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 to synthesize a product. Chemical analyses of A. leporis cultures revealed the presence of fumigaclavine A. Rugulovasine was only detected in a single sample, however, prompting a heterologous expression approach to confirm functionality of easQ and easH. An easA knockout strain of Metarhizium brunneum, which accumulates the rugulovasine precursor chanoclavine-I aldehyde, was chosen as expression host. Strains of M. brunneum expressing easQ and easH from A. leporis accumulated rugulovasine as demonstrated through mass spectrometry analysis. These data indicate that A. leporis is exceptional among fungi in having the capacity to synthesize products from three branches of the ergot alkaloid pathway and for utilizing an unusual satellite cluster approach to achieve that outcome.

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; thus, LAH values must be considered as relative to ergonovine as opposed to absolute. Cultures of A. leporis NRRL 3216 and easD knockout A. leporis were grown in 500 µL of sucrose yeast-extract medium (lacking agar) in 2-mL screw cap microcentrifuge tubes. Cultures were inoculated with 150,000 conidia, and triplicate cultures were harvested and assayed at three-day intervals for the time course. Culture filtrate was removed and measured by pipet while the solid fungal mass was harvested and dried by vacuum centrifugation till no change in mass could be detected. Culture fluids were diluted 1:1 with methanol and clarified by centrifugation prior to HPLC analysis. Solid portions were weighed and extracted by bead beating with five 3-mm diameter glass beads in 0.5 mL of methanol at 6 m/s for 30 s. The resulting extracts were rotated end-over-end for 30 min and clarified by centrifugation prior to HPLC analysis. Twenty µL of liquid or solid phase was analyzed by HPLC as described above. 

 Sets of sequences for phylogenetic analysis of Aspergillus leporis EasQ and EasH were assembled as follows. The respective protein sequences translated for EasQ from eas clusters of A. leporis, P. biforme, and P. camemberti eas gene cluster were used as the query in a BLASTp search for homologous proteins in the NCBI database for each organism. A cutoff of at least 30% identity over 70% query coverage was used to select matches from the returned list to be included in the data set for phylogenetic analysis. If more than two proteins from a given species met those criteria, only the top two matches were used. Since M. brunneum and A. fumigatus lack an eas cluster-associated version of EasQ, the EasQ sequences from A. leporis and P. camemberti were both used to search M. brunneum and A. fumigatus databases and the top three matches from both species were chosen (provided they met the criteria delineated above). When the BLASTp search resulted in fewer matches than outlined above, a tBLASTn search was performed against the whole-genome shotgun database for that organism. If hypothetical proteins queried in this manner met the cutoff criteria described above, the protein sequences were deduced by BLASTx comparison of the appropriate regions of the identified contigs. Accession numbers for homologs not already identified as a functional eas cluster-associated EasQ can be found in Figure 6. This process was repeated for EasH alleles with the only change being the inclusion of organisms having versions of EasH shown to catalyze key steps in the synthesis of cycloclavine (A. japonicus and Byssochlamys spectabilis) or ergopeptines (C. purpurea and Periglandula ipomoeae). Accession numbers for homologs not already identified as a functional EasH and associated with eas clusters can be found in Figure 7.

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.