Cleaving Ergot Alkaloids by Hydrazinolysis—A Promising Approach for a Sum Parameter Screening Method

Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40‒60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.

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Optimized Analysis of Ergot Alkaloids in Rye Products by Liquid Chromatography-Fluorescence Detection Applying Lysergic Acid Diethylamide as an Internal Standard

Toxins ◽

10.3390/toxins11040184 ◽

2019 ◽

Vol 11 (4) ◽

pp. 184 ◽

Cited By ~ 3

Author(s):

Iris Holderied ◽

Michael Rychlik ◽

Paul Elsinghorst

Keyword(s):

Solid Phase ◽

Ergot Alkaloids ◽

Internal Standard ◽

Lysergic Acid ◽

Lysergic Acid Diethylamide ◽

Food And Feed ◽

Internal Standardization ◽

Syringe Filter ◽

Optimized Conditions ◽

Medium Type

Analysis of ergot alkaloids remains a topic of importance and the European Food Safety Authority (EFSA) has encouraged laboratories to provide monitoring data for the further evaluation of their occurrence in food and feed. While LC-MS/MS has dominated developments in recent years, LC-FLD is still more widespread, especially in developing countries. To improve the analysis of ergot alkaloids by LC-FLD, we developed an improved protocol introducing lysergic acid diethylamide (LSD) for internal standardization. Several aspects such as the composition and pH of the extraction medium, type of sorbent and conditions applied for solid-phase extraction/clean-up, use of a keeper during final evaporation and the type of syringe filter used for filtration prior to injection were thoroughly investigated. Optimized conditions comprise extraction by ethyl acetate, methanol and 28% aqueous ammonia in combination with basic aluminum oxide for extract clean-up. Use of a keeper was found inappropriate as LC-FLD analysis was significantly affected by co-eluting keeper components. Similar observations were made with some of the investigated syringe filters, where polytetrafluoroethylene (PTFE) proved to be the most suitable. Validation and application of the optimized methodology to real samples provided limits of detection and quantification suitable for the evaluation of relevant ergot alkaloid contaminations in rye and bakery products with superior precision that was facilitated by the introduced internal standard, LSD.

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Several Metarhizium Species Produce Ergot Alkaloids in a Condition-Specific Manner

Applied and Environmental Microbiology ◽

10.1128/aem.00373-20 ◽

2020 ◽

Vol 86 (14) ◽

Cited By ~ 2

Author(s):

Caroline E. Leadmon ◽

Jessi K. Sampson ◽

Matthew D. Maust ◽

Angie M. Macias ◽

Stephen A. Rehner ◽

...

Keyword(s):

Yeast Extract ◽

Ergot Alkaloid ◽

Genomic Sequence ◽

Close Association ◽

Acid Amide ◽

Ergot Alkaloids ◽

Lysergic Acid ◽

Content Type ◽

Extract Agar ◽

Yeast Extract Agar

ABSTRACT Genomic sequence data indicate that certain fungi in the genus Metarhizium have the capacity to produce lysergic acid-derived ergot alkaloids, but accumulation of ergot alkaloids in these fungi has not been demonstrated previously. We assayed several Metarhizium species grown under different conditions for accumulation of ergot alkaloids. Isolates of M. brunneum and M. anisopliae accumulated the lysergic acid amides lysergic acid α-hydroxyethyl amide, ergine, and ergonovine on sucrose-yeast extract agar but not on two other tested media. Isolates of six other Metarhizium species did not accumulate ergot alkaloids on sucrose-yeast extract agar. Conidia of M. brunneum lacked detectable ergot alkaloids, and mycelia of this fungus secreted over 80% of their ergot alkaloid yield into the culture medium. Isolates of M. brunneum, M. flavoviride, M. robertsii, M. acridum, and M. anisopliae produced high concentrations of ergot alkaloids in infected larvae of the model insect Galleria mellonella, but larvae infected with M. pingshaense, M. album, M. majus, and M. guizhouense lacked detectable ergot alkaloids. Alkaloid concentrations were significantly higher when insects were alive (as opposed to killed by freezing or gas) at the time of inoculation with M. brunneum. Roots of corn and beans were inoculated with M. brunneum or M. flavoviride and global metabolomic analyses indicated that the inoculated roots were colonized, though no ergot alkaloids were detected. The data demonstrate that several Metarhizium species produce ergot alkaloids of the lysergic acid amide class and that production of ergot alkaloids is tightly regulated and associated with insect colonization. IMPORTANCE Our discovery of ergot alkaloids in fungi of the genus Metarhizium has agricultural and pharmaceutical implications. Ergot alkaloids produced by other fungi in the family Clavicipitaceae accumulate in forage grasses or grain crops; in this context they are considered toxins, though their presence also may deter or kill insect pests. Our data report ergot alkaloids in Metarhizium species and indicate a close association of ergot alkaloid accumulation with insect colonization. The lack of accumulation of alkaloids in spores of the fungi and in plants colonized by the fungi affirms the safety of using Metarhizium species as biocontrol agents. Ergot alkaloids produced by other fungi have been exploited to produce powerful pharmaceuticals. The class of ergot alkaloids discovered in Metarhizium species (lysergic acid amides) and their secretion into the growth medium make Metarhizium species a potential platform for future studies on ergot alkaloid synthesis and modification.

