UC Irvine

The use of an expanded genetic code for unnatural amino acid (UAA) incorporation in living cells has thus far enabled the precise modification of protein function. However, achieving programmed ribosomal synthesis of fully synthetic polymers requires both free space in the genetic code and mutually orthogonal aminoacyl tRNA synthetase (aaRS)/tRNA pairs, two conditions that scale with the number of distinctly encoded novel chemistries in the polypeptide. To address these constraints, we repurposed yeast mitochondria into centers for orthogonal translation, taking advantage of both the nonessentiality of mitochondrial DNA (mtDNA) and the already-distinct ribosome and tRNAs used for mitochondrial translation. By expressing engineered mito-aaRS variants in yeast, we demonstrate the sensitivity of mitochondrially-encoded proteins to multiple UAAs. We also observe tRNA charging via mutant synthetase in both yeast and mammalian mitochondria via bioorthogonal labeling and microscopy. Lastly, we implement a series of expression-boosting genetic optimizations and construct a mitochondrial protein purification-to-MS analysis pipeline. Taken together, these results lay the groundwork for further engineering of mitochondrial translation towards the goal of unnatural polymer synthesis.