Evolution inspired engineering of megasynthetases

  • Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

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Author:Kenan A. J. BozhüyükORCiDGND, Leonard Präve, Carsten KeglerGND, Sebastian Kaiser, Yan-Ni Shi, Wolfgang KuttenlochnerORCiD, Leonie Schenk, T. M. Mohiuddin, Michael GrollORCiDGND, Georg K. A. HochbergORCiD, Helge Björn BodeORCiDGND
Parent Title (English):bioRxiv
Document Type:Preprint
Date of Publication (online):2022/12/08
Date of first Publication:2022/12/08
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/09/14
Issue:2022.12.02.518901 Version 2
Edition:Version 2
Page Number:20
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International