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Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum

Applied and Environmental Microbiology ◽

10.1128/aem.01251-20 ◽

2020 ◽

Vol 86 (19) ◽

Author(s):

Kyle A. Davis ◽

Jessi K. Sampson ◽

Daniel G. Panaccione

Keyword(s):

Ergot Alkaloid ◽

High Performance ◽

Neurological Diseases ◽

Ergot Alkaloids ◽

Lysergic Acid ◽

Efficient Production ◽

Natural Sources ◽

Content Type ◽

Metarhizium Brunneum ◽

Alkaloid Synthesis

ABSTRACT Ergot alkaloids are important specialized fungal metabolites that are used to make potent pharmaceuticals for neurological diseases and disorders. Lysergic acid (LA) and dihydrolysergic acid (DHLA) are desirable lead compounds for pharmaceutical semisynthesis but are typically transient intermediates in the ergot alkaloid and dihydroergot alkaloid pathways. Previous work with Neosartorya fumigata demonstrated strategies to produce these compounds as pathway end products, but their percent yield (percentage of molecules in product state as opposed to precursor state) was low. Moreover, ergot alkaloids in N. fumigata are typically retained in the fungus as opposed to being secreted. We used clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) and heterologous expression approaches to engineer these compounds in Metarhizium brunneum, representing an alternate expression host from a different lineage of fungi. The relative percent yields of LA (86.9%) and DHLA (72.8%) were much higher than those calculated here for previously engineered strains of N. fumigata (2.6% and 2.0%, respectively). Secretion of these alkaloids also was measured, with averages of 98.4% of LA and 87.5% of DHLA being secreted into the growth medium; both values were significantly higher than those measured for the N. fumigata derivatives (both of which were less than 5.6% secreted). We used a similar approach to engineer a novel dihydroergot alkaloid in M. brunneum and, through high-performance liquid chromatography-mass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid α-hydroxyethylamide (dihydro-LAH). The engineering of these strains provides a strategy for producing novel and pharmaceutically important chemicals in a fungus more suitable for their production. IMPORTANCE Ergot alkaloids derived from LA or DHLA are the bases for numerous pharmaceuticals with applications in the treatment of dementia, migraines, hyperprolactinemia, and other conditions. However, extraction of ergot alkaloids from natural sources is inefficient, and their chemical synthesis is expensive. The ability to control and redirect ergot alkaloid synthesis in fungi may allow more efficient production of these important chemicals and facilitate research on novel derivatives. Our results show that Metarhizium brunneum can be engineered to efficiently produce and secrete LA and DHLA and, also, to produce a novel derivative of DHLA not previously found in nature. The engineering of dihydroergot alkaloids, including a novel species, is important because very few natural sources of these compounds are known. Our approach establishes a platform with which to use M. brunneum to study the production of other ergot alkaloids, specifically those classified as lysergic acid amides and dihydroergot alkaloids.

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Fate of Ergot Alkaloids during Laboratory Scale Durum Processing and Pasta Production

Toxins ◽

10.3390/toxins11040195 ◽

2019 ◽

Vol 11 (4) ◽

pp. 195 ◽

Cited By ~ 6

Author(s):

Sheryl Tittlemier ◽

Dainna Drul ◽

Mike Roscoe ◽

Dave Turnock ◽

Dale Taylor ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Ergot Alkaloid ◽

