scholarly journals Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I

2010 ◽  
Vol 76 (6) ◽  
pp. 1822-1830 ◽  
Author(s):  
Nicole Lorenz ◽  
Jana Olšovská ◽  
Miroslav Šulc ◽  
Paul Tudzynski
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Axenic Culture ◽  
Ergot Alkaloids ◽  
Claviceps Purpurea ◽  
Alkaloid Biosynthesis ◽  
Fusion Construct ◽  
Knockout Mutants ◽  
Clavine Alkaloids ◽  
Functional Analyses ◽  
Layer Chromatography

ABSTRACT Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

scholarly journals Comparative Ergot Alkaloid Elaboration by Selected Plectenchymatic Mycelia of Claviceps purpurea through Sequential Cycles of Axenic Culture and Plant Parasitism

Biology ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 41
Author(s):  
Peter Mantle
Keyword(s):  
Present Report ◽  
Axenic Culture ◽  
Ergot Alkaloids ◽  
Culture Conditions ◽  
Claviceps Purpurea ◽  
Classical Concept ◽  
Industrial Fermentation ◽  
Plant Parasitism ◽  
Key Factor ◽  
Selection Of

Ergot alkaloids have an established place in plant pathology and toxicology. As pharmaceuticals, their sourcing is via natural or managed agricultural occurrence of sclerotia of Claviceps purpurea (Fr.) Tul. or through industrial fermentation processes with other Claviceps. The key factor for biosynthesis is differentiation of a particular mycelial anatomy. Previous study of these fungi from two disparate English grass genera, Spartina and Phragmites, has shown that only mycelia expressing a plectenchymatic sclerotium-like anatomy in specific axenic culture conditions elaborated ergot alkaloids, and then only as far as lysergic acid. The present report describes sequential cycles of axenic and parasitic cultivation for wild isolates from Dactylis and Alopecurus with intervention of a single ascospore step. This confirms the homozygous character of C. purpurea and defines several potential experimental axenic and parasitic conditions within the species for comparing genomic aspects of partial or full biosynthesis of cyclic tri-peptide alkaloids. Whereas Alopecurus ergot isolates readily parasitized rye, use of Dactylis isolates as inoculum for rye ovaries failed to cause the usual sphacelial fructification but supported growth of exceptionally thin sclerotia, sometimes two in a floret, with low alkaloid content attributed to reduced medullary component. However, after two cycles of axenic and rye-parasitic cultivation, and consistent re-selection of the plectenchymatic character in axenic mycelia, typical growth of ergot sclerotia occurred on rye. Caution thus seems necessary in tests for putative host specificity in any taxonomic realignments within the classical concept of C. purpurea. A Dactylis ergot isolate was also uniquely shown to parasitise the plumule of germinating rye seeds confirming the susceptibility of apical tissues. A key biosynthetic feature of a mycelial glyceride oil, rich in ricinoleic acid, as a prelude to axenic and parasitic formation of ergot alkaloids by C. purpurea is emphasised.

scholarly journals Comparison of Ergot Alkaloid Biosynthesis Gene Clusters in Claviceps Species Indicates Loss of Late Pathway Steps in Evolution of C. fusiformis

2007 ◽  
Vol 73 (22) ◽  
pp. 7185-7191 ◽  
Author(s):  
Nicole Lorenz ◽  
Ella V. Wilson ◽  
Caroline Machado ◽  
Christopher L. Schardl ◽  
Paul Tudzynski
Keyword(s):  
Ergot Alkaloids ◽  
Gene Clusters ◽  
Lysergic Acid ◽  
Claviceps Purpurea ◽  
Alkaloid Biosynthesis ◽  
In Planta ◽  
Acid Biosynthesis ◽  
Peptide Synthetase ◽  
Biosynthesis Gene ◽  
Inactivating Mutation

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 Ψ ). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB Ψ and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB Ψ 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.

scholarly journals Ergot Alkaloids in Wheat and Rye Derived Products in Italy

Foods ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 150 ◽  
Author(s):  
Francesca Debegnach ◽  
Simona Patriarca ◽  
Carlo Brera ◽  
Emanuela Gregori ◽  
Elisa Sonego ◽  
...  
Keyword(s):  
Durum Wheat ◽  
Middle Ages ◽  
Plant Pathogen ◽  
Ergot Alkaloids ◽  
Cereal Grains ◽  
Wheat Bread ◽  
Claviceps Purpurea ◽  
Eu Commission ◽  
Ongoing Monitoring ◽  
The Eu

Genus Claviceps is a plant pathogen able to produce a group of toxins, ergot alkaloids (EAs), whose effects have been known since the Middle Ages (ergotism). Claviceps purpurea is the most important representative specie, known to infect more than 400 monocotyledonous plants including economically important cereal grains (e.g., rye, wheat, triticale). EAs are not regulated as such. Maximum limits are in the pipeline of the EU Commission while at present ergot sclerotia content is set by the Regulation (EC) No. 1881/2006 in unprocessed cereals (0.05% as a maximum). This study aimed to investigate the presence of the six principal EAs (ergometrine, ergosine, ergocornine, α-ergocryptine, ergotamine and ergocristine) and their relative epimers (-inine forms) in rye- and wheat-based products. Of the samples, 85% resulted positive for at least one of the EAs. Wheat bread was the product with the highest number of positivity (56%), followed by wheat flour (26%). Rye and wheat bread samples showed the highest values when the sum of the EAs was considered, and durum wheat bread was the more contaminated sample (1142.6 μg/kg). These results suggest that ongoing monitoring of EAs in food products is critical until maximum limits are set.

