Fig. 1

Schematic overview of the SCRaMbLE-in toolkit for metabolic engineering. Once designed, the synthetic pathway of interest will be rapidly prototyped in vitro. The pathway is pre-assembled in such a way that all genes are assembled with respective regulatory elements, except that one key gene is left “promoter-less”, having a recombination site upstream. Three recombination systems, Cre-loxP, VCre-Vlox, and Dre-rox, were developed to integrate a range of promoters upstream to the key gene in the pathway. The assembled library is transformed into yeast and potentially productive pathways are selected and integrated in vivo in the next step. The selected productive pathways are flanked with a pair of loxPsym sites and can subsequently be integrated into the synthetic chromosomes through SCRaMbLE, and simultaneously the synthetic yeast chassis undergoes whole-genome rearrangement to result in strain optimization. The successful integrated strains will be profiled with mass spectrometry analysis and next-generation sequencing