Role of Two Exceptional trans Adenylation Domains and MbtH-like Proteins in the Biosynthesis of the Nonribosomal Peptide WS9324A from Streptomyces calvus ATCC 13382.

Nonribosomal peptide synthetases (NRPS) are organized in a modular arrangement. Usually, the modular order corresponds to the assembly of the amino acids in the respective peptide, following the collinearity rule. The WS9326A biosynthetic gene cluster fromStreptomyces calvusshows deviations from this rule. Most interesting is the presence of twotransadenylation domains that are located downstream of the modular NRPS arrangement. Adenylation domains are responsible for the activation of their respective amino acids. In this study, we confirmed the involvement of thetransadenylation domains in WS9326A biosynthesis by performing gene knockout experiments and by observing the selective adenylation of their predicted amino acid substratesin vitro. We conclude that thetransadenylation domains are essential for WS9326A biosynthesis. Moreover, both adenylation domains are observed to have MbtH-like protein dependency. Overall, we conclude that thetransadenylation domains are essential for WS9326A biosynthesis.