Progress 02/26/14 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PhD student Katy Ryan completed her dissertation on ergotryptamine with assistance from capacity funds from this project. MS students Paige Bragg and Samantha Fabian completed their degrees in Genetics with assistance from capacity funds from this project. Current PhD student Matthew Maust received training in fungal molecular biology, biochemistry, and analytical methods while working toward objectives of this project. Each of these students presented their results at one or more scientific conferences and each have co-authored peer-reviewed articles published in scientific journals. How have the results been disseminated to communities of interest?Results have been published in 19 peer-reviewed scienitifc articles (refer to products page) and in numerous presentations at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Capacity funding from this Hatch project facilitated the generation of data that helped us achieve each of the three objectives of this project. To complete objectives 1 and 2, we focused on alleles of very different P450 monooxygenase-encoding genes that control previously uncharacterized steps in the ergot alkaloid pathway. Our previous work showed that agroclavine and festuclavine are alternate products of different versions of the enzyme EasA that provide the basis for divergence of the ergot alkaloid pathway to produce lysergic acid derivatives (from agroclavine) or dihydroergot alkaloids and fumigaclavines (both from festuclavine). In heterologous expression studies, we demonstrated that agroclavine is subject to a six-electron oxidation to lysergic acid through repeated activity of the enzyme CloA. We subsequently showed that festuclavine serves as the branch point between the dihydroergot alkaloid pathway (of rare Claviceps species) and fumigaclavine pathway (of certain Neosartorya and Penicillium species). Through gene knockout and complementation studies in Neosartorya fumigata, we showed that the gene easM encodes a P450 monooxygenase that oxidizes carbon 9 of festuclavine to send intermediates into the fumigaclavine branch of the pathway. Alternatively, festuclavine may be repeatedly oxidized at the methyl group (customarily labeled C15) by the product of an allele of cloA found in fungi that produce derivatives of dihydrolysergic acid. Another alkaloid, which we identified as a prenylated version of festuclavine, also accumulated to significantly higher levels in the easM knockout strain as compared to wild type. Other researchers have observed this prenylated version of festuclavine in low concentrations in wild-type strains of N. fumigata and have named it deacetoxyfumigaclavine C. We propose that this compound arises by prenylation of festuclavine by the prenyl transferase enzyme EasL. We have shown in previous work that EasL of N. fumigata accepts several different ergot alkaloids as substrates and prenylates them. In the easM knockout, relatively high levels of festuclavine accumulate, allowing it to serve as an abundant substrate for EasL. Complementation with a wild-type copy of easM restored the ability of the fungus to produce fumigaclavines. Concentrations of festuclavine and the prenylated form of festuclavine were reduced to wild-type levels in the strain complemented with the wild-type copy of easM. Collectively, these data provide mechanisms for movement of intermediates into lysergic acid, dihydrolysergic acid, and fumigaclavine branches of the ergot alkaloid pathway. Objective 3--the characterization of the ergot alkaloid ergotryptamine--was achieved by generating increased yields of the compound by heterologously expressing genes dmaW, easF, and easC in the ergot alkaloid-nonproducer Aspergillus nidulans. With these increased yields, we investigated the chemical structure of ergotryptamine and its role in the ergot alkaloid pathway. High-resolution mass spectrometry, precursor-labeling, and NMR studies showed that ergotryptamine differed from the known ergot alkaloid pathway compound N-methyl-dimethylallyltryptophan by the loss of the carboxyl group, addition of a hydroxyl group, and shift in position of a carbon-carbon double bond. Feeding studies with N. fumigata mutants did not show ergotryptamine turnover, suggesting it is a pathway byproduct as opposed to an authentic intermediate. Several natural Epichloe endophytes were found to produce ergotryptamine, and further mass spectrometry investigations revealed the equivalency of ergotryptamine with an Epichloe-derived ergot alkaloid provisionally described as 6,7-secolysergine in previous work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Durden, L., Wang, D., Panaccione, D.G., and Clay, K. 2019. Decreased root-knot nematode gall formation in roots of the morning glory Ipomoea tricolor symbiotic with ergot alkaloid-producing fungal Periglandula sp. Journal of Chemical Ecology 45:879-887.
