Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides

Abstract

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.

Figure 1

Flexizyme-enabled benchtop biosynthesis of thiopeptide scaffolds. a) Biosynthetic gene clusters of thiocillins, thiomuracin GZ, and aesturamide. Genes for key enzymes used in this work are highlighted with asterisks. b) Proposed hybrid route to the thiocillin core.

Figure 2

Flexizyme-enabled benchtop biosynthesis of a thiocillin scaffold. a) Sequence of the designer precursor peptide, including a pMCSG7-derived sequence, the LynD RS and TclM RS. The Trp-codon was reprogrammed to incorporate SecPh. EICs for SecPh and hexathiazole precursor peptide after LynD/TbtE treatment (b), hexathiazole and Dha-containing product after oxidative elimination (c), and fully cyclized thiocillin core (d).

Figure 3

Flexizyme-enabled benchtop biosynthesis of lactazole W2S, S11C. a) Sequence of designer precursor peptide. b) Proposed route to lactazole scaffolds. EICs for the hexathiazole and Dha-containing product of treatment with LynD/TbtE and H₂O₂ (c), and fully cyclized lactazole core (d).