Directed Evolution of the Nonribosomal Peptide Synthetase BpsA to Enable Recognition by the Human Phosphopantetheinyl Transferase for Counter-Screening Antibiotic Candidates

ACS Infect Dis. 2020 Nov 13;6(11):2879-2886. doi: 10.1021/acsinfecdis.0c00606. Epub 2020 Oct 29.


Bacterial type II phosphopantetheinyl transferases (PPTases), required for the activation of many cellular mega-synthases, have been validated as promising drug targets in several pathogens. Activation of the blue-pigment-synthesizing nonribosomal peptide synthetase BpsA by a target PPTase can be used to screen in vitro for new antibiotic candidates from chemical libraries. For a complete screening platform, there is a need to also counter-screen inhibitors for cross-reactivity with the endogenous human Type II PPTase (hPPTase), as this is a likely source of toxicity. As hPPTase is unable to recognize the PCP-domain of native BpsA, we used a combination of directed evolution and rational engineering to generate a triple-substitution variant that is able to be efficiently activated by hPPTase. Our engineered BpsA variant was able to readily detect inhibition of both hPPTase and the equivalent rat PPTase by broad-spectrum PPTase inhibitors, demonstrating its potential for high-throughput counter-screening of novel antibiotic candidates.

Keywords: 4′-phosphopantetheinyl transferase; NRPS; Type II PPTase; aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase; antibiotic screening; indigoidine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents* / pharmacology
  • Bacterial Proteins
  • Humans
  • Peptide Synthases / genetics
  • Rats
  • Transferases (Other Substituted Phosphate Groups)* / genetics


  • Anti-Bacterial Agents
  • Bacterial Proteins
  • phosphopantetheinyl transferase
  • Transferases (Other Substituted Phosphate Groups)
  • Peptide Synthases
  • non-ribosomal peptide synthase