Engineering DNA templated nonribosomal peptide synthesis

Nanocontainers or macromolecular scaffolds for artificial biocatalytic cascades facilitate sequential enzyme reactions but diffusive escape of intermediates limits rate enhancement. Nonribosomal peptide synthetases (NRPS) naturally form gigantic assembly lines and prevent escape by covalently tethering intermediates. Here, we have built DNA-templated NRPS (DT-NRPS) by adding zinc finger tags to split NRPS modules. The zinc fingers direct the NRPS modules to 9-bp binding sites on a DNA strand, where they form a catalytically active enzyme cascade. DT-NRPS outperform previously reported DNA templated enzyme cascades in terms of DNA acceleration which demonstrates that covalent intermediate channeling is possible along the DNA template. Attachment of assembly line enzymes to a DNA scaffold is a promising catalytic strategy for the sequence-controlled biosynthesis of nonribosomal peptides and other polymers.

Reprogramming Nonribosomal Peptide Synthesis by Surgical Mutation

Nonribosomal peptide synthetases produce highly modified bioactive peptides, many of which are used therapeutically. As such, they have been the target of intense protein engineering to enable biosynthetic access to peptide variants with improved drug properties or altered bioactivities. In this account, we describe our ongoing efforts to reprogram nonribosomal peptide synthesis by surgical mutation. In contrast to ribosomal biosynthesis, nonribosomal peptide synthesis has proven difficult to engineer, arguably due to a lack of suitable tools. To address this limitation, we have established a high-throughput assay that provides unprecedented control over the gatekeeper adenylation domains responsible for building block selection and incorporation. Expansion of this strategy to other building blocks and domains promises to make it a powerful evolutionary platform for tailoring assembly lines for custom synthesis of peptide therapeutics.1. Nonribosomal Peptides2. Reprogramming A Domains for Clickable Amino Acids3 A High-Throughput A Domain Assay4 Reprogramming A Domains for β-Amino Acids5 Downstream Processing6 Conclusions and Outlook

HAMA: a multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes

Adenylation enzymes are engineering targets in ribosomal and nonribosomal peptide synthesis. Through multiplexed LC-MS/MS measurement of hydroxamates, the HAMA assay records specificity profiles of these enzymes in a snap.