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 ofSaccharomyces cerevisiaein 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. Cre activity can be activated by single red light pulses and easily shut-off via far-red light, while L-SCRaMbLE does not interfere with the host metabolism or hinder growth performance of cell cultures. For the selection of SCRaMbLEd isolates without a selection pressure we developed an efficient and straightforward workflow to identify complex rearranged synthetic chromosomes. The screening method is based on novel genotyping primers, the loxPsym tags, which indicates 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.
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