Isolation and characterization of meridamycin biosynthetic gene cluster from Streptomyces sp. NRRL 30748

Abstract

Meridamycin is a non-immunosuppressive, FKBP12-binding natural macrolide with potential therapeutic applications in a variety of medical conditions. To set the stage for structural modification of meridamycin by genetic engineering, we have cloned and completely sequenced approximately 117 kb of DNA encompassing the meridamycin biosynthetic gene cluster from the producing strain, Streptomyces sp. NRRL 30748. Clustered in the center of the cloned DNA stretch are six genes responsible for the construction of the core structure of meridamycin, including merP encoding a non-ribosomal peptide synthase for pipecolate-incorporation, four PKS genes (merA-D) together encoding 1 loading module and 14 extension modules, and merE encoding a cytochrome P450 monooxygenase. A number of genes with potential pathway-specific regulatory or resistance functions have also been identified. The absence of the gene encoding lysine cyclodeaminase in the sequenced gene cluster and the rest of the genome of NRRL 30748 indicated the synthesis of pipecolate in this strain is not through the common lysine cyclodeamination route previously described for rapamycin and FK506/FK520 biosynthesis. An efficient conjugation method has been developed for Streptomyces sp. NRRL 30748 to facilitate the genetic manipulation of meridamycin biosynthetic gene cluster. Disruption of merP resulted in the complete abolition of meridamycin production, proving the identity of the gene cluster. A novel meridamycin analogue, C36-keto-meridamycin, has been successfully generated through deletion of a DNA fragment encoding KR1 domain of MerA from the chromosomal DNA.