Independent Evolution of a Lysergic Acid Amide in Aspergillus Species

Abstract

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. IMPORTANCE Lysergic acid derivatives are specialized metabolites with historical, agricultural, and medical significance and were known heretofore only from fungi in one family, the Clavicipitaceae. Our data show that several Aspergillus species, representing a different family of fungi, also produce lysergic acid derivatives and that the ability to put lysergic acid into its amide forms evolved independently in the two lineages of fungi. From microbiological and pharmaceutical perspectives, the Aspergillus species may represent better experimental and industrial organisms than the currently employed, lysergic acid producers of the plant-associated Clavicipitaceae. The observation that both lineages independently evolved the derivative lysergic acid α-hydroxyethylamide (LAH), among many possible lysergic acid amides, suggests a selection for this metabolite.

Contribution of a novel gene to lysergic acid amide synthesis in Metarhizium brunneum

“…To date all genes required for ergot alkaloid synthesis have been encoded in the eas cluster [ 2 , 3 , 7 , 8 ]. In a recent study relevant to this point, Jones et al [ 10 ] reported that three species of Aspergillus synthesize LAH and evolved the steps for incorporation of lysergic acid into LAH independently of M. brunneum (and other fungi in the Clavicipitaceae) yet had similarly constituted eas clusters with no additional genes present that might be redundant with easP . The possibility that two lineages evolved similar pathways for incorporating lysergic acid into LAH yet kept one unrelated and redundant gene outside the eas cluster appears unlikely.…”

“…In fungi that produce LAH as well as ergonovine (e.g., M. brunneum , the paspalum ergot fungus Claviceps paspali , and the morning glory symbiont Periglandula ipomoeae ) there are two additional genes in the eas cluster: easO , encoding a Baeyer–Villiger monooxygenase (BVMO) required for synthesis of LAH [ 6 ], and easP , encoding an α/β hydrolase fold protein, a role for which in ergot alkaloid synthesis has not yet been demonstrated but whose presence correlates perfectly with the ability to produce LAH [ 7 , 8 , 10 ]. As a BVMO, EasO is hypothesized to insert an oxygen between the alpha carbon and carbonyl carbon of the alanyl portion of lysergyl-alanine [ 6 ].…”

Derivation of the multiply‐branched ergot alkaloid pathway of fungi

“…One aspect of the genes involved in ergot alkaloid biosynthesis that greatly facilitates research is that the genes are clustered in the genomes of producing fungi (Figure 2 ). These gene clusters (referred to as eas clusters, for ergot alkaloid synthesis; Schardl et al, 2006 ) contain core genes that are common to multiple ergot alkaloid producers as well as branch‐specific genes required for producing the unique alkaloids found in different fungi (Fabian et al, 2018 ; Haarmann et al, 2005 ; Jones et al, 2021 ; Martín et al, 2017 ; Schardl, Young, Hesse, et al, 2013 ; Schardl, Young, Pan, et al, 2013 ; Tudzynski et al, 1999 ). Thus, as the functions of genes in various pathway branches are determined, the values of gene clusters increase as predictors of ergot alkaloid biosynthesis capacity and as sources of genes controlling certain biochemical reactions.…”

ERGOT Claviceps purpurea (Fries) Tulasne ALKALOID DIVERSITY AND VIRULENCE: EVOLUTION, GENETIC DIVERSIFICATION, AND METABOLIC ENGINEERING (review)

“…К л а с т е р г е н о в б и о п р о д у к ц и и а л к а л о и д о в. О кластерной организации генов биосинтеза алкалоидов у спорыньи впервые сообщили в 1999 году, и в частности было показано значение гена dmaW для биосинтеза (8,102). Кластеры генов биосинтеза эргоалкалоидов обнаружены у различных грибов (30), например у Clavicipitaceae (30,103,104), в частности у Claviceps (30,102,103), Epichloe (20,30), Periglandula (3), Metarhizium brunneum (86,105), Neotyphodium lolli (106), Balansia cyperi, Balansia obtecta (30); у Aspergillus (107,108), в частности у Aspergillus fumigatus (107,110), A. leporis, A. homomorphus, A. hancockii (111) и A. japonicus (112,113); у Clavulinopsis fusiformis (106); у Arthroderma benhamiae (114,115); у Penicillium camemberti и Penicillium biforme (116).…”