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.

In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues

“…Importantly, these results have showcased the utility of the in vitro bioengineering of the translation machinery and RiPP modifying enzymes to obtain artificial peptides with noncanonical structures. Although the proof‐of‐concept experiments demonstrated herein focused on the bioengineering of PatD‐catalyzed cyclodehydration, genetic code reprogramming by means of the reprogrammed FIT system can, in principle, be integrated with other RiPP modifying enzymes . Such combinations would further expand the molecular complexity of peptides produced by the artificial in vitro biosynthesis system strategy .…”

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

“…Synthesis of methyllantionine-containing cyclic peptides takes advantage of vinylglycine ( 98 ) (Goto et al, 2009 ). Synthesis of alkylamides uses Aha ( 94 ) or 2-mercaptoethanol-masking homocysteine ( 96 ), and phenyllactic acid ( 62 ) (Nakajima et al, 2009 ), while synthesis of thiopeptide scaffolds utilizes Se-phenylselenocysteine ( 99 ) (Fleming et al, 2019 ).…”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…In addition to the leader peptide, pyridine synthases require C‐terminal recognition elements, possibly to distinguish between multiple potential dehydroalanine reaction partners . Knowledge of the recognition elements enables the biocatalytic application of pyridine synthases on unnatural, possibly synthetic peptide substrates . Still mostly in the dark, however, are catalytic details regarding the [4+2] cycloaddition.…”

“…[78] Knowledge of the recognition elements enables the biocatalytic application of pyridine synthases on unnatural, possibly synthetic peptides ubstrates. [78,80] Still mostlyi nt he dark, however,a re catalytic details regarding the [4+ +2] cycloaddition.…”