Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering

Abstract

Graphical Abstract Highlights d The indigoidine synthetase BpsA was used to evolve functional recombinant PCP domains d Evolved PCP domains gained a general ability to interact effectively with TE domains d PCP-domain +4 and +24 residue positions are particularly important for TE interactions d Many inactive recombinant NRPSs may be just a few point mutations away from activity SUMMARY Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

Biofilm and Pathogenesis-Related Proteins in the Foodborne P. fluorescens ITEM 17298 With Distinctive Phenotypes During Cold Storage

“…As depicted in Figure 2, the biosynthesis of most proteins of this pathway was inhibited by the treatment with BLF derived peptides. In addition to these putative pigments, in our previous studies (Caputo et al, 2015;Quintieri et al, 2019b) we also found the colorless and reduced form of 3' ,3'-bipyridyl pigment indigoidine (leucoindigoidine, m/z = 251.0781), and its synthesis-related proteins (PROKKA_02722 and PROKKA_00418; Reverchon et al, 2002;Takahashi et al, 2007;Beer et al, 2014;Owen et al, 2016). Both these proteins were also identified in this study and their amounts did not change significantly in presence of HLF.…”

The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis

“…Using A-domains which accept methylated amino acids in domain swaps could also be used to introduce methylated amino acids, or unnatural analogues such as the fluorinated amino acids into other NRP natural products of agricultural of pharmaceutical interest. Indeed, there have been some major advances in NRPS structural biology and engineering recently, 21 – 23 which means that the prospect of swapping in domains/modules to introduce N -methylated amino acids into existing NRPs could become feasible. Detailed in vitro studies of the N -MeT enzyme and the N –Me amino acid activating A-domains and modules, including crystallography, could help to elucidate where the selectivity and specificity of such systems lie, and the roles that the individual NRPS domains play in controlling the output of such systems.…”

The many faces and important roles of protein–protein interactions during non-ribosomal peptide synthesis

“…However, studies from the Ackerley group have cast doubt on such assertions, as their results indicate that PCPs normally found to interact with a condensation domain are unable to interact effectively with a thioesterase (TE) domain in an reengineered NRPS machinery. 49 Thus, in order to understand the grounds for the specicity of PCPs, we will now discuss the interaction network between PCPs and other interacting domains based on the structural evidence currently available. To assist the reader, a table of all structures mentioned in this review is included in the text (Table 1).…”