Elsevier

Phytochemistry

Volume 66, Issue 11, June 2005, Pages 1312-1320
Phytochemistry

The ergot alkaloid gene cluster in Claviceps purpurea: Extension of the cluster sequence and intra species evolution

https://doi.org/10.1016/j.phytochem.2005.04.011Get rights and content

Abstract

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Hölter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133–141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5 kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

Graphical abstract

The extension of the ergot alkaloid cluster and the comparison of its structure in two chemical races of Claviceps purpurea are reported.

  1. Download : Download full-size image

Introduction

The ergot disease of grasses and cereals caused by the ergot fungus Claviceps purpurea is characterized by the formation of overwintering structures, so-called sclerotia, containing variable amounts of mycotoxins, the ergopeptines. This is a class of cyclol-structured alkaloid peptides containing D-lysergic acid. Since some of these substances have considerable pharmacological importance, physiology, biochemistry and genetics of their formation have been studied in detail (Stadler and Giger, 1984, Tudzynski et al., 2001). Tsai et al. (1995) identified the gene (dmaW) for the first specific enzyme of the pathway, dimethylallyltryptophan synthase (see Fig. 1), in C. fusiformis, and Tudzynski et al. (1999) described a putative ergot alkaloid gene cluster in the C. purpurea strain P1; the putative cluster included a homologue of dmaW, cpd1, and a trimodular NRPS gene, cpps1, encoding lysergylpeptidyl synthetase 1 (LPS1) involved in the attachment of the tripeptide moiety to dehydrolysergic acid (Riederer et al., 1996). Recently, a second NRPS gene in the cluster region, cpps2, was shown to encode LPS2, the D-lysergic acid activating NRPS module (Correia et al., 2003; see Fig. 1). The function of the other genes identified in the neighborhood of cpd1, cpps1 and cpps2 – among them several oxidase/oxygenase genes – remains to be elucidated. Here, we show that the putative cluster region extends on both sides of the originally described sequence, covering at least 68.5 kb. It contains two additional NRPS genes, cpps3 and cpps4, the functions of which remain to be determined. A second major aspect of the present paper is the comparison of the putative ergot alkaloid cluster in two chemical races: strain P1 (major alkaloid: ergotamine) and ECC93 (ergocristine) to study variability/evolution of the cluster sequence within the species C. purpurea and to determine if the difference in the primary ergopeptine produced is mainly due to internal amino acid pools (as postulated by Riederer et al., 1996) or to differences in critical substrate specificity conferring amino acids of the NRPS modules.

Section snippets

Defining the ergot alkaloid gene cluster in strain P1

The core region of the ergot alkaloid gene cluster (Correia et al., 2003) stretches from a putative oxidase gene (cpox3) to the LPS1 encoding gene cpps1 over 40 kb. Chromosome walking using clones of an EMBL3 genomic library of strain P1 extended the available genomic sequence downstream of cpox3 (“left” side) over 15 kb (see Fig. 2). This region contains an additional NRPS gene, cpps3, and a gene probably involved in primary metabolism, cpimd (encoding an isopropylmalate-dehydratase). Cpps3

Strains and culture conditions

Claviceps purpurea strains P1 and ECC93, which produce mainly ergotamine and ergocristine, respectively, have been described previously (Tudzynski et al., 1999, Keller et al., 1988), as were the standard media and culture conditions (Tudzynski et al., 1999).

For northern analyses, mycelia were cultivated in Mantle A medium with low (0.5 g/l KH2PO4) and high (2.0 g/l KH2PO4) amounts of phosphate, respectively. After 5 d mycelia were harvested and RNA was prepared using the RNAgents® Total RNA

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (Tu 50/13, SPP 1152: Evolution of metabolic diversity, to P.T.) and the United States Department of Agriculture (2001-35319-10930 and 58-6401-2-0025 to D.G.P. and C.L.S.). We thank N. Lorenz for discussion and for support in the northern analyses, and U. Keller (Berlin) for providing us with unpublished data.

References (24)

  • U. Keller et al.

    Ergot alkaloids

  • G. Mey et al.

    The biotrophic, non-appressoria forming grass pathogen Claviceps purpurea needs a Fus3/Pmk1 homologous MAP kinase for colonization of rye ovarian tissue

    Mol. Plant Microb. Interact.

    (2002)
  • Cited by (101)

    • Overexpression of Trp-related genes in Claviceps purpurea leading to increased ergot alkaloid production

      2021, New Biotechnology
      Citation Excerpt :

      Thus the role of lpsC in strain P1 is still questionable. In correlation with results obtained previously by northern blot analyses [11,37,56], all 10 EA biosynthetic genes were significantly upregulated in WT under inducing compared to non-inducing conditions (Table A.2). Under inducing conditions, dmaW, encoding the enzyme catalyzing the prenylation of L-Trp by dimethylallyl diphosphate [14,15] was the highest expressed gene in WT C. purpurea P1.

    • Secondary Metabolite Diversity of the Genus Aspergillus: Recent Advances

      2016, New and Future Developments in Microbial Biotechnology and Bioengineering: Aspergillus System Properties and Applications
    • Quantitative and qualitative transcriptome analysis of four industrial strains of Claviceps purpurea with respect to ergot alkaloid production

      2016, New Biotechnology
      Citation Excerpt :

      The NRPS genes, lpsA1 (CPUR_04074.1) and lpsA2 (CPUR_04073.1), possess regions containing acyl carrier and peptide synthetase domains. These domains are organized into three modules, where each module carries one amino acid; the composition of the amino acids determines the final ergopeptine (Table 1) [11]. Based on the hypothesis that the production of specific ergopeptines is determined by NRPS module specificity, we should observe amino acid changes in the binding pocket of the first two modules (the third is specific for proline) of lpsA1 or/and lpsaA2 genes in the strains Gal012, Gal130, and Gal310, but not in Gal404, which is an ergotamine producer similar to the reference strain.

    View all citing articles on Scopus
    View full text