Mutation of the eunicellane synthase Bnd4 alters its product profile and expands its prenylation ability

21 June 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Bnd4 catalyzes the first committed step in the biosynthesis of the bacterial diterpenoid benditerpenoic acid and was the first eunicellane synthase identified from nature. We investigated the catalytic roles of the aromatic residues in the active site of Bnd4 through a series of mutation studies. These experiments revealed that large hydrophobic or aromatic side chains are required at F162 and Y197 for eunicellane formation and that selected mutations at W316 converted Bnd4 into a cembrane synthase. In addition, the Bnd4Y197A mutant expanded the native prenylation ability of Bnd4 from accepting C5 and C10 prenyl donors to C15. This study supports the mechanism of eunicellane formation by Bnd4 and encourages further engineering of terpene synthases into practical and efficient prenyltransferases.


terpene synthase

Supplementary materials

Methods; strains, plasmids, and primers used in this study (Tables S1–S3); summary of NMR data for compounds 6 and 7 (Table S4); sequence alignments of relevant diterpene synthase (Figures S1 and S23); SDS-PAGE analysis of purified proteins (Figure S2); diterpene overproduction system in E. coli (Figure S3); NMR spectra of compounds 2–4, 6, 7, and 9 (Figures S4–S11, S13–22, and S24–27); HPLC traces of enzyme reactions (Figures S12 and S28); supporting references


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