Chemical analyses of three lysergic acid amide-producing Aspergillus species and sequences for phylogenetic analyses of associated enzymes

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.

Methods
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)