Contribution of a novel gene to lysergic acid amide synthesis in Metarhizium brunneum

Objective: The fungus Metarhizium brunneum produces ergot alkaloids of the lysergic acid amide class, most abundantly lysergic acid alpha-hydroxyethylamide (LAH). Genes for making ergot alkaloids are clustered in the genomes of producers. Gene clusters of LAH-producing fungi contain an alpha/beta hydrolase fold protein-encoding gene named easP whose presence correlates with LAH production but whose contribution to LAH synthesis in unknown. We tested whether EasP contributes to LAH accumulation through gene knockout studies. Results: We knocked out easP in M. brunneum via a CRISPR/Cas9-based approach, and accumulation of LAH was reduced to less than half the amount observed in the wild type. Because LAH accumulation was reduced and not eliminated, we identified and mutated the only close homolog of easP in the M. brunneum genome, a gene we named estA. An easP/estA double mutant did not differ from the easP mutant in lysergic acid amide accumulation, indicating estA had no role in the pathway. We conclude EasP contributes to LAH accumulation but is not absolutely required. Either a gene encoding redundant function and lacking sequence identity with easP resides outside the ergot alkaloid synthesis gene cluster, or EasP plays an accessory role in the synthesis of LAH.

Automated summary

In this study, a gene named easP in the fungus Metarhizium brunneum was found to contribute to the accumulation of lysergic acid alpha-hydroxyethylamide (LAH) but is not essential. Another gene estA in the same pathway was found to have no role in LAH synthesis.