DOI: 10.5061/dryad.4j0zpc8cd
title: Chemical analyses of three lysergic acid amide-producing Aspergillus species and sequences for phylogenetic analyses of associated enzymes

description: 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.
description: 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,
 <i>Aspergillus leporis</i>,
 <i>Aspergillus homomorphus</i>, and
 <i>Aspergillus hancockii</i> 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 <i>eas</i> pathway of all
 available LAH producers were assembled as follows. The protein encoded by
 each gene in an organism’s <i>eas</i> 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
 <i>eas</i>-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 “<i>eas</i>
 cluster” are as follows: <i>A. homomorphus</i> CBS 101889,
 PSTJ01000028; <i>A. leporis</i> NRRL 3216
 <i>eas</i> cluster 1, SWBU01000165; <i>A. leporis
 </i>NRRL 3216 <i>eas</i> cluster 2, SWBU01000104;
 <i>A. hancockii</i> CBS 142004 <i>eas</i> cluster
 1, MBFL02000298; <i>A. hancockii</i> CBS 142004
 <i>eas</i> cluster 2, MBFL02000239; <i>A.
 hancockii</i> CBS 142004 <i>eas</i> cluster 3,
 MBFL02000250; <i>M. brunneum</i> ARSEF 3297, AZNG01000019;
 <i>C. paspali </i>RRC 1481, AFRC01000012; and, <i>P.
 ipomoeae</i> IasaF13, AFRD01000277 and a table of the corresponding
 accession numbers for individual proteins is provided here:
 <b>Accession numbers for
 <i>eas</i> cluster genes of LAH-producing fungi included in
 the present study</b> Protein <i>M. brunneum</i><br> ASEF 3297 <i>P. ipomeae</i><br> IasaF13 <i>C. paspali</i><br> RRC-1481 <i>A. leporis</i><br> CBS 151.66 cluster 1 <i>A. leporis </i><br> CBS 151.66 cluster 2 <i>A. hancockii</i> FRR 3425 cluster 1 <i>A. hancockii</i> FRR 3425 cluster 2 <i>A. hancockii</i> FRR 3425 cluster 3 <i>A. homo-morphus</i><br> CBS 101889 DmaW XP_014540959 AEV21221 AET79202 KAB8071281 KAB8073422 pseudogene KAF7589021 KAF7588835 XP_025554348 EasF XP_014540957 AEV21223 AET79195 KAB8071283 KAB8073420 not present KAF7589019 pseudogene XP_025554350 EasE XP_014540956 AEV21224 deduced <sup>a</sup> KAB8071282 KAB8073421 pseudogene KAF7589020 pseudogene XP_025554349 EasC XP_014540954 AEV21226 AET79197 KAB8071280 KAB8073423 not present KAF7589022 not present XP_025554347 EasD XP_014540955 AEV21225 AET79196 KAB8071286 pseudogene not present KAF7589017 pseudogene XP_025554353 EasA XP_014540951 AEV21229 AET79198 KAB8071285 KAB8073418 KAF7588053 pseudogene not present XP_025554352 EasG XP_014540958 AEV21222 AET79194 KAB8071284 KAB8073419 KAF7588052 pseudogene not present XP_025554351 CloA XP_014540953 AEV21227 AET79203 KAB8071277 KAB8073414 KAF7588056 not present not present XP_025554344 LpsB XP_014540952 AEV21228 AET79204 not present not present not present not present not present not present LpsC XP_014540950 AEV21230 AET79199 not present not present not present not present not present not present LpsD not present not present not present KAB8071279 KAB8073424 KAF7588050 not present not present XP_025554346 EasO XP_014540960 AEV21220.2 AET79193 KAB8071278 KAB8073416 KAF7588054 not present not present XP_025554345 EasP XP_014540949 AEV21231.2 AET79200 KAB8071276 KAB8073415 KAF7588055 not present not present XP_025554343 EasT not present not present not present not present KAB8073417 not present pseudogene not present not present <sup>a</sup> deduced by translating coordinates 2214-2409, 2532-3050, and 3168-4130 in GenBank accession JABAJK010000166 representing <i>C. paspali</i> isolate ILB432, since <i>C. paspali </i>RRC-1481 is reported as having a non-functional copy of <i>easE</i> (Schardl et al. 2013 PLoS Pathogens 9:e1003323) 
description: Chemical data are contained in an excel file with separate tabs
 for each of the three <i>Aspergillus</i> 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.