With the completion of Sc2.0 chromosomes, synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) becomes more critical for in-depth investigation of fundamental biological questions and screening of industrially valuable characteristics. Further applications, however, are hindered due to the lack of facile and tight regulation of the SCRaMbLE process, and limited understanding of key factors that may affect the rearrangement outcomes. Here we propose an approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion (GCE) technology with low basal activity. By systematically analyzing 1380 derived strains and six yeast pools subjected to GCE-SCRaMbLE, we find that Cre enzyme abundance, genome ploidy and chromosome conformation play key roles in recombination frequencies and determine the SCRaMbLE outcomes. With these insights, the GCE-SCRaMbLE system will serve as a powerful tool in the future exploitation and optimization of the Sc2.0-related technologies.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNA Pyl .

With the completion of Sc2.0 chromosomes, SCRaMbLE becomes more critical for in-depth investigation of fundamental biological questions and screening of industrially valuable characteristics. Further applications, however, are hindered due to the lack of facile and tight regulation of the SCRaMbLE process, and limited understanding of key factors that may affect the rearrangement outcomes. Here we propose a new approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion (GCE) technology with low basal activity. By systematically analyzing 1380 derived strains and six yeast pools subjected to GCE-SCRaMbLE, we find that Cre enzyme abundance, genome ploidy and chromosome conformation play key roles in recombination frequencies and determine the SCRaMbLE outcomes. With these insights, the GCE-SCRaMbLE system will serve as a powerful tool in the future exploitation and optimization of the Sc2.0-related technologies.