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Progress 10/01/18 to 01/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PhD student Matthew Maust received training in manuscript preparation and report writing. How have the results been disseminated to communities of interest?Publications as listed in the "Products" section of this report and previous annual reports. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
All project goals have been met, and the final few months of the project were spent writing manuscripts and reports. A summary of the accomplishments relative to the three project goals is contained in the "Accomplishments" section of the "final report" for this project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Durden, L., Wang, D., Panaccione, D.G., and Clay, K. 2019. Decreased root-knot nematode gall formation in roots of the morning glory Ipomoea tricolor symbiotic with ergot alkaloid-producing fungal Periglandula sp. Journal of Chemical Ecology 45:879-887.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?MS student Samantha Fabian received training in biochemistry, microbiology, and recombinant DNA techniques. She presented her research in an invited symposium talk at the International Mycological Congress in San Juan, Puerto Rico. She published her work in a first -author publication. She complated her degree in May, 2018. PhD student Matthew Maust received training in instrumental analytical methods and recombinant DNA techniques. He presented her research in a poster at the International Mycological Congress in San Juan, Puerto Rico. He was second author on two refereed journal articles. Undergraduate student Caroline Leadmon received training in biochemistry, microbiology, and recombinant DNA techniques. She presented her research in an invited symposium talk at the International Mycological Congress in San Juan, Puerto Rico and in a poster at a regional meeting for the Society of Toxicology. Undergraduate student Jessi Tyo also received training in biochemistry, microbiology, and recombinant DNA techniques. She presented her research in a poster at the International Mycological Congress in San Juan, Puerto Rico. How have the results been disseminated to communities of interest?Results have been published in peer-reviewed scientific literature and presentated in poster and oral formats at conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
With the stated objectives of the proposal met, fundamental research on the ability of Metarhizium species to produce ergot alkaloids was continued. This work is relevant to agriculture, because Metarhizium species colonize plant roots in a nondetrimental way. These fungi also kill insects and have thus been used as commercial biocontrol agents. The accumulation of ergot alkaloids in Metarhizium species is also relevant to medicine, because the class of ergot alkaloids that these fungi accumulate is used to treat dementia, migraines, and hyperprolactinemia, among other conditions. The Metarhizium species are rare as culturable and genetically manipulable sources of these chemicals. For these reasons, the data described herein were used as preliminary data in seeking an NIH grant. Our preliminary data indicated that an isolate of M. anisopliae (ARSEF 9354) and several isolates of M. brunneum produced ergot alkaloids lysergic acid alpha-hydroxyethylamide (LAH), ergonovine, and ergine in culture, whereas isolates of M. robertsii, M. flavoviride, and M. pingshaense did not accumulate ergot alkaloids in culture. Over 80% of the ergot alkaloids (on a molar basis) were secreted into the growth medium. Because multilocus DNA sequencing studies we conducted subsequently indicated that M. anisopliae ARSEF 9354 was in fact an isolate of M. brunneum, we obtained three independent isolates of M. anisopliae and tested them for ergot alkaloid accumulation in culture. None of the three isolates produced ergot alkaloids in culture. Representatives of each of the species were tested for their ability to accumulate ergot alkaloids in infected larvae of the insect Galleria mellonella. All tested isolates of M. brunneum, M. flavoviride, and M. robertsii produced LAH, ergonovine, and ergine in insect larvae. Two of three tested isolates of M. anisopliae accumulated ergot alkaloids in insects. The isolate of M. pingshaense we tested did not accumulate ergot alkaloids in larvae. In the case of M. brunneum, we tested whether the vital status of the larva at infection affected accumulation of ergot alkaloids. Whereas the fungus colonized and emerged from larvae that were alive or dead at the time of inoculation, significantly more ergot alkaloids accumulated in larvae that were alive at inoculation. Isolates of M. brunneum and M. flavoviride also were grown on roots of corn and common bean. Roots and shoots of Metarhizium-colonized plants lacked ergot alkaloids. In summary, the data show that Metarhizium species tested, except for M. pingshaense, produce ergot alkaloids of the lysergic acid amide class in insects, only M. brunneum accumulates ergot alkaloids in culture, and neither of two Metarhizium species tested accumulated ergot alkaloids in plants. The induction of ergot alkaloids when Metarhizium species are inoculated into live insects suggests a role for the alkaloids in insect pathogenesis. The potential for ergot alkaloids to contribute to insect pathogenesis will be an important topic for future research.