10.5061/DRYAD.4J0ZPC8CD
Panaccione, Daniel
0000-0002-4159-164X
West Virginia University
Jones, Abigail
West Virginia University
Chemical analyses of three lysergic acid amide-producing Aspergillus
species and sequences for phylogenetic analyses of associated enzymes
Dryad
dataset
2021
FOS: Biological sciences
NIH NIGMS*
2R15-GM114774-2
NIH NIGMS
2R15-GM114774-2
2021-09-27T00:00:00Z
2021-09-27T00:00:00Z
en
114953 bytes
4
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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 Protein M. brunneum ASEF 3297 P. ipomeae IasaF13 C.
paspali RRC-1481 A. leporis CBS 151.66 cluster 1 A. leporis CBS 151.66
cluster 2 A. hancockii FRR 3425 cluster 1 A. hancockii FRR 3425 cluster 2
A. hancockii FRR 3425 cluster 3 A. homo-morphus 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 a 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 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.