Two Satellite Gene Clusters Enhance Ergot Alkaloid Biosynthesis Capacity of Aspergillus leporis.

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. However, rugulovasine was only detected in a single sample, 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. IMPORTANCE Ergot alkaloids are chemicals produced by several species of fungi and are notable for their impacts on agriculture and medicine. The ability to make ergot alkaloids is typically encoded by a clustered set of genes that are physically adjacent on a chromosome. Different ergot alkaloid classes are formed via branching of a complex pathway that begins with a core set of the same five genes. Most ergot alkaloid-producing fungi have a single cluster of genes that is complete, or self-sufficient, and produce ergot alkaloids from one or occasionally two branches from that single cluster. Our data show that Aspergillus leporis is exceptional in having the genetic capacity to make products from three pathway branches. Moreover, it uses a satellite cluster approach, in which gene products of partial clusters rely on supplementation with a chemical intermediate produced via another gene cluster, to diversify its biosynthetic potential without duplicating all the steps.

Ergot Alkaloids Contribute to the Pathogenic Potential of the Fungus Aspergillus leporis.

Opportunistically pathogenic fungi have varying potential to cause disease in animals. Factors contributing to their virulence include specialized metabolites, which in some cases evolved in contexts unrelated to pathogenesis. Specialized metabolites that increase fungal virulence in the model insect Galleria mellonella include the ergot alkaloids fumigaclavine C in Aspergillus fumigatus (syn. Neosartorya fumigata) and lysergic acid α-hydroxyethylamide (LAH) in the entomopathogen Metarhizium brunneum. Three species of Aspergillus recently found to accumulate high concentrations of LAH were investigated for their pathogenic potential in G. mellonella. Aspergillus leporis was most virulent, A. hancockii was intermediate, and A. homomorphus had very little pathogenic potential. Aspergillus leporis and A. hancockii emerged from and sporulated on dead insects, thus completing their asexual life cycles. Inoculation by injection resulted in more lethal infections than did topical inoculation, indicating that A. leporis and A. hancockii were preadapted for insect pathogenesis but lacked an effective means to breach the insect's cuticle. All three species accumulated LAH in infected insects, with A. leporis accumulating the most. Concentrations of LAH in A. leporis were similar to those observed in the entomopathogen M. brunneum. LAH was eliminated from A. leporis through a CRISPR/Cas9-based gene knockout, and the resulting strain had reduced virulence to G. mellonella. The data indicate that A. leporis and A. hancockii have considerable pathogenic potential and that LAH increases the virulence of A. leporis. IMPORTANCE Certain environmental fungi infect animals occasionally or conditionally, whereas others do not. Factors that affect the virulence of these opportunistically pathogenic fungi may have originally evolved to fill some other role for the fungus in its primary environmental niche. Among the factors that may improve the virulence of opportunistic fungi are specialized metabolites--chemicals that are not essential for basic life functions but provide producers with an advantage in particular environments or under specific conditions. Ergot alkaloids are a large family of fungal specialized metabolites that contaminate crops in agriculture and serve as the foundations of numerous pharmaceuticals. Our results show that two ergot alkaloid-producing fungi that were not previously known to be opportunistic pathogens can infect a model insect and that, in at least one of the species, an ergot alkaloid increases the virulence of the fungus.

Derivation of the multiply-branched ergot alkaloid pathway of fungi.

Ergot alkaloids are a large family of fungal specialized metabolites that are important as toxins in agriculture and as the foundation of powerful pharmaceuticals. Fungi from several lineages and diverse ecological niches produce ergot alkaloids from at least one of several branches of the ergot alkaloid pathway. The biochemical and genetic bases for the different branches have been established and are summarized briefly herein. Several pathway branches overlap among fungal lineages and ecological niches, indicating activities of ergot alkaloids benefit fungi in different environments and conditions. Understanding the functions of the multiple genes in each branch of the pathway allows researchers to parse the abundant genomic sequence data available in public databases in order to assess the ergot alkaloid biosynthesis capacity of previously unexplored fungi. Moreover, the characterization of the genes involved in the various branches provides opportunities and resources for the biotechnological manipulation of ergot alkaloids for experimentation and pharmaceutical development.

