Ergot alkaloids derived from lysergic acid have impacted humanity as contaminants of crops and as the bases of pharmaceuticals prescribed to treat dementia, migraines, and other disorders. Several plant-associated fungi in the Clavicipitaceae produce lysergic acid derivatives, but many of these fungi are difficult to culture and manipulate. Some Aspergillus species, which may be more ideal experimental and industrial organisms, contain an alternate branch of the ergot alkaloid pathway but none were known to produce lysergic acid derivatives. We mined genomes of Aspergillus species for ergot alkaloid synthesis (eas) gene clusters and discovered three species––A. leporis, A. homomorphus, and A. hancockii––had eas clusters indicative of the capacity to produce a lysergic acid amide. In culture, A. leporis, A. homomorphus, and A. hancockii produced lysergic acid amides, predominantly lysergic acid α-hydroxyethylamide (LAH). Aspergillus leporis and A. homomorphus produced high concentrations of LAH and secreted most of their ergot alkaloid yield into the culture medium. Phylogenetic analyses indicated genes encoding enzymes leading to the synthesis of lysergic acid were orthologous to those of the lysergic acid amide-producing Clavicipitaceae; however, genes to incorporate lysergic acid into an amide derivative evolved from different ancestral genes in the Aspergillus species. Our data demonstrate fungi outside the Clavicipitaceae produce lysergic acid amides and indicate the capacity to produce lysergic acid evolved once, but the ability to insert it into LAH evolved independently in Aspergillus species and the Clavicipitaceae. The LAH-producing Aspergillus species may be useful for study and production of these pharmaceutically important compounds.

Ergot alkaloid data were collected by high performance liquid chroatorgraphy with fluorescence detection. The stationary phase was a C18 column (Prodigy ODS3, 150 mm length x 4.6 mm i.d., 5 µM particle size; Phenomenex, Torrance, CA), and the mobile phase was a multilinear gradient from 5% acetonitrile in 50 mM ammonium acetate to 75% acetonitrile in 50 mM ammonium acetate over 55 min. Fluorescence was detected by exciting at 310 nm and measuring emission at 410 nm. To measure ergot alkaloid accumulation over time and to quantify moles secreted into the medium as compared to moles retained in the hyphae, Aspergillus leporis, Aspergillus homomorphus, and Aspergillus hancockii were grown in 500 µL of SYE (lacking agar) in 2-mL screw cap microcentrifuge tubes at room temperature. Cultures were inoculated with 150,000 conidia, and triplicate cultures were harvested and assayed at three-day intervals. Culture filtrate was removed and measured by pipetting, diluted with an equal volume of methanol, and then clarified by centrifugation before HPLC analysis as described above. After careful removal of all liquid, the solid phase of the culture was dried by vacuum centrifugation till no change in mass could be detected. The mass of the solid phase was measured, and alkaloids were extracted by bead beating with five 3-mm diameter glass beads in 1 mL of methanol at 6 m/s for 30 s. The resulting extract was rotated end-over-end for 30 min and clarified by centrifugation. Twenty µL of liquid or solid phase was analyzed by HPLC as described above. Quantitative data are based on peak areas compared to an external standard curve of ergonovine, which contains the identical fluorophore found in all lysergic acid derivatives; therefore, concentrations should be considered as relative to ergonovine as opposed to absolute.

Sets of sequences for phylogenetic analysis of each of the genes in the eas pathway of all available LAH producers were assembled as follows. The protein encoded by each gene in an organism’s eas cluster was used as query in a blastp search of the proteins in the NCBI database for that same organism. The top two matches that met the criteria of at least 30% identity over 70% query coverage were included in the data set for phylogenetic analysis. If an organism’s database contained fewer than two matches that met the 30% identity/70% coverage criteria, then the eas-related protein from that organism was used as query in a tblastn search of the same organism’s whole genome shotgun database. If hypothetical proteins queried in this manner met the criteria described above, then proteins corresponding to up to two top matches were deduced by blastx comparison of the appropriate region of the identified contig and included in the set of proteins for phylogenetic analysis. Homologs meeting the criteria of 30% identity over 70% query coverage are labeled by NCBI accession number in Fig. 5 and Fig. S3. Accession numbers for contigs containing sequences listed simply as “eas cluster” are as follows: A. homomorphus CBS 101889, PSTJ01000028; A. leporis NRRL 3216 eas cluster 1, SWBU01000165; A. leporis NRRL 3216 eas cluster 2, SWBU01000104; A. hancockii CBS 142004 eas cluster 1, MBFL02000298; A. hancockii CBS 142004 eas cluster 2, MBFL02000239; A. hancockii CBS 142004 eas cluster 3, MBFL02000250; M. brunneum ARSEF 3297, AZNG01000019; C. paspali RRC 1481, AFRC01000012; and, P. ipomoeae IasaF13, AFRD01000277 and a table of the corresponding accession numbers for individual proteins is provided here:

Accession numbers for eas cluster genes of LAH-producing fungi included in the present study

^a deduced by translating coordinates 2214-2409, 2532-3050, and 3168-4130 in GenBank accession JABAJK010000166 representing C. paspali isolate ILB432, since C. paspali RRC-1481 is reported as having a non-functional copy of easE (Schardl et al. 2013 PLoS Pathogens 9:e1003323) 

Chemical data are contained in an excel file with separate tabs for each of the three Aspergillus species.  The “hplc data” tabs correspond to the summary data shown in Figure 4 of the Applied and Environmental Microbiology article.  Variables are ergot alkaloids listed in column headers, where the abbreviation LAH means lysergic acid alpha-hydroxyethylamide.  The data are arrayed as µg of specified alkaloid/0.5 mL culture and then again as nmol of specified alkaloid/0.5 mL culture for each of the five listed ergot alkaloids.  Rows provide data for hyphae (solid phase) versus culture fluid (liquid phase) for each alkaloid by sampling day post inoculation (recorded in the sample-day column).  The data in each tab are arrayed in a format ready to be copied and pasted into a JMP worksheet. 

Amino acid sequence data used to create the phylogenetic trees shown in Figure 5 and Figure S3 of the Applied and Environmental Microbiology article and its supplement are provide in a Word file.  Data for individual trees are separated by page breaks and labeled by enzyme. Data are ready to be copied and pasted for alignment and phylogenetic analyses.