Do the Shuffle: Expanding the Synthetic Biology Toolkit for Shufflon-like Recombination Systems.

Naturally occurring DNA inversion systems play an important role in the generation of genetic variation and adaptation in prokaryotes. Shufflon invertase (SI) from plasmid R64, recognizing asymmetric sites, has been adopted as a tool for synthetic biology. However, the availability of a single enzyme with moderate rates of recombination has hampered the more widespread use of SIs. We identified 14 previously untested SI genes and their sites in public databases. We established an assay based on single-molecule sequencing that allows the quantification of the inversion rates of these enzymes and determined cross-recognition to identify orthogonal SI/ pairs. We describe SI enzymes with substantially improved shuffling rates when expressed in an inducible manner in . Our findings will facilitate the use of SIs in engineering biology where synthetic shufflons enable the generation of millions of sequence variants for applications such as barcoding or experimental selection.