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Bragg, P.E., Maust, M.D., and Panaccione, D.G. 2017. Ergot alkaloid biosynthesis in the maize (Zea mays) ergot fungus Claviceps gigantea. Journal of Agricultural and Food Chemistry 65:10703-10710.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Fabian, S.J., Maust, M.D., and Panaccione, D.G. 2018. Ergot alkaloid synthesis capacity of Penicillium camemberti. Applied and Environmental Microbiology 84:e01583-18.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?MS student Paige Bragg received training in genetics, microbiology, and biochemistry, and earned her MS degree in May, 2017. She presented her work at a regional conference and published an MS thesis. BS student Stephanie Arnold receivedtraining in molecular biology and biochemistry.She presented her work at aregional conference and published two refereed journal articles. She earned her BS degree in May, 2017. PhD studentMatthew Maust and MS student Samantha Fabian received training in molecular biology and analytical chemistry. They each made presentations at a national conference. Undergraduate students Caroline Leadmon and Jessi Tyo receivedtraining in molecular biology and biochemistry. . How have the results been disseminated to communities of interest?The results were reported in refereed journal articles, in presentations at national, international, and local conferences, and in a thesis. What do you plan to do during the next reporting period to accomplish the goals?We will continue work on Objective 1, which is to characterize unsolved oxidation steps in the ergot alkaloid pathway. Immediate work will focus on the gene easO and its role in the synthesis of lysergic acid amides.
Impacts
What was accomplished under these goals?
In support of Objective 1, which involves discovery of genes encoding unsolved oxidation steps in the ergot alkaloid pathway, we began experiments aimed at determining the function of the ergot alkaloid synthesis cluster gene labeled easO. Based on its sequence, this gene has the capacity to encode a flavin-containing monooxygenase. The presence of the gene in different fungi correlates with the ability of fungi to produce the lysergic acid amide known as lysergic acid alpha-hydroxyethylamide (abbreviated LAH). To test the function of this gene, we inactivated it by gene knockout in an LAH-producing fungus. The knockout mutant failed to accumulate LAH and instead accumulated the lysergic acid amide ergonovine, which is an important lysergic acid amide in medicine and is also proposed to be two enzymatic steps prior to LAH in the LAH branch of the ergot alkaloid pathway. The data indicate that easO encodes an enzyme that oxidizes ergonovine; the product of that oxidation would then need to be acted on by another enzyme (hypothesized to be EasP) to produce LAH. Further work on easO and its significance will be pursued in the coming year. In support of objective 2, we thoroughly characterized the roles of different alleles of cloA in the synthesis of lysergic acid (abbreviated LA) compared to its dihydrogenated variant dihydrolysergic acid (abbreviated DHLA). LA and DHLA are structurally similar fungal secondary metabolites, but they have different activities related to cognitive function and other clinical applications. LA has been produced by genetic modification of the model fungus Neosartorya fumigata (synonym Aspergillus fumigatus), but the biosynthetic pathway to DHLA has not been thoroughly characterized. Our previous studies showed that the enzyme CloA from the LA-producing fungus Epichloë typhina x festucae oxidized the substrate agroclavine to LA. We transformed cloA from E. typhina x festucae into a N. fumigata strain that accumulates festuclavine, a precursor to DHLA, but it failed to oxidize festuclavine to DHLA. Substrate (agroclavine) feeding studies demonstrated that the enzyme was active in the modified fungus but simply failed to recognize festuclavine as substrate. We hypothesized that CloA from Claviceps africana, a DHLA-producing fungus, would oxidize festuclavine to DHLA. Introns in a genomic clone of C. africana cloA were not processed correctly in N. fumigata, so coding sequence only versions of cloA alleles from C. africana and closely related C. gigantea were synthesized and expressed in the festuclavine-accumulating mutant background. HPLC and mass spectrometry analyses demonstrated that transformants expressing CloA from either of these Claviceps spp. produced DHLA. Substrate feeding studies indicated that the C. africana and C. gigantea versions of CloA accepted agroclavine as well as festuclavine as substrates. Our results show that production of DHLA requires a specialized allele of cloA. Objective 3 has been completed, and the results have been summarized in previous progress reports and published.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Panaccione, D.G. and Arnold, S.L. 2017. Ergot alkaloids contribute to virulence in an insect model of invasive aspergillosis. Scientific Reports 7:8390.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Arnold, S.L. and Panaccione, D.G. 2017. Biosynthesis of the pharmaceutically important fungal ergot alkaloid dihydrolysergic acid requires a specialized allele of cloA. Applied and Environmental Microbiology 83:e00805-17.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Florea, S., Panaccione, D.G. and Schardl, C.L. 2017. Ergot alkaloids of the Clavicipitaceae. Phytopathology 107:504-518.