Orthologous peramine and pyrrolopyrazine‐producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi

Abstract

Summary Peramine is a non‐ribosomal peptide‐derived pyrrolopyrazine (PPZ)‐containing molecule with anti‐insect properties. Peramine is known to be produced by fungi from genus Epichloë, which form mutualistic endophytic associations with cool‐season grass hosts. Peramine biosynthesis has been proposed to require only the two‐module non‐ribosomal peptide synthetase (NRPS) peramine synthetase (PerA), which is encoded by the 8.3 kb gene perA, though this has not been conclusively proven. Until recently, both peramine and perA were thought to be exclusive to fungi of genus Epichloë; however, a putative perA homologue was recently identified in the genome of the insect‐pathogenic fungus Metarhizium rileyi. We use a heterologous expression system and a hydrophilic interaction chromatography‐based analysis method to confirm that PerA is the only pathway‐specific protein required for peramine biosynthesis. The perA homologue from M. rileyi (MR_perA) is shown to encode a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë. Furthermore, perA is part of a larger seven‐gene PPZ cluster in M. rileyi, Metarhizium majus and the stalked‐cup lichen fungus Cladonia grayi. These PPZ genes encode proteins predicted to derivatize peramine into more complex PPZ metabolites, with the orphaned perA gene of Epichloë spp. representing an example of reductive evolution.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS

“…In the absence of the R 2 domain, it is conceivable that release of PPZ-1,4-diones from truncated PpzA variants could be catalyzed by separate protein, such as a type II thioesterase. However, candidate genes for this function are not found coclustered with any ppzA variant (2, 16, 23). Furthermore, expression of these truncated ppzA variants alone was sufficient to achieve PPZ-1,4-dione production in the naive host P. paxilli at levels comparable to 1a production by PN2013/ Efe_ppzA-1 strains (Figs.…”

“…The fungus Penicillium paxilli strain PN2013 was utilized as a naive heterologous expression host to identify the metabolite products of the enzymes encoded by different ppzA alleles, with 1a -producing PN2013/ Efe_ppzA-1 control strains obtained from a previous study (23). As expected, PN2013 transformants expressing Efe_ppzA-2 from E. festucae strain E189 or Ety_ppzA-2 from E. typhina subsp.…”

“…was thought to be restricted to 1a , especially given that PPZ clusters containing ppzA-1 orthologs alongside at least 6 accessory genes were recently identified in Metarhizium and Cladonia spp. (23). However, the results presented here show that allelic variants of a single NRPS can generate metabolite diversity comparable to many gene clusters, with 4 functionally distinct ppzA alleles described here in addition to the previously characterized ppzA-1 (2, 23).…”

“…(23). However, the results presented here show that allelic variants of a single NRPS can generate metabolite diversity comparable to many gene clusters, with 4 functionally distinct ppzA alleles described here in addition to the previously characterized ppzA-1 (2, 23). Of these, the ppzA-2 , ppzA-3 , and ppzA-4 alleles all derive from the same 3′ sequence deletion caused by a transposable element insertion (14, 15, 18), whereas ppzA-5 contains a premature stop codon near the 3′ end of the gene (14).…”

Secondary metabolites from hypocrealean entomopathogenic fungi: genomics as a tool to elucidate the encoded parvome

“…77 Peramine 35 (Scheme 10) is an NRP-derived pyrrolopyrazine-containing SM with insecticidal activities. 78 Peramine was first identified from Epichloë spp. that form mutualistic endophytic associations with cool-season grasses, whereby this compound contributes to the protection of the fungus-grass association against herbivory.…”