Advances in DNA synthesis technology have led to rapid growth in the field of synthetic biology, heralding a nascent era of synthetic genomics. Sc2.0 (Saccharomyces cerevisiae version 2.0) is an international consortium with the aim of designing and constructing a fully‐synthetic eukaryotic genome. Fundamental design changes to the synthetic genome include the removal of unstable tRNA genes and their intended collation onto a “tRNA neochromosome”, with the aim of producing a more robust and stable synthetic genome structure. To maintain viability of a synthetic yeast, the tRNA neochromosome is therefore considered an important if not essential aspect of this project. The application of engineering principles is synonymous with synthetic biology, regularly employing the recursive Design‐Build‐Test cycle to improve experimental approach. This doctoral study explores the design, construction and characterisation of a tRNA neochromosome in Saccharomyces cerevisiae. A series of design principles influenced by engineering concepts were used to rationalise the complexities of de novo chromosome engineering, maximise its stability and ensure function in vivo. A methodology based on in vivo homologous recombination was then developed and shown to reliably construct the neochromosome from its constituent parts. Experimental characterisation revealed that genetic elements function as expected, and that the parental strain can tolerate the sole presence of one each of three single‐copy, essential tRNA genes (SUP61, TRT2 and TRR4), although Northern blot revealed potential precursor accumulation of the SUP61 tRNA caused by the presence of a synthetic 5’ flanking sequence. Following the addition of synthetic telomere seed sequences, pulsed‐field gel electrophoresis (PFGE) and deep sequencing revealed complex structure variations in two independent strain backgrounds. Except for these structural variations, successful neochromosome construction demonstrated the applicability of the approaches used and the remarkable ability of the yeast model to support the presence of a 17th chromosome housing an additional 275 tRNA genes. The research in this thesis has for the first time described the design, construction and characterisation of a eukaryotic neochromosome de novo. It is hoped that the findings presented will further our understanding of tRNA biology and enhance the aims of the Sc2.0 project.

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Le complexe bc₁ de la chaîne respiratoire mitochondriale est une bonne cible thérapeutique pour traiter le paludisme car cette enzyme est essentielle au parasite. Ses deux sites actifs, Qo et Qi, formés par le cytochrome b, ne sont pas totalement conservés entre les espèces, facilitant la découverte d’inhibiteurs à affinité différentielle, ce qui est important dans le développement de médicaments. L’atovaquone est le seul antipaludique ciblant le complexe bc₁ utilisé en médecine. L’émergence de résistance rend urgente l’étude de nouveaux inhibiteurs. Les ELQs (Endochin-like Quinolones) sont une classe d’antipaludiques particulièrement prometteuse.Pour étudier la liaison des inhibiteurs dans les sites actifs et l’effet de mutations de résistance, nous utilisons la levure et des méthodes biochimiques et bio-informatiques. Dans ce travail, nous avons étudié la relation entre mutations de résistance à l’atovaquone dans le site Qo et perte de fonction. Nous avons aussi modifié le site Qo de la levure pour qu’il mime mieux le site de l’enzyme du parasite. Les résidus «Plasmodium» altèrent le fonctionnement du site, résultant en une surproduction d’ions superoxides et une perte de croissance respiratoire, qui est restaurée par la modification d’une autre sous-unité du complexe, ISP, partenaire du site Qo, suggérant que les deux sous-unités doivent s’ajuster pour un fonctionnement correct. Nous avons analysé des polymorphismes de la région Qo observés chez l’Homme et trouvé qu’ils peuvent modifier la sensibilité du complexe à l’atovaquone, ce qui pourrait avoir un impact sur les effets secondaires du traitement. Nous avons ensuite étudié le mode d’action d’ELQ-400 et montré que ce nouvel antipaludique cible les deux sites Qo et Qi, ce qui rend l’apparition de résistance peu probable. Enfin, nous avons commencé la reconstruction du site Qi de la levure pour mimer le site du parasite.Les mutants de levure avec un complexe bc₁ «Plasmodium» semblent être de bons outils pour l’étude des inhibiteurs. Leur étude a aussi permis de comprendre mieux la structure et le fonctionnement du complexe bc₁. / The bc₁ complex of the mitochondrial respiratory chain is a good therapeutic target for the treatment of malaria as the enzyme is essential for pathogen proliferation. The two catalytic sites, Qo and Qi, formed by cytochrome b, are not fully conserved between species, facilitating the development of inhibitors with differential saffinity, which is important for the development of new drugs. At present, Atovaquone is the only antimalarial drug targeting the bc₁ complex used in medicine. The emergence of resistance makes it important to find new inhibitors, and the ELQs (Endochin-like Quinolones) are promising antimalarial candidates.In order to study the inhibitor binding to the active sites and the effect of resistance mutations, we have used yeast and a combination of biochemical and bioinformatic methods. We have studied the relationship between atovaquone resistance mutations in the Qo site and loss of function. We have also modified the yeast Qo site to make it more like the parasite site. The “Plasmodium” residues in the yeast Qo site altered its activity, which resulted in the overproduction of superoxide and the loss of respiratory growth. This could be restored by the modification of another bc₁ complex subunit interacting with the Qo site, ISP, suggesting that both these subunits need to be readjusted for correct activity. We then analyzed polymorphisms of the Qo region reported in Humans and found that they could alter the enzyme sensitivity to atovaquone, which could impact the side-effects linked to atovaquone treatment. We have also studied the mode of action of ELQ-400 and showed that this new antimalarial drug targets both the Qo and Qi sites, which would make the emergence of resistance less likely. Finally, we have started the reconstruction of yeast Qi site to make it resemble the parasite site.The yeast mutants with a “Plasmodium-like” bc₁ complex could be useful tools for the study of antimalarial drugs. These analyses have also resulted in a better understanding of the structure and function of the bc₁ complex.

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