title: Directed evolution of aryl carrier proteins in the enterobactin synthetase.
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 approximately 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhou
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Lai
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution; D004758:Enterobactin / Q000096:biosynthesis; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000737:chemistry / Q000378:metabolism; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D015010:Yersinia pestis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development
chemical_list: D002352:Carrier Proteins; D002396:Catechols; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010080:Oxazoles; D010636:Phenols; D013844:Thiazoles; C104398:yersiniabactin; D004758:Enterobactin; C040227:vibriobactin; D008025:Ligases; C066234:enterobactin synthetase