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Arnold, S.A., and Panaccione, D.G. 2017. Roles of alternate alleles of the gene cloA in ergot alkaloid biosynthesis. Phytopathology 107:S4.1.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Bragg, P.E., and Panaccione, D.G. 2017. Genetic analysis of the ergot alkaloid biosynthetic capacity of the maize ergot fungus Claviceps gigantea. Phytopathology 107:S4.2.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Bragg, P.E. 2017. Genetic analysis of the biosynthesis of pharmaceutically important dihydroergot alkaloids by heterologous expression of Claviceps gigantea genes in the fungus Neosartorya fumigata. West Virginia University, ProQuest Dissertations Publishing, 2017. West Virginia University, ProQuest Dissertations Publishing, 2017. 10275387.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students Samantha Fabian and Paige Bragg were trained in genetic manipulation of fungi to produce specialized metabolites. The students were trained in analytical methods to detect, identify, and quantify specialized metabolites. How have the results been disseminated to communities of interest?A series of journal articles has been written that describe the results. These articles are listed in the appropriate section of this report. Results were presented at an international meeting in Pucon, Chile. What do you plan to do during the next reporting period to accomplish the goals?In support of objective 1, efforts will continue on determining function of novel genes involved in syntehtizong ergot alkaloids. In particular the individual activities of the two genes involved in rugulovasine synthesis will be separated and studied in detail. In support of objective 2, efforts will focus on increasing dihydroergot alkaloid accumualtion by targeting genes to different cellular compartments.
Impacts
What was accomplished under these goals?
In support of objective 1, two novel genes have been identified in the fungus Penicillium camemberti that are required for the synthesis of the unusual ergot alkaloid rugulovasine. Expression of these genes appears to be suppressed in P. camemberti; however, when they are expressed jointly in a mutant of the model fungus Neosartorya fumigata that accumulates a certain ergot alkaloid intermediate, the products of the two genes convert that intermediate into the ergot alkaloid rugulovasine. Each of the two genes appears to contribute an oxidation step that results in an oxidized intermediate that undergoes a spontaneous dehydration to result in rugulovasine. A wild relative of P. camemberti was acquired that contains the same set of genes and produces rugulovasine in culture. Genes encoding five additional steps in rugulovasine synthesis are clustered with the two novel oxidase genes in the P. camemberti genome. These five genes are homologous to those catalyzing the first five steps in the ergot alkaloid pathways of all known ergot alkaloid-producing fungi. In support of objective 2, we have found that P450 monooxygenases involved in synthesizing lysergic acid are incapable of oxidizing the dihydroergot alkaloid substrate festuclavine into dihydrolysergic acid. Alleles of this particular oxidase gene need to come from dihydroergot alkaloid-producing fungi to oxidize intermediates into dihydroergot alkaloids. When such genes are expressed in a festuclavine-accumulating fungus, dihydroergot alkaloids result. The data indicate unexpected substrate specificity in this particular enzyme. Objective 3 has been completed, and the results have been summarized in previous progress reports and published.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Bilovol, Y., and Panaccione, D.G. 2016. Functional analysis of the gene controlling hydroxylation of festuclavine in the ergot alkaloid pathway of Neosartorya fumigata. Current Genetics 62:853-860.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Florea, S., Phillips, T.D., Panaccione, D.G., Farman, M.L., and Schardl, C.L. 2016. Chromosome-end knockoff strategy to reshape alkaloid profiles of a fungal endophyte. G3 Genes Genomes Genetics 6:2601-2610.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Mulinti, P., Florea, S., Schardl, C.L., and Panaccione, D.G. 2016. Modulation of ergot alkaloids in a grass-endophyte symbiosis by alteration of mRNA concentrations of an ergot alkaloid synthesis gene. Journal of Agricultural and Food Chemistry 64:4982-4989.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student Yulia Bilovol (Female, US permanent resident) completed the requirements for an MS degreee during 2015. Undergraduate students Stephanie Arnold (Female, US citizen) and Tyson Currence (US citizen) also received training and conducted research supported in part by this project. All students received training and practice in modern molecular biological approaches to manipulating genes. How have the results been disseminated to communities of interest?A series of journal articles has been written that describe the results. These articles are listed in the apporpriate section of this report. What do you plan to do during the next reporting period to accomplish the goals?Objective 3 has been successfully completed. Work will continue on the oxidation steps that are part of both Objectives 1 and 2. In particular emphasis will be placed on modifying DNA constructs to yield more active enzymes.
