Synthetic Zippers as an Enabling Tool for Engineering of Non-Ribosomal Peptide Synthetases

Abstract

14Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural 15 products, including many clinically used drugs. Engineering of these often giant 16 biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) at high titre 17 is an ongoing challenge. Here we describe a strategy to functionally combine NRPS 18 fragments of Gram-negative and -positive origin, synthesising novel peptides at titres 19 up to 290 mg l -1 . Extending from the recently introduced definition of eXchange Units 20 (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into up to three 21 independently expressed and translated polypeptide chains. These synthetic type of 22 NRPS (type S) enables easier access to engineering, overcomes cloning limitations, 23 and provides a simple and rapid approach to building peptide libraries via the 24 combination of different NRPS subunits. 25 26 27 One Sentence Summary: 28 Divide and Conquer: A molecular tool kit to reprogram the biosynthesis of non-29 ribosomal peptides.30 31 32 33 Non-ribosomal peptide synthetases (NRPSs) are multifunctional enzymes, producing 34 a broad range of structural diverse and valuable compounds with diverse applications 35 in medicine and agriculture (1) making them key targets for bioengineering. The 36 structural diversity of non-ribosomal peptides (NRPs) arises from the assembly line 37 architecture of their biosynthesis. According to their biosynthetic logic, known NRPS 38 systems are classified into three groups, linear (type A), iterative (type B), and 39 nonlinear NRPSs (type C) (2). Type A NRPSs are composed of sequential catalytically 40 active domains organised in modules, each responsible for the incorporation and 41 modification of one specific amino acid (AA). The catalytic activity of a canonical 42 module is based upon the orchestrated interplay of an adenylation (A) domain for AA 43 selection and activation, a condensation (C) domain to catalyse peptide bond 44 formation, and a thiolation/ peptidyl-carrier protein (T) onto which the AAs or 45 intermediates are covalently tethered (3). In addition, tailoring domains, including 46 epimerization (E), methylation, and oxidation domains can be part of a module, or a 47 heterocyclization (Cy) domain instead of a C-domain can be present. Finally, most 48 NRPS termination modules harbour a TE-domain, usually responsible for the release 49 of linear, cyclic or branched cyclic peptides (4).50Type A NRPSs (Fig. 1a) follow the collinearity rule, i.e. the number of NRPS modules 51 corresponds directly to the number of monomers incorporated into the associated 52 product, and the arrangement of the modules directly follows the peptides primary 53 sequence (5). Whereas in in cis type A NRPSs all modules are arranged on a single 54 polypeptide chain (e.g. ACV-synthetase (6)), in trans assembly-lines comprise a 55 number of individual proteins (Daptomycin-synthetase (7)). Mutual protein-protein 56 interactions of the latter are mediated by specialized C-(donor) and N-...

Bifurcation drives the evolution of assembly-line biosynthesis

“…Finally, the question arises as to whether our knowledge of the NRPS evolution can also be used to develop new engineering approaches. Recently, several efficient and/or highly productive engineering strategies have been published [58][59][60][61] . Yet efficient engineering of these often-huge biosynthetic machineries to produce novel bioactive NRPs is an ongoing challenge.…”

Nonribosomal Peptide Synthesis

“…Smaller heterocyclic rings, such as thiazole in 9, thiazoline in 13, or oxazoline in 14, are common structural properties of nonribosomal peptides. In addition, fatty acids (3), glycosylations (5), N-methylations (7), and N-formylations (18) may also be present, as well as the addition of propionate units (8) or acetate [14].…”