Elucidation of the Biosynthesis Machinery of Calyxamide A from the Sponge Symbiotic Microorganisms

The Japanese marine sponge Discodermia calyx contains a cytotoxic cyclic peptide, calyxamide A, which possesses N-formyl, α-ketoamide, thiazole, and trans-olefin functionalities. Since α-ketoamide group is widely occurring in microbial metabolites, calyxamide was considered to be produced by an unknown bacterium associated with D. calyx.  In this study, in order to identify the true producer of calyxamide, we tried to isolate the biosynthetic gene cluster of calyxamide A from the sponge-bacterial symbiont metagenome. To clone the biosynthetic gene cluster, we searched for type I polyketide synthetase (PKS), since the trans-olefin apparently originates from the polyketide pathway. As a result, we found the putative biosynthetic gene cluster of calyxamide A, containing non-ribosomal peptide synthetase (NRPS) and PKS modules. Moreover, the homology-based analysis of the biosynthetic gene cluster suggested that -ketoamide was generated from α-ketoamide precursor through a chain shortening process catalyzed by a flavin-utilizing monooxygenase. In addition, this gene cluster contained other unique enzymes, such as a sulfotransferase and a sulfatase. Taking into account that calyxamide A has no sulfonyl group in its structure, we proposed that these enzymes are involved in the protection-deprotection mechanism via sulfonyl group, as is the case of organic synthesis. Although some natural compounds biosynthesized from PKS-NRPS gene cluster have been reported to use natural protection-deprotection mechanisms, the mechanism using a sulfonamide functionality has never been reported. Therefore, we are now trying to examine the functions of these enzymes. Finally, single cell analysis revealed that calyxamide PKS-NRPS was encoded by an uncultivated bacterium Entotheonella sp.