Impacts
What was accomplished under these goals?
In support of objective 1, we continued work on the gene designated easM and the role of the enzyme encoded by this gene in oxidizing festuclavine. Festuclavine is an important intermediate in the fumigaclavine pathway of Neosartorya fumigata and the dihydroergot alkaloid pathway of certain Claviceps species. Knockout of easM in Neosartorya fumigata by homologous recombination of a mutated copy of that gene resulted in accumulation of festuclavine to levels significantly higher than those observed in the wild type fungus (P<0.0001) and elimination of fumigaclavine B (the immediate oxidation product of festuclavine) and fumigaclavine C (the ultimate end product of the fumigaclavine pathway). Another alkaloid, which we identified as a prenylated version of festuclavine, also accumulated to significantly higher levels in the easM knockout strain as compared to wild type (P=0.0002). Other researchers have observed this prenylated version of festuclavine in low concentrations in wild-type strains of N. fumigata and have named it deacetoxyfumigaclavine C. We propose that this compound arises by prenylation of festuclavine by the prenyl transferase enzyme EasL. We have shown in previous work that EasL of N. fumigata accepts several different ergot alkaloids as substrates and prenylates them. In the easM knockout, relatively high levels of festuclavine accumulate, allowing it to serve as an abundant substrate for EasL. Complementation with a wild-type copy of easM restored the ability of the fungus to produce fumigaclavines. Concentrations of festuclavine and the prenylated form of festuclavine were reduced to wild-type levels in the strain complemented with the wild-type copy of easM. These data indicate that easM encodes an enzyme that oxidizes festuclavine to fumigaclavine B. The strain of N. fumigata carrying the easM knockout may be a valuable resource in future research (outlined under Objective 2) on the mechanism of oxidation of festuclavine to dihydroergot alkaloids. The easM knockout accumulates abundant festuclavine which, in certain Claviceps species, serves as a critical intermediate in the biosynthesis of dihydroergot alkaloids. Work on Objective 3 was concluded with the publication of the formal description of ergotryptamine in the Journal of Agriculural and Food Chemistry.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Robinson, S.L., and Panaccione, D.G. 2015. Diversification of ergot alkaloids in natural and modified fungi. Toxins 7:201-218.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ryan, K.L., Akhmedov, N.G., and Panaccione, D.G. 2015. Identification and structural elucidation of ergotryptamine, a new ergot alkaloid produced by genetically modified Aspergillus nidulans and natural isolates of Epichloe species. Journal of Agricultural and Food Chemistry 63:61-67.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Beaulieu, W.T., Panaccione, D.G., Ryan K.L., Kaonongbua, W., and Clay, K. 2015. Phylogenetic and chemotypic diversity of Periglandula species in eight new morning glory hosts (Convolvulaceae). Mycologia 107:667-678.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Young, C.A., Schardl, C.L., Panaccione, D.G., Florea, S., Takach, J.E., Charlton, N.D., Moore, N., Webb, J.S, and Jaromczyk, J. 2015. Genetics, genomics and evolution of ergot alkaloid diversity. Toxins 7:1273-1302.
