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) Rci from plasmid R64, recognizing
asymmetric sfx 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 sfx 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/sfx pairs. We describe
SI enzymes with substantially improved shuffling rates when expressed
in an inducible manner in E. coli.
Our findings will facilitate the use of SIs in engineering biology
where synthetic shufflons enable the generation of millions of sequence
variants in vivo for applications such as barcoding
or experimental selection.