Author ORCID Identifier

https://orcid.org/0009-0000-1387-4223

Semester

Summer

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Division of Plant and Soil Sciences

Committee Chair

Daniel Panaccione

Committee Member

Jennifer Hawkins

Committee Member

Matthew Kasson

Committee Member

Teiya Kijimoto

Abstract

Ergot alkaloids are a complex family of tryptophan-derived mycotoxins produced by a diverse range of fungi that occupy a wide variety of ecological niches including soil saprotrophs, plant endophytes, pathogens of plants or insects, and opportunistic pathogens of humans and other mammals. Ergot alkaloids are a similarly diverse family of chemicals that elicit a variety of pharmacological activities in animals due to their resemblance to neurotransmitters and high binding affinity for neurological receptors, including those that bind adrenaline, dopamine, and 5-hydroxytryptamine receptors. These structural similarities allow us to create medicines aimed at treating a range of neurological diseases and disorders including dementia, migraines, and Parkinson’s. The genes encoding ergot alkaloid biosynthesis are found clustered together in the genomes of the different fungi that make them. The fungus Metarhizium brunneum produces lysergic acid α-hydroxyethylamide (LAH), an ecologically and pharmaceutically relevant compound, as its main ergot alkaloid and secretes most of this compound into the surrounding environment. The first objective of this study involved engineering M. brunneum to produce the dihydrogenated versions of its natural ergot alkaloids, that is dihydrolysergic acid (DHLA) and dihydroLAH. The results showed that the fungus can produce both products and was also found to secrete most of both compounds at levels comparable to their unsaturated counterparts. The fungus Aspergillus leporis is a soil saprotroph that has been previously shown to have evolved the capacity for LAH production independently of species in the Clavicipitaceae. Two partial, fragmented gene clusters encoding different clavine-type ergot alkaloid branches were discovered in separate areas of the A. leporis genome and formed the basis for a second study. Chemical analyses indicated that fumigaclavine A production is encoded by one of the fragmented gene clusters. Concentrations of fumigaclavine A peaked around 15 days, following a decrease in LAH levels. The other partial cluster encoded two enzymes necessary to complete production of rugulovasines A and B, but rare production in A. leporis indicated some unknown environmental stimuli required for their production. Expression of these two genes in an appropriate background of M. brunneum allowed for confirmation of their function. The fungus Aspergillus fumigatus, an opportunistic human pathogen, is a known producer of fumigaclavines, another branch of clavine ergot alkaloids, and a distant relative to A. leporis. Due to this relation, A. fumigatus was chosen as platform with which to study the activity and localization of a novel gene from A. leporis, named easT, that encodes a putative major facilitator superfamily transporter. The results indicate that the transporter encoded by easT localizes to discrete regions of fungal hyphae independent of mCherry-tagged peroxisomes and plays a role in transport of ergot alkaloids and/or their precursors. Collectively, the results presented here showcase different ways that ergot alkaloid production can be diversified in both natural and engineered fungal systems.

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