Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

Abstract

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE

“…Although we observed the widespread occurrence of genomic rearrangements in all four synthetic chromosomes, the SCRaMbLE recombination frequency seemed to be relatively low. Thus, our finding highlights the importance of applying selective pressure or phenotypic screening to identify cells with genomic rearrangements subjected to the SCRaMbLE system for a broader range of applications, as demonstrated in previous studies 32,33 . We also envision that the recombination frequency mediated by GCE-SCRaMbLE would be further enhanced by improving the suboptimal incorporation efficiency of OMeY into Cre enzyme via the engineering of LeuOmeRS/tRNA CUA pair 34 and eukaryotic release factor 1 (eRF1) that competes with unnatural amino acid insertion in response to stop codons 35 .…”

Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE

“…The other reason might be that no selection was introduced. We predicted that the proportion of SCRaMbLEd cells by GCE-SCRaMbLE would be further increased by applying selective screening as demonstrated in previous studies 32,33 . We con rmed that genome ploidy played a key role in determining SCRaMbLE outcomes, which is in agreement with previous studies using diploids for SCRaMbLE 13 .…”

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

“…The extra wild-type chromosomes increased the survival of the semi-synthetic diploid over a synthetic haploid strain which had a 60 % reduction in cell density after 10 h of Cre-recombinase induction. Although the S and WS strain population densities recovered over longer growth periods, the initial high cell death rates allow cells with limited SCRaMbLE events (which are more fit) to dominate, resulting in an undesired final population with low genotypic diversity (Wightman et al, 2020). The tetraploid strains displayed improved viability during SCRaMbLE, with the WSSS strain containing the largest number of synthetic chromosomes, only losing ∼30% viability.…”