Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicates loss of late pathway steps in evolution of C. fusiformis

Abstract

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB(Psi)). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB(Psi) and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB(Psi) were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.

FIG. 1.

Diagrams of EA biosynthesis in C. fusiformis (end product, elymoclavine) and C. purpurea (ergopeptides). Genes of interest are enclosed in boxes. DMAPP, dimethylallylpyrophosphate; DMAT, dimethylallyltryptophan; MeDMAT, methyl dimethylallyltryptophan. (Modified from reference with permission.)

FIG. 2.

Schematic comparison of the EAS clusters of C. purpurea (top) and C. fusiformis (bottom). Homologous genes in the two species are indicated by dark gray arrows, whereas genes identified in only one species are indicated by light gray arrows. The arrows indicate the orientation of transcription. The genes, as well as the intergenic regions, are drawn to scale. Rearrangement in the locus, indicated by dashed lines, is associated with truncation of lpsB in C. fusiformis. ap is a putative aminopeptidase gene adjacent to the C. fusiformis EAS cluster. The accession no. for the C. fusiformis cluster sequence is EU006773.

FIG. 3.

Schematic comparison of CplpsB and its homologue CflpsB^Ψ. Adenylation (A), thiolation (T), and condensation (C) domains are indicated by shaded boxes, and the numbers indicate the domain borders (amino acids) for CplpsB. The arrows indicate frameshift mutations in CflpsB^Ψ resulting in a premature stop codon at position 94. The open box indicates the portion of CflpsB^Ψ downstream of the stop codon that aligns with CplpsB.

FIG. 4.

Southern blot analysis of genomic DNA from several EA-producing fungi. The membrane was first hybridized to a probe from CpdmaW (A) and then stripped and hybridized to probe from CplpsA (B). Samples (4 μg) of total fungal DNA were digested with EcoRI to completion, fractionated by electrophoresis in a 0.9% agarose gel, and transferred to a nylon membrane. The lanes contained DNA from C. purpurea P1 (lane 1), C. fusiformis SD58 (lane 2), Neotyphodium coenophialum ATCC 90664 (lane 3), N. lolii × Epichloë typhina Lp1 (lane 4), and B. obtecta B249 (lane 5). The arrows in panel B indicate the positions of a residual signal from the CpdmaW probe; the lower band (8.3 kb) was close to the position of an authentic lpsA signal.

FIG. 5.

Heterologous expression of CfcloA in C. purpurea strains, including the wild type (P1) (WT), a ΔCpcloA mutant, and a transformant carrying the full-length CfcloA gene.

FIG. 6.

TLC analyses of ergot alkaloids from the C. fusiformis wild type (WT) and a transformant carrying the CpcloA gene. Culture extracts were chromatographed on TLC plates, visualized by using van Urk's reagent, and identified by comparison with pure standards.

FIG. 7.

Liquid chromatography-mass spectroscopy analyses of ergot alkaloids from the C. fusiformis wild type (WT) and a transformant carrying the CpcloA gene. The inset shows the migration of the lysergic acid standard.

FIG. 8.

Diagram of ergot alkaloid biosynthesis in the C. fusiformis wild type (WT) and a transformant carrying the CpcloA gene. In the C. fusiformis wild type the pathway ends at elymoclavine due to a nonfunctional CfcloA gene. In addition to elymoclavine, agroclavine accumulates, perhaps due to feedback inhibition. In the C. fusiformis transformant carrying a functional CpcloA gene, lysergic acid and elymoclavine accumulate. The absence of functional lpsA and lpsB genes in C. fusiformis apparently precludes the synthesis of ergopeptines in this transformant.