: Orthogonal Evolution in theSaccharomyces cerevisiaeMitochondrion

Abstract

The synthesis capabilities of the translation system (TS) provide engineering and directed evolution potential as well as a system for generating novel polymers. Orthogonal translation systems offer a parallel platform for translation engineering, enabling primary and secondary translation systems to operate independently without interfering with each other. However, current translation platforms for engineering lack complete orthogonality between the primary and secondary systems. Using a combination of indirect modifications through antibiotics, truncations of mito-rProteins mL50 and uL23, and replacement of mito-rProtein uL2, we develop and test the mitochondrial translation system in Saccharomyces cerevisiae as a fully orthogonal platform for in vivo directed evolution of translation. Our results show that continuous, comprehensive, creative, directed-evolution of a fully orthogonal TS in vivo appears to be a useful approach for technical control of the TS. This study emphasizes the power of system-wide evolutionary approaches over rational design. Evolution is creative and non-linear and can find unexpected solutions to problems.