Functional and Genetic Analysis of Esterase Genes Involved in Synthesis of Ergot Alkaloids

Kelcie N. Britton*, Chey R. Steen, Jessi K. Sampson, Daniel G. Panaccione, Ph.D.
Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26505

Presentation Category: Science & Techonology (Oral Presentation)

Student’s Major: Immunology and Medical Microbiology

Ergot alkaloids are unique nitrogenous metabolites produced by several species of fungi that have been associated with many adverse historical events, including the Salem Witch Trials. Despite their toxicity, modified and appropriately dosed ergot alkaloid derivatives are effective pharmaceutical treatments for dementia, migraines, and hyperprolactinemia. Pathways to some ergot alkaloids have been studied both biochemically and genetically, but critical steps in the synthesis of lysergic acid amides remain elusive. These gaps are significant because many of the pharmaceutically relevant ergot alkaloids are semi-synthetically derived from lysergic acid amides. Lysergic acid ⍺-hydroxyethylamide (LAH) is the ergot alkaloid produced by the fungus Metarhizium brunneum. We hypothesize two genes, named easP and estA, encode esterases involved in the final step of LAH biosynthesis. To test this hypothesis, CRISPR mutants were engineered with easP alone mutated and with both estA and easP mutated. The product of easP has a significant effect on the production of LAH; the easP mutant only accumulated half of the LAH measured in wild type. The quantity of LAH in strains was normalized relative to fungal biomass as estimated by measuring the fungal metabolite ergosterol by liquid chromatography-mass spectrometry. The double mutant has been prepared, and the effects on ergot alkaloids will be measured in ongoing experiments. The hypothesized activity of EasP as an esterase is being tested by heterologously expressing the protein in E. coli. The phenotype of our CRISPR mutant demonstrates that easP is an integral part of the pathway to LAH.

Funding: The Arnold and Mabel Beckman Foundation, NIH

Program/mechanism supporting research/creative efforts: Arnold and Mabel Beckman Foundation