DOI: 10.5061/dryad.7pvmcvdzt
title: Analyses of ergot alkaloid production in Aspergillus leporis and an easD knockout and sequences for phylogenetic analyses of rugulovasine-associated genes

description: 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.
description: 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
 C<sub>18</sub> 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
 <em>A. leporis</em> NRRL 3216 and <em>easD</em>
 knockout <em>A. leporis</em> 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 <em>A. leporis, P. biforme,
 </em>and<em> P. camemberti eas </em>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 <em>M. brunneum </em>and<em> A.
 fumigatus</em> lack an <em>eas</em> cluster-associated
 version of EasQ, the EasQ sequences from <em>A. leporis
 </em>and<em> P. camemberti</em> were both used to search
 <em>M. brunneum </em>and<em> A. fumigatus</em>
 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
 (<em>A. japonicus </em>and<em> Byssochlamys
 spectabilis</em>) or ergopeptines (<em>C. purpurea
 </em>and<em> Periglandula ipomoeae</em>). Accession
 numbers for homologs not already identified as a functional EasH and
 associated with <em>eas</em> clusters can be found in Figure
 7.
description: 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.