Functional analysis of calD involved in calcimycin postsynthetic modification by Streptomyces chartreusis NRRL 3882

Abstract:

[objective] Calcimycin, a divalent cation specific ionophore, is a pyrrole moiety containing polyther antibiotic, widely used to study the biological function of divalent cation in eukaryotic cells. The biosynthetic mechanism of calcimycin remains to be fully elucidated. calD is a gene with unknown function in the calcimycin biosynthetic gene cluster. The aim of this study is to identify the function of calD in calcimycin biosynthesis pathway. [Methods] Bioinformatic analysis was used to predict the function of calD. PCR targeting was used to disrupt calD in Streptomyces chartreusis NRRL 3882. HPLC/MS was used to analyze the fermentation product. [Results] Bioinformatic analysis shows that CalD belongs to the family of alcohol dehydrogenase. The calD gene disruption mutant was obtained. The mutant was also complemented with calD. HPLC analysis shows that inactivating of calD decreases but not abolishes calcimycin production. The yield of N-demethylcalcimycin is also decreased. The calD mutant accumulates hydroxyl-cezomycin. [Conclusion] calD participates in calcimycin biosynthesis. It might be responsible for the oxidative conversion of the hydroxy-cezomycin to keto-cezomycin. This result paved way for the further analysis of the biochemical function of calD.