The genome evolves by changing its sequence, content, and arrangement over time, but the extent to which these aspects can be changed remains unknown.
More...The genome evolves by changing its sequence, content, and arrangement over time, but the extent to which these aspects can be changed remains unknown. Synthetic genomics provides a bottom-up strategy to explore these questions beyond long-term natural evolution. Here, we use the left arm of chromosome XII as a pilot for a new way to probe yeast genome plasticity. A neochromosome was designed to facilitate the relocation of essential genes dispersed throughout chrXIIL. We demonstrated that their chromosomal location and transcriptional direction do not restrain the function of these genes after constructing several versions of this essential gene neochromosome. Utilizing a partially synthetic chromosome XII, we systematically probe the essentiality of sequences in chrXIIL by targeted DNA deletion, telomere capping, and random deletion using SCRaMbLE. And just 12 genes on this arm were sufficient for cell viability. However, additional genes are required for maximal fitness. We found that reducing the entire left arm to only 25 genes gives near wild-type fitness.
Furthermore, we tested whether these 25 genes could be reconstructed using aggressively altered coding sequences and completely synthetic promoters and terminators. We demonstrated that ORFs with each amino acid encoded only by its optimized codon remained functional. In addition, we showed that although each reconstructed transcription unit (TU) could replace its native counterpart individually when assembled together, an essential neochromosome with only 12 reconstructed TUs failed to support cell viability, suggesting the presence of subtle differences between the neochromosome of reconstructed TUs and the one with corresponding native sequences. Finally, by adding back additional genes, we succeeded in constructing a completely refactored neochromsome, which could substitute for chrXIIL for cell viability. Our work not only highlights the previously unimaginable plasticity of the yeast genome but also illustrates the feasibility of computationally designed living systems. Less...