L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes Article Swipe
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· 2022
· Open Access
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· DOI: https://doi.org/10.1101/2022.12.12.519280
· OA: W4312197826
Optimization of the metabolic flux through heterologous pathways to improve bioproduction or utilization of alternative substrates requires both fine-tuning of non-native gene expression levels and improvement of the host genome. The SCRaMbLE system incorporated into synthetic Sc2.0 yeast strains enables a rapid approach to rearrange the genome of Saccharomyces cerevisiae in order to create optimized chassis. Here, we show that the light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or circular synthetic chromosome III. We present an efficient and straightforward workflow for the identification of complex rearranged synthetic chromosomes from SCRaMbLEd isolates without selection pressure. The screening method is based on novel genotyping primers, the loxPsym tags, which indicate not only deletions but also inversions and translocations. Long-read Nanopore sequencing is used to decode the selected genotypes and shows in conjunction with flow cytometry that large-scale karyotype alterations can be a consequence of SCRaMbLE.