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

OBJECTIVE: The fungus Metarhizium brunneum produces ergot alkaloids of the lysergic acid amide class, most abundantly lysergic acid α-hydroxyethylamide (LAH). Genes for making ergot alkaloids are clustered in the genomes of producers. Gene clusters of LAH-producing fungi contain an α/ß hydrolase fold protein-encoding gene named easP whose presence correlates with LAH production but whose contribution to LAH synthesis in unknown. We tested whether EasP contributes to LAH accumulation through gene knockout studies. RESULTS: We knocked out easP in M. brunneum via a CRISPR/Cas9-based approach, and accumulation of LAH was reduced to less than half the amount observed in the wild type. Because LAH accumulation was reduced and not eliminated, we identified and mutated the only close homolog of easP in the M. brunneum genome, a gene we named estA. An easP/estA double mutant did not differ from the easP mutant in lysergic acid amide accumulation, indicating estA had no role in the pathway. We conclude EasP contributes to LAH accumulation but is not absolutely required. Either a gene encoding redundant function and lacking sequence identity with easP resides outside the ergot alkaloid synthesis gene cluster, or EasP plays an accessory role in the synthesis of LAH.

Diversification of ergot alkaloids and heritable fungal symbionts in morning glories.

Heritable microorganisms play critical roles in life cycles of many macro-organisms but their prevalence and functional roles are unknown for most plants. Bioactive ergot alkaloids produced by heritable Periglandula fungi occur in some morning glories (Convolvulaceae), similar to ergot alkaloids in grasses infected with related fungi. Ergot alkaloids have been of longstanding interest given their toxic effects, psychoactive properties, and medical applications. Here we show that ergot alkaloids are concentrated in four morning glory clades exhibiting differences in alkaloid profiles and are more prevalent in species with larger seeds than those with smaller seeds. Further, we found a phylogenetically-independent, positive correlation between seed mass and alkaloid concentrations in symbiotic species. Our findings suggest that heritable symbiosis has diversified among particular clades by vertical transmission through seeds combined with host speciation, and that ergot alkaloids are particularly beneficial to species with larger seeds. Our results are consistent with the defensive symbiosis hypothesis where bioactive ergot alkaloids from Periglandula symbionts protect seeds and seedlings from natural enemies, and provide a framework for exploring microbial chemistry in other plant-microbe interactions.

Independent Evolution of a Lysergic Acid Amide in Aspergillus Species.

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 the genomes of Aspergillus species for ergot alkaloid synthesis (eas) gene clusters and discovered that 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 that 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 that fungi outside the Clavicipitaceae produce lysergic acid amides and indicate that 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 the 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 selection for this metabolite.

A Baeyer-Villiger Monooxygenase Gene Involved in the Synthesis of Lysergic Acid Amides Affects the Interaction of the Fungus Metarhizium brunneum with Insects.

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum, produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)-by far the most abundant lysergic acid amide in M. brunneum-are unknown. Ergot alkaloid synthesis (eas) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes (easO and easP) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of the alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella, indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. IMPORTANCE Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species, including M. brunneum, were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum.

Ergot alkaloids are important specialized fungal metabolites that are used to make potent pharmaceuticals for neurological diseases and disorders. Lysergic acid (LA) and dihydrolysergic acid (DHLA) are desirable lead compounds for pharmaceutical semisynthesis but are typically transient intermediates in the ergot alkaloid and dihydroergot alkaloid pathways. Previous work with Neosartorya fumigata demonstrated strategies to produce these compounds as pathway end products, but their percent yield (percentage of molecules in product state as opposed to precursor state) was low. Moreover, ergot alkaloids in N. fumigata are typically retained in the fungus as opposed to being secreted. We used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and heterologous expression approaches to engineer these compounds in Metarhizium brunneum, representing an alternate expression host from a different lineage of fungi. The relative percent yields of LA (86.9%) and DHLA (72.8%) were much higher than those calculated here for previously engineered strains of N. fumigata (2.6% and 2.0%, respectively). Secretion of these alkaloids also was measured, with averages of 98.4% of LA and 87.5% of DHLA being secreted into the growth medium; both values were significantly higher than those measured for the N. fumigata derivatives (both of which were less than 5.6% secreted). We used a similar approach to engineer a novel dihydroergot alkaloid in M. brunneum and, through high-performance liquid chromatography-mass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid α-hydroxyethylamide (dihydro-LAH). The engineering of these strains provides a strategy for producing novel and pharmaceutically important chemicals in a fungus more suitable for their production.IMPORTANCE Ergot alkaloids derived from LA or DHLA are the bases for numerous pharmaceuticals with applications in the treatment of dementia, migraines, hyperprolactinemia, and other conditions. However, extraction of ergot alkaloids from natural sources is inefficient, and their chemical synthesis is expensive. The ability to control and redirect ergot alkaloid synthesis in fungi may allow more efficient production of these important chemicals and facilitate research on novel derivatives. Our results show that Metarhizium brunneum can be engineered to efficiently produce and secrete LA and DHLA and, also, to produce a novel derivative of DHLA not previously found in nature. The engineering of dihydroergot alkaloids, including a novel species, is important because very few natural sources of these compounds are known. Our approach establishes a platform with which to use M. brunneum to study the production of other ergot alkaloids, specifically those classified as lysergic acid amides and dihydroergot alkaloids.

