The gene cluster involved in producing the cyclic heptadepsipeptide micropeptin was cloned from the genome of the unicellular cyanobacterium Microcystis aeruginosa K-139. Sequencing revealed four genes encoding non-ribosomal peptide synthetases (NRPSs) that are highly similar to the gene cluster involved in cyanopeptolins biosynthesis. According to predictions based on the non-ribosomal consensus code, the order of the mcnABCE NPRS modules was well consistent with that of the biosynthetic assembly of cyclic peptides. The biochemical analysis of a McnB(K-139) adenylation domain and the knock-out of mcnC in a micropeptin-producing strain, M. viridis S-70, revealed that the mcn gene clusters were responsible for the production of heptadepsipeptide micropeptins. A detailed comparison of nucleotide sequences also showed that the regions between the mcnC and mcnE genes of M. aeruginosa K-139 retained short stretches of DNA homologous to halogenase genes involved in the synthesis of halogenated cyclic peptides of the cyanopeptolin class including anabaenopeptilides. This suggests that the mcn clusters of M. aeruginosa K-139 have lost the halogenase genes during evolution. Finally, a comparative bioinformatics analysis of the congenial gene cluster for depsipetide biosynthesis suggested the diversification and propagation of the NRPS genes in cyanobacteria.
IntroductionMicrocystis species are among the most common waterbloom-forming cyanobacteria and a valuable source of various secondary metabolites (Carmichael, 1994). Microcystis aeruginosa is known to produce microcystin, a potent cyclic heptapeptide hepatotoxin that inhibits protein phosphatases 1 and 2A and has more than 60 components (MacKintosh et al., 1990;Sivonen and Jones, 1999). Microcystins are recognized as being causative of many animal poisonings and human illnesses (Sivonen and Jones, 1999). Moreover, M. aeruginosa produce several small peptides including micropeptin, aeruginosin and microviridin, which are inhibitors of protease (Rohrlack et al., 2003). Microviridin J causes a lethal molting disruption in Daphnia pulicaria, suggesting that other cyanobacterial protease inhibitors are toxic to zooplankton (Rohrlack et al., 2004). In order to assess the health hazard posed by cyanobacteria to humans and livestock, it is necessary to achieve a better understanding of the secondary metabolites produced by bloomforming cyanobacteria. These cyanopeptides are predicted to be produced nonribosomally by the multifunctional peptide synthetases (Finking and Marahiel, 2004
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