Ergot Alkaloids ◽

Laboratory Scale ◽

Biologically Active ◽

Tandem Mass ◽

Whole Grain ◽

Food And Feed

The fate of ergot alkaloids during the milling of durum and subsequent production and cooking of pasta was examined. Durum samples containing varying amounts of ergot sclerotia (0.01–0.1% by mass) were milled, and all milling product was analyzed for 10 ergot alkaloids using liquid chromatography with tandem mass spectrometry. Spaghetti was prepared from the semolina obtained during milling. Ergocristine, ergocristinine, and ergotamine were the predominant ergot alkaloids observed in the milling fractions and spaghetti. Approximately 84% of the total ergot alkaloid mass of the whole grain durum resided in the milling product fractions associated with the outer kernel layers (bran, shorts, feeds). No consistent loss of ergot alkaloids was observed during the production or cooking of spaghetti. However, changes in the ratio of R- to S-enantiomers occurred during the milling and cooking of spaghetti. Products containing bran, shorts, and feeds, as well as cooked spaghetti, contained a higher proportion of the less biologically active S-enantiomers. The results of this study emphasize the need to monitor R- and S-enantiomers, and to consider food and feed products, as opposed to whole grain, when assessing any exposure of consumers to ergot alkaloids.

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Ergot Alkaloids of the Family Clavicipitaceae

Phytopathology ◽

10.1094/phyto-12-16-0435-rvw ◽

2017 ◽

Vol 107 (5) ◽

pp. 504-518 ◽

Cited By ~ 28

Author(s):

Simona Florea ◽

Daniel G. Panaccione ◽

Christopher L. Schardl

Keyword(s):

Ergot Alkaloid ◽

Gene Loss ◽

Ergot Alkaloids ◽

Gene Clusters ◽

Lysergic Acid ◽

Forage Grasses ◽

Alkaloid Biosynthesis ◽

The Family ◽

Gene Recruitment ◽

Alkaloid Profiles

Ergot alkaloids are highly diverse in structure, exhibit diverse effects on animals, and are produced by diverse fungi in the phylum Ascomycota, including pathogens and mutualistic symbionts of plants. These mycotoxins are best known from the fungal family Clavicipitaceae and are named for the ergot fungi that, through millennia, have contaminated grains and caused mass poisonings, with effects ranging from dry gangrene to convulsions and death. However, they are also useful sources of pharmaceuticals for a variety of medical purposes. More than a half-century of research has brought us extensive knowledge of ergot-alkaloid biosynthetic pathways from common early steps to several taxon-specific branches. Furthermore, a recent flurry of genome sequencing has revealed the genomic processes underlying ergot-alkaloid diversification. In this review, we discuss the evolution of ergot-alkaloid biosynthesis genes and gene clusters, including roles of gene recruitment, duplication and neofunctionalization, as well as gene loss, in diversifying structures of clavines, lysergic acid amides, and complex ergopeptines. Also reviewed are prospects for manipulating ergot-alkaloid profiles to enhance suitability of endophytes for forage grasses.

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Independent evolution of a lysergic acid amide in Aspergillus species

Applied and Environmental Microbiology ◽

10.1128/aem.01801-21 ◽

2021 ◽

Author(s):

Abigail M. Jones ◽

Chey R. Steen ◽

Daniel G. Panaccione

Keyword(s):

