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Background: The cyclic peptide toxins of Amanita mushrooms, such as α-amanitin and phalloidin, are encoded by the "MSDIN" gene family and ribosomally biosynthesized. Based on partial genome sequence and PCR analysis, some members of the MSDIN family were previously identified in Amanita bisporigera, and several other members are known from other species of Amanita. However, the complete complement in any one species, and hence the genetic capacity for these fungi to make cyclic peptides, remains unknown. Results: Draft genome sequences of two cyclic peptide-producing mushrooms, the "Death Cap" A. phalloides and the "Destroying Angel" A. bisporigera, were obtained. Each species has ~30 MSDIN genes, most of which are predicted to encode unknown cyclic peptides. Some MSDIN genes were duplicated in one or the other species, but only three were common to both species. A gene encoding cycloamanide B, a previously described nontoxic cyclic heptapeptide, was also present in A. phalloides, but genes for antamanide and cycloamanides A, C, and D were not. In A. bisporigera, RNA expression was observed for 20 of the MSDIN family members. Based on their predicted sequences, novel cyclic peptides were searched for by LC/MS/MS in extracts of A. phalloides. The presence of two cyclic peptides, named cycloamanides E and F with structures cyclo(SFFFPVP) and cyclo(IVGILGLP), was thereby demonstrated. Of the MSDIN genes reported earlier from another specimen of A. bisporigera, 9 of 14 were not found in the current genome assembly. Differences between previous and current results for the complement of MSDIN genes and cyclic peptides in the two fungi probably represents natural variation among geographically dispersed isolates of A. phalloides and among the members of the poorly defined A. bisporigera species complex. Both A. phalloides and A. bisporigera contain two prolyl oligopeptidase genes, one of which (POPB) is probably dedicated to cyclic peptide biosynthesis as it is in Galerina marginata. Conclusion: The MSDIN gene family has expanded and diverged rapidly in Amanita section Phalloideae. Together, A. bisporigera and A. phalloides are predicted to have the capacity to make more than 50 cyclic hexa-, hepta-, octa-, nona- and decapeptides. [ABSTRACT FROM AUTHOR]

Abstract: Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerina marginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerina hybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G. marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear. [Copyright &y& Elsevier]