Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides

Thiopeptides are
natural antibiotics that are fashioned from short
peptides by multiple layers of post-translational modification. Their
biosynthesis, in particular the pyridine synthases that form the macrocyclic
antibiotic core, has attracted intensive research but is complicated
by the challenges of reconstituting multiple-pathway enzymes. By combining
select RiPP enzymes with cell free expression and flexizyme-based
codon reprogramming, we have developed a benchtop biosynthesis of
thiopeptide scaffolds. This strategy side-steps several challenges
related to the investigation of thiopeptide enzymes and allows access
to analytical quantities of new thiopeptide analogs. We further demonstrate
that this strategy can be used to validate the activity of new pyridine
synthases without the need to reconstitute the cognate prior pathway
enzymes.