Date of Graduation
Davis College of Agriculture, Natural Resources and Design
Daniel G Panaccione
Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi in the Clavicipitaceae (order Hypocreales) and Trichocomaceae (order Eurotiales). Ergot alkaloid pathways vary among producers due to genetic variation within ergot alkaloid synthesizing (eas) gene clusters. However, the early steps of the pathway leading to the formation of chanoclavine-I appear to be evolutionarily conserved among producers. Chanoclavine-I synthesis occurs in at least five steps, and knockout and heterologous expression studies have demonstrated that four genes, dmaW, easF, easE, and easC, are required for pathway steps prior to chanoclavine-I formation. It was unknown whether these genes were sufficient to encode synthesis of chanoclavine-I or if another gene not located within the eas cluster was involved. DmaW performs the determinant step of alkaloid synthesis by prenylating tryptophan to produce dimethylallyltryptophan (DMAT). The easF gene encodes a methyltransferase responsible for the conversion of DMAT to N-Me-DMAT. The specific roles of EasE and EasC have not been established, though bioinformatics suggests that easE encodes an oxidoreductase and EasC is a catalase. The proposed biochemical scheme for chanoclavine-I synthesis involves two oxidation reactions for conversion of N-Me-DMAT to chanoclavine-I; thus, involvement of additional genes is plausible since easE encodes the only oxidase among the genes listed. Four genes, dmaW, easF, easE, and easC were amplified from the human pathogen Aspergillus fumigatus and transformed into Aspergillus nidulans, which does not contain any of the ergot alkaloid synthesis genes and produces no ergot alkaloids. HPLC and LC/MS analyses demonstrated that dmaW, easF, easE, and easC were sufficient for chanoclavine-I synthesis in A. nidulans; however, the possibility that uncharacterized activities already present in A. nidulans contributed to chanoclavine-I synthesis cannot be excluded. Combinations of the four genes listed above were amplified and transformed in A. nidulans to gain insights into the roles of EasE and EasC. Aspergillus nidulans transformant WFC (containing the genes dmaW, easF, and easC) accumulated a novel ergot alkaloid not detected in control strains or A. nidulans WFE (containing the genes dmaW, easF, and easE). The structure of the newly discovered compound was elucidated by labeling studies, high-resolution LC/MS, and NMR and given the trivial name ergotryptamine. Ergotryptamine feeding studies conducted with A. nidulans WFE and easC knockout A. fumigatus strains revealed that this metabolite is not an intermediate in chanoclavine-I synthesis. Ergotryptamine also was detected in several species of Epichloe, and further investigations established that ergotryptamine is the same molecule provisionally characterized as 6,7-secolysergine in previous work with Epichloe species. I conclude that the four genes listed above are sufficient for the synthesis of chanoclavine-I in A. nidulans and reveal the chemical structure of a novel metabolite produced during the early steps of ergot alkaloid synthesis. The approach of expressing ergot alkaloid pathway genes in A. nidulans provides a mechanism for better understanding of the early steps in ergot alkaloid synthesis.
Ryan, Katy, "Elucidation of the early steps of ergot alkaloid synthesis by pathway reconstruction in Aspergillus nidulans" (2014). Graduate Theses, Dissertations, and Problem Reports. 6545.