Ergot Alkaloid ◽

Phylogenetic Analyses ◽

Acid Amide ◽

Ergot Alkaloids ◽

Gene Clusters ◽

Lysergic Acid ◽

Alkaloid Synthesis ◽

Genes Encoding ◽

Selection For ◽

High Concentrations

Ergot alkaloids derived from lysergic acid have impacted humanity as contaminants of crops and as the bases of pharmaceuticals prescribed to treat dementia, migraines, and other disorders. Several plant-associated fungi in the Clavicipitaceae produce lysergic acid derivatives, but many of these fungi are difficult to culture and manipulate. Some Aspergillus species, which may be more ideal experimental and industrial organisms, contain an alternate branch of the ergot alkaloid pathway but none were known to produce lysergic acid derivatives. We mined genomes of Aspergillus species for ergot alkaloid synthesis ( eas ) gene clusters and discovered three species–– A. leporis, A. homomorphus, and A. hancockii ––had eas clusters indicative of the capacity to produce a lysergic acid amide. In culture, A. leporis, A. homomorphus, and A. hancockii produced lysergic acid amides, predominantly lysergic acid α-hydroxyethylamide (LAH). Aspergillus leporis and A. homomorphus produced high concentrations of LAH and secreted most of their ergot alkaloid yield into the culture medium. Phylogenetic analyses indicated genes encoding enzymes leading to the synthesis of lysergic acid were orthologous to those of the lysergic acid amide-producing Clavicipitaceae; however, genes to incorporate lysergic acid into an amide derivative evolved from different ancestral genes in the Aspergillus species. Our data demonstrate fungi outside the Clavicipitaceae produce lysergic acid amides and indicate the capacity to produce lysergic acid evolved once, but the ability to insert it into LAH evolved independently in Aspergillus species and the Clavicipitaceae. The LAH-producing Aspergillus species may be useful for study and production of these pharmaceutically important compounds. IMPORTANCE Lysergic acid derivatives are specialized metabolites with historical, agricultural, and medical significance and were known heretofore only from fungi in one family, the Clavicipitaceae. Our data show that several Aspergillus species, representing a different family of fungi, also produce lysergic acid derivatives and that the ability to put lysergic acid into its amide forms evolved independently in the two lineages of fungi. From microbiological and pharmaceutical perspectives, the Aspergillus species may represent better experimental and industrial organisms than the currently employed, lysergic acid producers of the plant-associated Clavicipitaceae. The observation that both lineages independently evolved the derivative lysergic acid α-hydroxyethylamide (LAH), among many possible lysergic acid amides, suggests a selection for this metabolite.

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A Baeyer-Villiger monooxygenase gene involved in the synthesis of lysergic acid amides affects the interaction of the fungus Metarhizium brunneum with insects

Applied and Environmental Microbiology ◽

10.1128/aem.00748-21 ◽

2021 ◽

Author(s):

Chey R. Steen ◽

Jessi K. Sampson ◽

Daniel G. Panaccione

Keyword(s):

Ergot Alkaloid ◽

Galleria Mellonella ◽

Insect Pests ◽

Biological Activities ◽

Plant Root ◽

Total Concentration ◽

Acid Amide ◽

Ergot Alkaloids ◽

Structural Similarity ◽

Lysergic Acid

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum , produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)––by far the most abundant lysergic acid amide in M. brunneum ––are unknown. Ergot alkaloid synthesis ( eas ) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes ( easO and easP ) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella , indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. Importance Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species including M. brunneum were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

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

Canadian Journal of Animal Science ◽

10.1139/cjas-2017-0098 ◽

2018 ◽

Vol 98 (4) ◽

pp. 688-700 ◽

Cited By ~ 2

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.

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Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

Applied and Environmental Microbiology ◽

10.1128/aem.02137-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6465-6472 ◽

Cited By ~ 21

Author(s):

Sarah L. Robinson ◽

Daniel G. Panaccione

Keyword(s):

Aspergillus Fumigatus ◽

Candidate Genes ◽

Mass Spectra ◽

Ergot Alkaloids ◽

Lysergic Acid ◽

Precursor Feeding ◽

Gene Encoding ◽

Content Type ◽

Multiple Reactions ◽

Important Pathway

ABSTRACTDifferent lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungusEpichloë festucaevar.lolii×Epichloë typhinaisolate Lp1 (henceforth referred to asEpichloësp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate geneseasHandcloAwere expressed in a mutant strain of the moldAspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with theEpichloësp. Lp1 allele ofeasA, which encodes an enzyme that catalyzed the synthesis of agroclavine from anA. fumigatusintermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressingeasAandcloAfromEpichloësp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result ofEpichloësp. Lp1easAexpression in the absence ofcloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

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Developments in mycotoxin analysis: an update for 2012-2013

World Mycotoxin Journal ◽

10.3920/wmj2013.1637 ◽

2014 ◽

Vol 7 (1) ◽

pp. 3-33 ◽

Cited By ~ 52

Author(s):

F. Berthiller ◽

P.A. Burdaspal ◽

C. Crews ◽

M.H. Iha ◽

R. Krska ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Sample Preparation ◽

Ergot Alkaloids ◽

Tandem Mass ◽

Wide Range ◽

Rapid Spread ◽

Food And Feed ◽

Mycotoxin Analysis ◽

The Individual

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2012 and mid-2013. It covers the major mycotoxins: aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone. A wide range of analytical methods for mycotoxin determination in food and feed were developed last year, in particular immunochemical methods and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-based methods. After a section on sampling and sample preparation, due to the rapid spread and developments in the field of LC-MS/MS multimycotoxin methods, a separate section has been devoted to this area of research. It is followed by a section on mycotoxins in botanicals and spices, before continuing with the format of previous reviews in this series with dedicated sections on method developments for the individual mycotoxins.

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