Summary We have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron ( NOG2 ) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Laboratory evolution and polyploid SCRaMbLE reveal genomic plasticity to synthetic chromosome defects and rearrangements