Molecular Cloning and Analysis of the Ergopeptine Assembly System in the Ergot Fungus Claviceps purpurea

Abstract

Claviceps purpurea produces the pharmacological important ergopeptines, a class of cyclol-structured alkaloid peptides containing D-lysergic acid. These compounds are assembled from D-lysergic acid and three different amino acids by the nonribosomal peptide synthetase enzymes LPS1 and LPS2. Cloning of alkaloid biosynthesis genes from C. purpurea has revealed a gene cluster including two NRPS genes, cpps 1 and cpps 2. Protein sequence data had assigned earlier cpps1 to encode the trimodular LPS1 assembling the tripeptide portion of ergopeptines. Here, we show by transcriptional analysis, targeted inactivation, analysis of disruption mutants, and heterologous expression that cpps 2 encodes the monomodular LPS2 responsible for D-lysergic acid activation and incorporation into the ergopeptine backbone. The presence of two distinct NRPS subunits catalyzing formation of ergot peptides is the first example of a fungal NRPS system consisting of different NRPS subunits.

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

“…This system of ergot-peptide formation was the first identified fungal-NRP-synthesis system consisting of different NRP synthesis subunits. [24] Other examples of type C NRP synthetases include PesA of Metarhizium anisolpiae and AbrePsy1 of Alternaria brassicae. [14,15] The functional assessment of 4'-PPTase activity and the identification of a novel NRP synthetase provide new insights into non-ribosomal peptide synthesis in A. fumigatus.…”

“…Northern hybridisation and RT-PCR analysis of 4'-PPTase and Afpes1 gene expression A. fumigatus cultures were harvested at defined time points (24,48,72 and 96 h), mycelia collected, washed with ice-cold phosphate-buffered saline and total-RNA extracted by using TRI reagent (Sigma-Aldrich). Northern analyses (60 mg total RNA per well) were carried out by using 32 P-labelled regions of Afpes1 (NT: 9716-10535) and 4'-PPTase (NT: 1-1123) to detect relevant transcripts according to standard protocols.…”

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…Subsequent complementation experiments resulted in the ultimate confirmation of the postulated role of DMATS in the biosynthetic process in question (Wang et al 2004). Analogous experimental procedure allowed for the characterization of two consecutive genes, displaying the capacity to encode nonribosomal peptide synthetases (NRPS), namely: lpsA (Pannacione et al 2001,2003) and lpsB Fleetwood et al 2007), playing a vital role in the ergopeptine formation (Figure 13b) (Riederer et al 1996;Walzel et al 1997;Correia et al 2003). The ultimate objective: to determine whether ergot alkaloid biosynthetic genes were clustered in Neotyphodium lolii was pursued by using chromosome walking and Southern blot analysis and successfully attained (Fleetwood et al 2007).…”

“…Fuelled by the conviction of secondary metabolite pathway genes clustering in fungal genomes (Keller and Hohn 1997), large-scale studies have been taken up, resulting in the identification of gene clusters for ergot alkaloid biosynthesis in C. purpurea (Tudzynski et al 1999;Correia et al 2003;Haarmann et al 2005Haarmann et al , 2006 and A. fumigatus (Coyle and Panaccione 2005;Unso¨ld and Li 2005;Li and Unso¨ld 2006); they contain 13 and 14 genes, respectively. It has been recently hypothesized that the fundamental ability to synthesize some type of ergot alkaloid was present in the most recent common ancestor of these Ascomycetes, accounting for the current phylogenetically discontinuous distribution of these secondary metabolites (Panaccione 2005).…”

Genome mining reveals the presence of a conserved gene cluster for the biosynthesis of ergot alkaloid precursors in the fungal family Arthrodermataceae

“…The complete cluster contains 14 genes which are coordinately induced under ergot alkaloid production conditions (Correia et al, 2003). Twelve homologous genes have been identified in the ryegrass pathogen Neotyphodium lolii Lp19 and its sexual counterpart Epichloë festucae (Panaccione et al, 2001).…”