A new set of mutations in the second transmembrane helix of the Cox2p-W56R substantially improves its allotopic expression in <i>Saccharomyces cerevisiae</i>

The dual genetic control of mitochondrial respiratory function, combined with the high mutation rate of the mitochondrial genome (mtDNA), makes mitochondrial diseases among the most frequent genetic diseases in humans (1 in 5,000 in adults). With no effective treatments available, gene therapy approaches have been proposed. Notably, several studies have demonstrated the potential for nuclear expression of a healthy copy of a dysfunctional mitochondrial gene, referred to as allotopic expression, to help recover respiratory function. However, allotopic expression conditions require significant optimization. We harnessed engineering biology tools to improve the allotopic expression of the COX2-W56R gene in the budding yeast Saccharomyces cerevisiae. Through conducting random mutagenesis and screening of the impact of vector copy number, promoter, and mitochondrial targeting sequence, we substantially increased the mitochondrial incorporation of the allotopic protein and significantly increased recovery of mitochondrial respiration. Moreover, CN-PAGE analyses revealed that our optimized allotopic protein does not impact cytochrome c oxidase assembly, or the biogenesis of respiratory chain supercomplexes. Importantly, the most beneficial amino acid substitutions found in the second transmembrane helix (L93S and I102K) are conserved residues in the corresponding positions of human MT-CO2 (L73 and L75), and we propose that mirroring these changes could potentially help improve allotopic Cox2p expression in human cells. To conclude, this study demonstrates the effectiveness of using engineering biology approaches to optimise allotopic expression of mitochondrial genes in the baker's yeast.