SCRaMbLE: A study of its robustness and challenges through enhancement of hygromycin B resistance in a semi-synthetic yeast.

Recent advances in synthetic genomics launched an ambitious project of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, arguably is the most functional synthetic genome-wide alteration to date. The SCRaMbLE system has the capability of performing rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting the long read nanopore sequencing, we demonstrate that all structural variations are due to the recombination between loxP sites thus no off-target effects. During the study, we highlighted a phenomenon imposed on SCRaMbLE termed "essential raft", where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to its essentiality. Despite that, SCRaMbLE was able to explore the genome space and produce alternative structural variations that resulted in an increased hygromycin B resistance in the synII strain. In this study, we showed among the rearrangements generated via SCRaMbLE, deleting YBR219C and YBR220C contribute to hygromycin B resistance phenotype. However, the hygromycin B resistance in SCRaMbLEd were significantly higher compared to the individual single deletions, thus demonstrating the role and the necessity of complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its further improvements would be an invaluable tool to evaluate the emergence of antibiotic resistance in yeast.