scholarly journals Assessment of ergot (Claviceps purpurea) exposure in pregnant and postpartum beef cows

2018 ◽  
Vol 98 (4) ◽  
pp. 688-700 ◽  
Author(s):  
T. Grusie ◽  
V. Cowan ◽  
J. Singh ◽  
J. McKinnon ◽  
B. Blakley
Keyword(s):  
Ergot Alkaloid ◽  
Post Partum ◽  
Ergot Alkaloids ◽  
Maximum Size ◽  
Beef Cows ◽  
Plasma Prolactin ◽  
Claviceps Purpurea ◽  
Ambient Temperatures ◽  
Beef Cow ◽  
The Impact

Cows were fed ration for 9 wk containing 5, 48, 201, and 822 μg kg−1 ergot alkaloids. The objective was to evaluate the impact of ergot consumption in beef cow–calf operations. Ergot alkaloids up to 822 μg kg−1 did not alter the weight of peripartum and postpartum beef cows (P = 0.93) or nursing calves (P = 0.08), rectal temperature (P = 0.16), or plasma prolactin concentrations (P = 0.30) at moderate ambient temperatures. Ergot did not influence the time (>1 ng mL−1; P = 0.79) or the progesterone concentration (P = 0.38) at the time of first postpartum rise or the size of the first (14 ± 0.6 mm; P = 0.40) and second (13 ± 0.5 mm; P = 0.41) follicles to ovulate. The maximum size of the first postpartum corpus luteum (CL) was 4 mm larger in the 822 μg kg−1 ergot group compared with the control (P = 0.03) for the first ovulation post partum, but not for the second (P = 0.11). There was no effect of ergot exposure on the number of days until the appearance of the first (43 ± 4 d; P = 0.95) or second (52 ± 4 d; P = 0.98) CL post partum. Ergot alkaloid concentrations up to 822 μg kg−1 did not affect pregnancy rates (X2 = 0.36). In conclusion, ergot alkaloid exposure for 9 wk to concentrations as high as 822 μg kg−1 did not alter performance in pregnant and postpartum beef cattle at moderate ambient temperatures.

Covalently Bound Flavin in D-6-Hydroxynicotine Oxidase from Arthrobacter oxidans

1972 ◽  
Vol 27 (9) ◽  
pp. 1073-1074 ◽  
Author(s):  
M. Brühmüller ◽  
H. Möhler ◽  
K. Decker
Keyword(s):  
Amino Acid ◽  
Flavin Adenine Dinucleotide ◽  
Ph Dependence ◽  
Spectral Characteristics ◽  
Amino Acid Derivative ◽  
Aminopeptidase M ◽  
Arthrobacter Oxidans ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Covalently Bound

D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.

Occurrence of ergot and its alkaloids in winter rye harvested in Poland

2018 ◽  
Vol 11 (4) ◽  
pp. 635-646 ◽  
Author(s):  
M. Bryła ◽  
E. Ksieniewicz-Woźniak ◽  
G. Podolska ◽  
A. Waśkiewicz ◽  
K. Szymczyk ◽  
...  
Keyword(s):  
European Commission ◽  
Level Set ◽  
Fungal Pathogen ◽  
Ergot Alkaloids ◽  
Winter Rye ◽  
Claviceps Purpurea ◽  
Maximum Permissible Level ◽  
Cereal Species ◽  
The Mean ◽  
Mean Concentration

Ergot (Claviceps purpurea) is a fungal pathogen that infects various grass and small grain cereal species, most often open-pollinated grasses, including rye and triticale. We tested 122 samples of rye grains harvested in three different regions of Poland in 2016 and 2017 for ergot and its alkaloids. Ergot sclerotia were found in all samples. The mean content of ergot sclerotia in grain ranged between 0.74 and 1.06 g/kg, and the mean concentration of ergot alkaloids in grain ranged between 270.1 and 580. 4 μg/kg, depending on the region of cultivation. 37% of the samples were infected with ergot below the 0.5 g/kg level set by the European Commission as the maximum permissible level for ergot, and in those samples the mean ergot alkaloids concentration was 155.8 μg/kg (range 4.7-667.9 μg/kg). A statistically significant correlation (R2=0.6941) between ergot content and concentration of ergot alkaloids was found. Ergot alkaloids concentration in grain was re-calculated into ergot alkaloids concentration in sclerotia, and was found to vary widely from 114 to 3,167 mg/kg. Ergot alkaloids profiles were most frequently dominated by R-configured ergopeptides, such as ergocryptine, ergocornine and ergocristine (31, 29 and 16% of the samples, respectively).

Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum

1998 ◽  
Vol 44 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Seanna L Annis ◽  
Daniel G Panaccione
Keyword(s):  
Tall Fescue ◽  
Claviceps Purpurea ◽  
Neotyphodium Coenophialum ◽  
Experimental Conditions ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Different Ages ◽  
Gene Transcripts ◽  
Functional Analyses

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hybridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as in ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.Key words: Claviceps purpurea, endophyte, ergot, ergotamine, Neotyphodium coenophialum.

scholarly journals Total synthesis, biosynthesis and biological profiles of clavine alkaloids

2016 ◽  
Vol 14 (25) ◽  
pp. 5894-5913 ◽  
Author(s):  
Stephanie R. McCabe ◽  
Peter Wipf
Keyword(s):  
Total Synthesis ◽  
Ergot Alkaloids ◽  
Clavine Alkaloids ◽  
Biological Aspects

This review highlights noteworthy synthetic and biological aspects of the clavine subfamily of ergot alkaloids.

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