Artificial Splitting of a Non‐Ribosomal Peptide Synthetase by Inserting Natural Docking Domains

Kegler, Carsten; Bode, Helge B.

doi:10.1002/ange.201915989

Cited by 15 publications

(9 citation statements)

References 39 publications

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“…Artificial splitting of NRPS proteins into modules and reconnection with docking domains has yielded good amounts of protein and high peptide yields upon heterologous co-expression before. 34 Therefore, we retrieved C-terminal (Dc) and Nterminal (Dn) docking domains mediating module interactions in xenortide biosynthesis from InxAB (InxA-Dc/Dn-InxB). 35,36 These docking domains are fully portable to the gramicidin S NRPS modules.…”

Section: Resultsmentioning

confidence: 99%

Engineering DNA templated nonribosomal peptide synthesis

Huang

Stephan

Kries

2020

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Engineering DNA templated nonribosomal peptide synthesis

Huang

Stephan

Kries

2020

Preprint

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“…At present, the combinatorial biosynthesis of synthases such as surfactin [ 21 ], fengycin [ 22 ] and tyrocidine [ 23 ] has been widely reported, but the combinatorial biosynthesis strategy for bacillomycin D, a hybrid enzyme system, has not been studied. Previous researchers have successfully generated new analogs through the mutation, deletion and replacement of COM domains between NRPS subunits and suggested that the COM domain plays a decisive role in the interaction between NRPS subunits [ 18 , 22 , 24 ]. This makes it possible to uses COM domains to mediate module interactions and promote diverse polypeptide production.…”

Section: Introductionmentioning

confidence: 99%

Deletion of COM donor and acceptor domains and the interaction between modules in bacillomycin D produced by Bacillus amyloliquefaciens

Zhang

et al. 2022

Synthetic and Systems Biotechnology

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“…Mutual protein-protein interactions of the latter are mediated by specialized C-(donor) and N-terminally (acceptor) attached ~30 AAs long a-helical structural elements, so called communication mediating (COM) or docking domains (DDs) (8). DDs typically are located in between two modules and only interact with weak affinities (4-25 µM) (9-13), but are crucial to ensure biosynthesis of the desired product(s) (8,11,14). Despite recent progress on applying DD substitutions to program new assembly lines, in most cases structural information is lacking to effectively apply DDs for general engineering purposes (11,15,16).…”

Section: Introductionmentioning

confidence: 99%

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

Bozhueyuek

Watzel

Abbood

et al. 2020

Preprint

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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-...

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Artificial Splitting of a Non‐Ribosomal Peptide Synthetase by Inserting Natural Docking Domains

Cited by 15 publications

References 39 publications

Engineering DNA templated nonribosomal peptide synthesis

Engineering DNA templated nonribosomal peptide synthesis

Deletion of COM donor and acceptor domains and the interaction between modules in bacillomycin D produced by Bacillus amyloliquefaciens

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

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