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Biosynthesis of Non‐Ribosomal Peptide Beta‐Lactones by Plant‐Associated Pseudomonas fluorescens
Author(s) -
Schaffer Jason,
Wencewicz Timothy
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.656.25
Subject(s) - thioesterase , pseudomonas fluorescens , biosynthesis , biochemistry , amino acid , gene cluster , biology , serine , chemistry , enzyme , stereochemistry , bacteria , gene , genetics
Secondary metabolites are molecules produced by bacteria that grant a competitive advantage in their native environment. Many life‐saving antibiotics are directly taken from or inspired by these biologically active and structurally diverse molecules, such as vancomycin, daptomycin, and penicillin. In addition to the compounds themselves, the biosynthetic enzymes that produce these structurally complex molecules are of interest, due to their ability to assemble these molecules in an efficient and stereochemically controlled manner. We report the biosynthetic gene cluster of the antibiotic obafluorin, a β‐lactone produced by plant associated species of Pseudomonas fluorescens . We present a detailed genetic and biochemical characterization of the entire obafluorin biosynthetic pathway which includes the biosynthesis of the non‐proteinogenic amino acid β‐OH‐ p ‐NO 2 ‐homophenylalanine, produced en route to the final compound. The crucial stereochemistry of the obafluorin β‐lactone is set during the synthesis of the 1,2 amino‐alcohol moiety by ObiH, a threonine transaldolase. The biosynthesis is completed on the non‐ribosomal peptide synthetase ObiF, which contains a rare serine to cysteine mutation in the type I thioesterase domain active site. This mutation plays a direct role in β‐lactone ring formation during final cleavage of the antibiotic from the enzyme. Our biosynthetic studies culminate with the in vitro reconstitution of five enzymes that enable total chemoenzymatic synthesis of the b‐lactone antibiotic obafluorin and analogs from simple, readily available building blocks. We show that homologous gene clusters are present in environmental bacteria and human pathogens, including the multi‐drug resistant opportunistic pathogen Burkholderia diffusa of growing concern for immunocompromised cystic fibrosis patients. We also report the utilization of key biosynthetic enzymes in a chemoenzymatic synthesis of obafluorin analogs and probe molecules. Support or Funding Information Research Corporation for Science Advancement, Cottrell Scholars AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .