Directed evolution of aryl carrier proteins in the enterobactin synthetase

Zhe Zhou, Jonathan R. Lai, Christopher T. Walsh

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

The recognition of carrier proteins by multiple catalytic partners occurs in every cycle of chain elongation in the biosynthesis of fatty acids and of the pharmacologically important polyketide and nonribosomal peptide natural products. To dissect the features of carrier proteins that determine specific recognition at distinct points in assembly lines, we have used the two-module Escherichia coli enterobactin synthetase as a model system. Using an entB knockout strain, we developed a selection for growth on iron-limiting medium to evolve aryl carrier protein domains. The aryl carrier proteins from VibB of Vibrio cholerae vibriobactin and HMWP2 of Yersinia pestis yersiniabactin assembly lines were evolved by random mutagenesis to support growth under selection conditions, yielding a convergent set of mutations. Subsequent in vitro biochemical characterizations with partner enzymes EntE, EntF, and Sfp on the evolved VibB aryl carrier protein revealed a ≈500-fold improvement in reconstituted enterobactin production activity. Mechanistic characterization identified three distinct specific recognition surfaces of VibBArCP for three catalytic partners in enterobactin biosynthesis. Our results suggest that heterologous carrier protein interactions can be engineered with a small number of mutations given a suitable selection scheme and provide insights for reprogramming nonribosomal peptide biosynthesis.

Original languageEnglish (US)
Pages (from-to)11621-11626
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number28
DOIs
StatePublished - Jul 10 2007
Externally publishedYes

Keywords

  • Nonribosomal peptide synthetase
  • Recognition
  • Siderophores

ASJC Scopus subject areas

  • General

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