Characterization of the Nocardiopsin Biosynthetic Gene Cluster Reveals Similarities to and Differences from the Rapamycin and FK-506 Pathways

Abstract

Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal. Expanding nature's macrolide-pipecolate biosynthetic repertoire: Cloning, sequencing, and analysis of the nocardiopsin biosynthetic pathway revealed that Nocardiopsis sp. CMB-M0232 employs a new strategy, featuring both a P450 epoxidase and an epoxide hydrolase, to transform the alkene group of a linear polyketide precursor into the hydroxy group required for macrolactonization.