Analyses of ergot alkaloid production in Aspergillus leporis and an easD knockout and sequences for phylogenetic analyses of rugulovasine-associated genes

Ergot alkaloids are fungal specialized metabolites that are important in agriculture and serve as sources of several pharmaceuticals. Aspergillus leporis is a soil saprotroph that possesses two ergot alkaloid biosynthetic gene clusters encoding lysergic acid amide production. We identified two additional, partial biosynthetic gene clusters within the A. leporis genome containing some of the ergot alkaloid synthesis (eas) genes required to make two groups of clavine ergot alkaloids, fumigaclavines and rugulovasines. Clavines possess unique biological properties compared to lysergic acid derivatives. Bioinformatic analyses indicated the fumigaclavine cluster contained functional copies of easA, easG, easD, easM, and easN. Genes resembling easQ and easH, which are required for rugulovasine production, were identified in a separate gene cluster. The pathways encoded by these partial, or satellite, clusters would require intermediates from the previously described lysergic acid amide pathway..., Ergot alkaloid data were collected by high-performance liquid chromatography with fluorescence detection. The column was a 150- by 4.6-mm inner-diameter, 5-µm particle size Prodigy C18 column (Phenomenex, Torrance, CA), and the mobile phase was a 55-min, binary, multilinear gradient of 5% acetonitrile to 75% acetonitrile in 50 mM aqueous ammonium acetate. Chanoclavine and fumigaclavine A were detected using excitation and emission wavelengths of 272 nm and 372 nm, respectively, and LAH was detected by excitation at 310 nm and recording emission at 410 nm. Chanoclavine-I and fumigaclavine A were quantified by comparing peak areas to an external standard curve prepared from fumigaclavine A (Alexis Biochemicals, San Diego, CA); thus, the values for chanoclavine-I must be considered as approximations relative to fumigaclavine A. LAH was quantified by comparing peak areas to an external standard curve prepared from ergonovine (Sigma, St. Louis, MO), which contains the identical fluorophore; ..., Alkaloid data are contained in a Microsoft Excel file. Amino acid sequence data are contained in a Microsoft Word file. Data are arrayed to be copied and pasted into applications for statistical analysis or phylogenetic analysis.Â