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Progress 02/26/14 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Ph.D. student Katy Ryan and M.S. student Yulia Bilovol were employed as graduate research assistants. Both students received training and practice in modern molecular biological approaches to manipulating genes. How have the results been disseminated to communities of interest? A series of journal articles has been written that describe the results. These articles are listed in the apporpriate section of this report. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we intend to continue to study the gene involved in oxidizing festuclavine to fumigaclavine B (which is the remaining part of objective 1). We also will intensify efforts to identify the oxidase-encoding gene responsible for oxidizing festuclavine to dihydrolysergic acid (objective 2).
Impacts
What was accomplished under these goals?
The gene required for oxidation of agroclavine (part of objective 1) was identified by following a heterologous expression strategy. Our previous work showed that different lineages of ergot alkaloid producers carry different alleles of easA that determine the first branch point of the ergot alkaloid pathway. By expressing the isomerase alleles of easA found in Claviceps purpurea, Epichloë festucae var. lolii (formerly Neotyphodium lolii), or Epichloë sp. Lp1 in a strain of Aspergillus fumigatus in which its native easA allele had been knocked out, we engineered A. fumigatus to produce agroclavine as opposed to festuclavine, which starts A. fumigatus down the pathway to lysergic acid-based ergot alkaloids. To take the next step in the pathway, agroclavine needs to be oxidized. We have used the bidirectional easA/easG promoter of A. fumigatus to drive expression of each of several candidate agroclavine oxidase genes (easD, easH, or cloA from E. sp. Lp1) along with expression of the isomerase allele of easA from E. sp. Lp1 in the A. fumigatus easA ko background. Strains expressing EasA/EasD or EasA/EasH accumulated agroclavine (as a result of expression of the isomerase allele of easA) but strains expressing EasA/CloA produced lysergic acid and its diastereoisomer isolysergic acid. This result was confirmed by MS/MS analyses. The data indicate that the ergot alkaloid cluster gene cloA encodes an enzyme that catalyzes all the oxidations of agroclavine to lysergic acid. Moreover, since only lysergic acid (and not its 8,9 double bond isomer paspalic acid) was produced in our EasA/CloA strain, the double bond isomerization appears to be catalyzed by one of the expressed enzymes (EasA or CloA) as opposed to happening spontaneously over time. The EasA/CloA strain of A. fumigatus easA ko is the first fungus known to produce lysergic acid as its biosynthetic endpoint in culture, and our data clearly show that expression of two genes from a lysergic acid-producing fungus is sufficient (in conjunction with the product of the native allele of easG in A. fumigatus) to produce lysergic acid from the common pathway intermediate chanoclavine aldehyde. Under objective 3, we investigated the chemical structure of ergotryptamine and its role in the ergot alkaloid pathway. High-resolution mass spectrometry, precursor-labeling, and NMR studies showed that ergotryptamine differed from the known ergot alkaloid pathway compound N-methyl-dimethylallyltryptophan by the loss of the carboxyl group, addition of a hydroxyl group, and shift in position of a carbon-carbon double bond. Feeding studies with Aspergillus mutants did not show ergotryptamine turnover, suggesting it is a pathway byproduct as opposed to an authentic intermediate. Several natural Epichlo? endophytes were found to produce ergotryptamine, and further mass spectrometry investigations revealed the equivalency of ergotryptamine with an Epichlo?-derived ergot alkaloid provisionally described as 6,7-secolysergine in previous work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Robinson, S.L., and Panaccione, D.G. 2014. Heterologous expression of lysergic acid and novel ergot alkaloids in Aspergillus fumigatus. Applied and Environmental Microbiology 80:6465-6472.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Panaccione, D.G., Beaulieu, W.T., and Cook, D. 2014. Bioactive alkaloids in vertically transmitted fungal endophytes. Functional Ecology 28:299-314.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Mulinti, P., Allen, N.A., Coyle, C.M., Gravelat, F.N., Sheppard, D.C., and Panaccione, D.G. 2014. Accumulation of ergot alkaloids during conidiophore development in Aspergillus fumigatus. Current Microbiology 68:1-5.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Morton, J.B., Benedito, V.A., Panaccione, D.G., and Jenks, M.A. 2014. Potential for industrial application of microbes in symbioses that influence plant productivity and sustainability in agricultural, natural or restored ecosystems. Industrial Biotechnology 10:347-353.
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