Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS

Abstract

18Several peptides in clinical use are derived from non-ribosomal peptide synthetases 19 (NRPS). In these systems multiple NRPS subunits interact with each other in a 20 specific linear order mediated by docking domains (DDs) to synthesize well-defined 21 peptide products. In contrast to these classical NRPSs, the subunits of 22 23 resulting in libraries of peptide products. In order to define the structural and 24 thermodynamic basis for their unusual interaction patterns, we determined the 25 structures of all N-terminal DDs ( N DDs) as well as of an N DD-C DD complex and 26 characterized all putative DD interactions thermodynamically for one such system. 27Key amino acid residues for DD interactions were identified that upon their exchange 28 not only changed the DD affinity but also resulted in rationally predictable changes in 29 peptide production. A simple set of 'recognition rules' for DD interactions was 30 identified that also operates in other megasynthase complexes. 31 32 42 C-terminal docking domains (DDs). Stachelhaus and coworkers have demonstrated 43 that a matching pair of DDs or COM (communication-mediating) domains at the N-44 terminus of peptidyl-donating NRPS ( N DD) and the C-terminus of accepting NRPS 45 ( C DD) play a decisive role in defining the order of interactions between subunits in 46 vitro and in vivo by swapping COM domains. 3 They also constructed a "universal 47 3 COM system" in vitro by comparison of COM domains in tyrocidin A and surfactin-like 48 NRPSs, which led to enzyme crosstalk between different biosynthetic systems that 49 promoted the combinatorial biosynthesis of different peptides. 4,5 However, the 50 structures of these DDs have not been elucidated so far but interactions between 51 DDs have been mapped based on photo crosslinking experiments. 6 More information 52 on DDs is available for polyketide synthase (PKS) or PKS/NRPS hybrid systems. 53 While for PKS three different types of DD pairs have been described structurally so 54 far, two structures for N DDs without a bound C DD have been solved for NRPS/PKS 55 hybrid systems. 7-12 56 Classic NRPSs are multimodular -each protein subunit normally consists of the 57 processing modules for multiple amino acids arranged in a linear fashion. 58 Furthermore, the different protein subunits interact with each other in a strictly 59 defined linear order yielding a single peptide product with a sequence that faithfully 60 reproduces the linear order of the processing modules along the protein chains. A 61 novel class of monomodular NRPSs involved in the production of libraries of 62 rhabdopeptide/xenortide peptides (RXPs) has been recently described from 63 entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus. 13 The 64RXP product spectrum in a single strain differs mainly in peptide length and 65 sequence, which requires an iterative use of certain NRPS modules and is 66 dependent on protein stoichiometry between NRPS modules acting in elongation and 67 termination ( Supplementary Figure 1). This suggests th...

Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

“…In this report, we demonstrate the cell-free production of the NRPS BpsA from Streptomyces lavendulae 19-20 and the RXP (rhabdopeptide-like peptide) producing NRPS KJ12ABC from Xenorhabdus KJ12.1 21 in the PURE system, as well as the direct production of their natural products indigoidine and rhabdopeptides, respectively (Figure 1A & B). We further show that other megasynthases (including the PKS-related fatty acid synthases (FASs)) can be produced and activated by post-translational modification.…”

“…In the light of the successful proof of concept, a set of megasynthases (NRPSs, PKSs, FASs) was screened for synthesis by the PURE cell-free system with coupled phosphopantetheinylation (sequential protocol, see Figure S1). We were able to synthesize and CoA-647-label all megasynthases applied in this screen, Penicillium patulum MSAS 30 , RAPS module 14 31 , PikA module 5 (PikAIII) 32 , DEBS module 4 7 , three variants of murine FAS (wild-type, KS-MAT-ACP-TE and with C-terminal GFP (FAS-GFP)) 33-34 , GrsA 35 , TycB1 36 , and the Xhenorhabdus KJ12.1 KJ12ABC 21 (Figure S5A & B). The murine FAS construct DH-KR-TE and the NRPS module KJ12C, which do not harbor a carrier protein domain, were not fluorescently labeled, as expected (Figure S5B).…”

“…In seeking to probe the PURE cell-free system for the production of another megasynthase, we decided to work with the RXP-synthesizing NRPS KJ12ABC from the bacterium Xhenorhabdus KJ12.1 21 . The RXP biosynthetic gene cluster of Xhenorhabdus KJ12.1 encodes three NRPS modules – kj12A encoding a C-A-T module (137.8 kDa), kj12B encoding a C-A/MT-T module (181.5 kDa), and kj12C encoding a stand-alone C term domain (62.3 kDa) (see Figure 1B).…”

“…The second module KJ12B is iteratively used in this assembly line with a relaxed methyltransferase (MT) activity. KJ12ABC produces a broad spectrum of compounds 21 .…”

Engineering DNA templated nonribosomal peptide synthesis

“…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. In a disconnected, trimodular system for fPO* formation (GrsA, TycB1, GrsB3) a Dc/Dn domain pair added to connect the second and third module increases activity more than 70-fold compared to modules without docking domains ( Figure S2).…”