Skip to main content
Dryad logo

Chemical analyses and insect interactions of an easO mutant of Metarhizium brunneum

Citation

Panaccione, Daniel; Steen, Chey (2021), Chemical analyses and insect interactions of an easO mutant of Metarhizium brunneum, Dryad, Dataset, https://doi.org/10.5061/dryad.0rxwdbs0t

Abstract

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum, produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)––by far the most abundant lysergic acid amide in M. brunneum––are unknown. Ergot alkaloid synthesis (eas) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes (easO and easP) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella, indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine.

Methods

Data were collected as described in Steen et al. 2021. Applied and Environmental Microbiology 88:e00748-21. 

Usage Notes

Data are contained in an excel file with separate tabs for each of the major data set.  The tab labeled “hplc data” contains data contributing to Table 1 of the Applied and Environmental Microbiology article.  The “deu ala” tab contains data supporting the statistical analyses in Fig. 3.  The tab labeled “insect survival” contains insect survival data used to prepare Figure 4.  The “spores” tab contains data used to prepare Figure 5.  The data in each tab are arrayed to be copied and pasted into a JMP worksheet. 

Funding

National Institutes of Health, Award: 2R15-GM114774-2