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

Cell Chem Biol. 2016 Nov 17;23(11):1395-1406. doi: 10.1016/j.chembiol.2016.09.014. Epub 2016 Oct 27.


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.

Keywords: BpsA; NRPS; PvdD; directed evolution; domain substitution; epimerization domain; error-prone PCR; indigoidine; pyoverdine; thiolation domain.

MeSH terms

  • Amino Acid Sequence
  • Bacteria / chemistry
  • Bacteria / genetics*
  • Bacteria / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Directed Molecular Evolution / methods
  • Peptide Synthases / chemistry
  • Peptide Synthases / genetics*
  • Peptide Synthases / metabolism
  • Piperidones / metabolism
  • Protein Engineering / methods*
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism


  • Bacterial Proteins
  • Piperidones
  • Recombinant Proteins
  • indigoidine
  • Peptide Synthases
  • non-ribosomal peptide synthase