Functional Analysis of an Esterase Gene Involved in Synthesis of Ergot Alkaloids

Kelcie Britton*, Chey Steen*, Jessi Sampson* and Daniel Panaccione, Davis College of Agriculture, Natural Resources and Design, West Virginia University, Morgantown, WV 26506

Field (Broad Category): Biochemistry (Biological & Biochemical Sciences) 

Student’s Major: Immunology and Medical Microbiology 

Best known for their roles in human and animal toxicoses, represented by waves of hysteria and gangrenous appendages, ergot alkaloids represent a broad class of nitrogenous metabolites produced across a range of fungal species. Despite their toxicity, fungal alkaloid derivatives have shown as 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 main lysergic acid amide produced by the fungus Metarhizium brunneum. We hypothesize a gene named easP encodes a carboxylesterase involved in the final step of LAH biosynthesis. To test this hypothesis a CRISPR mutant was engineered. An easP sgRNA/Cas9 construct containing phosphinothricin resistance was introduced into M. brunneum protoplasts. Molecular studies showed that the construct replaced the easP gene, which prompted analysis of alkaloid profiles on high performance liquid chromatography (HPLC). Alkaloid profiles produced on HPLC from the media-grown cultures showed modest accumulation of typical alkaloids, whereas the injection of spores into Galleria mellonella larvae resulted in increased quantities for analysis. When quantified relative to fungal biomass, the concentration of LAH was reduced by ~50% relative to the concentration observed in the wild-type fungus, this indicates significantly reduced capacity for conversion of lysergyl-alanine to LAH. Altogether, these data indicate that EasP plays a significant role in producing LAH. 

Funding: NIH 

Program/mechanism supporting research/creative efforts: WVU 497-level course