Characterization of the Nodularin Synthetase Gene Cluster and Proposed Theory of the Evolution of Cyanobacterial Hepatotoxins

Abstract

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

“…(33) and in members of the family Enterobacteriaceae (34). This unusual amino acid residue also occurs in the lipopeptide antibiotic friulimicin (35) and in the cyanobacterial hepatotoxins microcystin and nodularin (3,7). In the mesaconate and friulimicin pathways, the route to MeAsp occurs via rearrangement of Glu to 3-MeAsp involving glutamate mutase.…”

“…The recently sequenced nodularin gene cluster of Nodularia spumigena NSOR10 also encodes a 2-hydroxy-acid dehydrogenase homolog, NdaH. Like McyI, this enzyme was originally predicted to catalyze a dehydration reaction: the conversion of threonine to dehydrobutyrine (7). Although the microcystin and nodularin biosynthesis pathways may involve serine/ threonine dehydration reactions, it is unlikely that these reactions are catalyzed by McyI and NdaH, as originally predicted.…”

The cylindrospermopsin gene cluster of Aphanizomenon sp. strain 10E6: organization and recombination

“…In addition, recombination between different domains within the mcy gene clusters, point mutations and deletion of gene regions, as well as lack of substrate specificity of A-domains have been shown to contribute to the synthesis of different microcystin isoforms (Fewer et al, 2008;Mikalsen et al, 2003;Tooming-Klunderud et al, 2008). A deletion within the second module of mcyA and the first module of mcyB resulted in the synthesis of another cyanotoxin, nodularin (Moffitt & Neilan, 2004;Rantala et al, 2004). The number of genes within the sxt gene clusters of Aphanizomenon, Anabaena and Cylindrospermopsis varies, but five regions containing homologous sxt genes have been identified in all three genera.…”

Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp.

“…Nodularins and microcystins are biosynthesized via modular megasynthases (Figure 2A), which consists of both non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules Moffitt and Neilan, 2004). Each module contains enzymatic domains responsible for substrate selection and activation, modification and condensation.…”