Year: 2023
Type: dissertation
Abstract: Specialized metabolites produced by fungi impact human health. A large portion of the pharmaceuticals currently on the market are derived from metabolites biosynthesized by microbes. Ergot alkaloids are a class of fungal metabolites that are important in the interactions of environmental fungi with insects and mammals and also are used in the production of pharmaceuticals. In animals, ergot alkaloids can act as partial agonists or antagonists at receptors for 5-hydroxytryptamine (serotonin), dopamine, and noradrenaline as ergot alkaloids have chemical structures similar to those neurotransmitters. Therefore, they affect insects and mammals that consume them and can be used to produce drugs that can aid in treatment of migraines, dementia, Parkinson’s and gynecological issues. The conserved ergot alkaloid synthesis pathway is responsible for the biosynthesis of these tryptophan-derived toxins in ergot alkaloid-producing fungi. Clusters of ergot alkaloid synthesis (eas) genes present in genomes of ergot alkaloid-producing fungi encode enzymes that catalyze synthesis of these chemicals. The fungus Metarhizium brunneum produces lysergic acid α-hydroxyethylamide (LAH), an ecologically and pharmaceutically important compound. The final steps of LAH synthesis involve two monomodular nonribosomal peptide synthetases: lysergyl peptide synthetase 2 (Lps2); encoded by lpsB and Lps3,encoded by lpsC. A novel two-module Lps gene, lpsD, was discovered via genome mining in three species of lysergic acid amide-producing Aspergillus. Heterologous expression of lpsD from Aspergillus leporis in a lysergic acid-accumulating strain of Aspergillus fumigatus yielded ergonovine and lysergyl-alanine, end products typically derived from Lps2 and Lps3 in Clavicipitaceous fungi. Addition of the A. leporis easO gene to this strain resulted in accumulation of LAH, the pathway end product observed in both A. leporis and M. brunneum. This study established that the product of the lpsD gene is the functional equivalent of the two monomodular peptide synthetases of the Clavicipitaceae. While most biosynthetic steps in the ergot alkaloid pathway are well characterized, the regulation of the genes is unknown. Understanding the regulatory mechanisms may aid in insect biocontrol strategies and may be of value for the pharmaceutical industry, as upregulation of the pathway could result in increased biosynthetic yields. In this study, the role of two novel genes with regulatory potential, easR and easS were studied to determine their role in ergot alkaloid biosynthesis. CRISPR-mediated knockout of easR in M. brunneum resulted in a strain with no detectable ergot alkaloids. Infection of Galleria mellonella larvae with the easR knockout of M. brunneum compared to a wild-type control showed a
DOI: 10.33915/etd.11837
Author: Samantha Joy Fabian
Institution: West Virginia University
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4376647976