title: Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering.
abstract: 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.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6140, New Zealand.
authors.forename: Jeremy G
authors.initials: JG
authors.lastname: Owen
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Calcott
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Robins
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6140, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1023, New Zealand. Electronic address: david.ackerley@vuw.ac.nz.
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010881:Piperidones / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010881:Piperidones; D011994:Recombinant Proteins; C446045:indigoidine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: BpsA; NRPS; PvdD; directed evolution; domain substitution; epimerization domain; error-prone PCR; indigoidine; pyoverdine; thiolation domain