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. 2017 Dec 13;65(49):10703-10710.
doi: 10.1021/acs.jafc.7b04272. Epub 2017 Dec 5.

Ergot Alkaloid Biosynthesis in the Maize (Zea Mays) Ergot Fungus Claviceps Gigantea

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Free PMC article

Ergot Alkaloid Biosynthesis in the Maize (Zea Mays) Ergot Fungus Claviceps Gigantea

Paige E Bragg et al. J Agric Food Chem. .
Free PMC article

Abstract

Biosynthesis of the dihydrogenated forms of ergot alkaloids is of interest because many of the ergot alkaloids used as pharmaceuticals may be derived from dihydrolysergic acid (DHLA) or its precursor dihydrolysergol. The maize (Zea mays) ergot pathogen Claviceps gigantea has been reported to produce dihydrolysergol, a hydroxylated derivative of the common ergot alkaloid festuclavine. We hypothesized expression of C. gigantea cloA in a festuclavine-accumulating mutant of the fungus Neosartorya fumigata would yield dihydrolysergol because the P450 monooxygenase CloA from other fungi performs similar oxidation reactions. We engineered such a strain, and high performance liquid chromatography and liquid chromatography-mass spectrometry analyses demonstrated the modified strain produced DHLA, the fully oxidized product of dihydrolysergol. Accumulation of high concentrations of DHLA in field-collected C. gigantea sclerotia and discovery of a mutation in the gene lpsA, downstream from DHLA formation, supported our finding that DHLA rather than dihydrolysergol is the end product of the C. gigantea pathway.

Keywords: CloA; P450 monooxygenase; dihydrolysergic acid; dihydrolysergol; gene cluster.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Pathways to selected lysergic acid-derived ergot alkaloids and DHLA-derived ergot alkaloids. Enzymes responsible for catalysis are listed at appropriate points. Double arrows indicate omission of one or more intermediates. Relevant ring and carbon labeling (referred to in text) is indicated on the lysergic acid, 9, structure. Ergovaline, 10, is shown as a representative of several possible 9-derived ergopeptines; dihydroergosine, 6, is the only known dihydroergopeptine. Abbreviations: DMAPP, dimethylallylpyrophosphate; dma, dimethylallyl; eas, ergot alkaloid synthesis; clo, clavine oxidase; lps, lysergyl peptide synthetase.
Figure 2
Figure 2
HPLC analysis of N. fumigata easM knockout strain and representative transformant expressing C. gigantea cloA. Ergot alkaloids were detected by fluorescence with excitation and emission wavelengths of 272 and 372 nm, respectively. Abbreviations: easM ko, easM knockout; Trp, tryptophan; C.g., Claviceps gigantea; 3, festuclavine; 5, dihydrolysergic acid.
Figure 3
Figure 3
Parent ions and fragments of 5 and analyte accumulating in N. fumigata easM knockout expressing C. gigantea cloA. Spectra were collected with electrospray ionization in positive mode. Abbreviations: easM ko, easM knockout; C.g., Claviceps gigantea.
Figure 4
Figure 4
Analysis of C. gigantea cloA mRNA expressed in N. fumigata easM knockout. (A) Qualitative reverse transcriptase-PCR analysis of mRNA from genomic clone of C. gigantea cloA transformed into N. fumigata easM knockout (lane 1) or fully processed clone of C. gigantea cloA expressed in N. fumigata easM knockout (lane 2). Sizes of relevant fragments of BstEII-digested bacteriophage λ DNA are indicated to the left of the image. (B) Representations of structures of C. gigantea cloA genomic DNA and the two versions of cloA mRNA that accumulated upon expression of the genomic C. gigantea cloA in N. fumigata easM knockout.
Figure 5
Figure 5
HPLC analysis of a C. gigantea sclerotium. Analytes were detected by fluorescence with excitation and emission wavelengths of 272 and 372 nm, respectively. Peaks corresponding to characterized ergot alkaloids are indicated: 1, chanoclavine-I; 3, festuclavine; 4, dihydrolysergol; 5, dihydrolysergic acid.
Figure 6
Figure 6
High resolution LC–MS fragmentation spectra for (A) 5 standard and (B) coeluting analyte from C. gigantea sclerotia and (C) 4 standard and (D) coeluting analyte from C. gigantea sclerotia.
Figure 7
Figure 7
Ergot alkaloid synthesis (eas) clusters from C. purpurea, and C. gigantea. Abbreviations: dma, dimethylallyl; eas, ergot alkaloid synthesis; clo, clavine oxidase; lps, lysergyl peptide synthetase. The symbol Ψ indicates a pseudogene.
Figure 8
Figure 8
Nucleotide and deduced amino acid sequences from the region immediately surrounding a frameshift mutation in C. gigantea lpsA. In the second sequence, nucleotides AG were deleted to restore the reading frame to align with that of the closely related fungus C. africana, which produces 6. These particular nucleotides were chosen to maximize amino acid sequence identity of the deduced products; removal of other combinations of two nucleotides also would restore the frame. Spaces were inserted between codons to improve clarity of presentation.

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