An evolutionary approach to enable reprogramming of non-ribosomal peptide enzymology

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Non-ribosomal peptides are naturally produced by many bacteria and have been an excellent source of therapeutics, most notably new antibiotics. The synthesis of many of these peptides involves a class of enzymes called non-ribosomal peptide synthetases. These enzymes function in a manner analogous to an assembly line with enzyme workstations or 'modules' that coordinately build the peptide one amino acid at a time. The goal of this project is to learn the rules for mixing and matching the workstations from different non-ribosomal peptide synthetases. Ultimately, this would allow researchers to construct hybrid enzymes that synthesize novel peptides and then screen the peptides for therapeutic properties. This research will provide cross-disciplinary training due to the collaborative effort between faculty in the biological sciences and engineering sciences. This cross-disciplinary approach will be integrated into a formal undergraduate capstone course, an undergraduate summer research program, and outreach to K-12 students. This approach will also expose the students to basic scientific questions (e.g. substrate recognition) and show the students how this information can be applied to an important goal (e.g. production of designer molecules). During non-ribosomal peptide assembly, the adenylation (A) domains recognize specific amino acids and tethers them to partner peptidyl carrier protein (PCP) domains. Condensation (C) domains subsequently form amide bonds between two neighboring PCP-tethered amino acids in a directional manner, forming the peptide backbone. Structural and biochemical studies have identified A domain specificity codes that define the amino acid recognized by the domain. While this code has proved enormously useful in predicting the amino acid activated, a number of studies have shown that altering the residues that define this code nearly always fail to switch substrate specificity. Furthermore, complete domain or module swaps generating chimeric non-ribosomal peptide synthetases generally fail to function efficiently, likely due to improper protein-protein interactions or substrate specificity of the associated C domains. This project will investigate the reprogramming of both A and C domains of non-ribosomal peptide synthetases using in vivo directed evolution approaches. Using these approaches, this study aims to define a means for generating functional chimeric A domains, identify new A domain specificity codes for altering amino acid recognition, and understand the residues governing substrate recognition by C domains. Results from these studies will aid our ability to rationally reprogram the biosynthesis of this important class of natural products to generate designer molecules for a variety of applied purposes, while also gaining insights into how nature has accomplished this goal.