Several Metarhizium Species Produce Ergot Alkaloids in a Condition-Specific Manner.

Genomic sequence data indicate that certain fungi in the genus Metarhizium have the capacity to produce lysergic acid-derived ergot alkaloids, but accumulation of ergot alkaloids in these fungi has not been demonstrated previously. We assayed several Metarhizium species grown under different conditions for accumulation of ergot alkaloids. Isolates of M. brunneum and M. anisopliae accumulated the lysergic acid amides lysergic acid α-hydroxyethyl amide, ergine, and ergonovine on sucrose-yeast extract agar but not on two other tested media. Isolates of six other Metarhizium species did not accumulate ergot alkaloids on sucrose-yeast extract agar. Conidia of M. brunneum lacked detectable ergot alkaloids, and mycelia of this fungus secreted over 80% of their ergot alkaloid yield into the culture medium. Isolates of M. brunneum, M. flavoviride, M. robertsii, M. acridum, and M. anisopliae produced high concentrations of ergot alkaloids in infected larvae of the model insect Galleria mellonella, but larvae infected with M. pingshaense, M. album, M. majus, and M. guizhouense lacked detectable ergot alkaloids. Alkaloid concentrations were significantly higher when insects were alive (as opposed to killed by freezing or gas) at the time of inoculation with M. brunneum Roots of corn and beans were inoculated with M. brunneum or M. flavoviride and global metabolomic analyses indicated that the inoculated roots were colonized, though no ergot alkaloids were detected. The data demonstrate that several Metarhizium species produce ergot alkaloids of the lysergic acid amide class and that production of ergot alkaloids is tightly regulated and associated with insect colonization.IMPORTANCE Our discovery of ergot alkaloids in fungi of the genus Metarhizium has agricultural and pharmaceutical implications. Ergot alkaloids produced by other fungi in the family Clavicipitaceae accumulate in forage grasses or grain crops; in this context they are considered toxins, though their presence also may deter or kill insect pests. Our data report ergot alkaloids in Metarhizium species and indicate a close association of ergot alkaloid accumulation with insect colonization. The lack of accumulation of alkaloids in spores of the fungi and in plants colonized by the fungi affirms the safety of using Metarhizium species as biocontrol agents. Ergot alkaloids produced by other fungi have been exploited to produce powerful pharmaceuticals. The class of ergot alkaloids discovered in Metarhizium species (lysergic acid amides) and their secretion into the growth medium make Metarhizium species a potential platform for future studies on ergot alkaloid synthesis and modification.

Diversity and function of fungi associated with the fungivorous millipede, Brachycybe lecontii.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Biodiversity of Convolvulaceous species that contain Ergot Alkaloids, Indole Diterpene Alkaloids, and Swainsonine.

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n=30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.

Ergot Alkaloid Synthesis Capacity of Penicillium camemberti.

Ergot alkaloids are specialized fungal metabolites with potent biological activities. They are encoded by well-characterized gene clusters in the genomes of producing fungi. Penicillium camemberti plays a major role in the ripening of Brie and Camembert cheeses. The P. camemberti genome contains a cluster of five genes shown in other fungi to be required for synthesis of the important ergot alkaloid intermediate chanoclavine-I aldehyde and two additional genes (easH and easQ) that may control modification of chanoclavine-I aldehyde into other ergot alkaloids. We analyzed samples of Brie and Camembert cheeses, as well as cultures of P. camemberti, and did not detect chanoclavine-I aldehyde or its derivatives. To create a functioning facsimile of the P. camembertieas cluster, we expressed P. camemberti easH and easQ in a chanoclavine-I aldehyde-accumulating easA knockout mutant of Neosartorya fumigata The easH-easQ-engineered N. fumigata strain accumulated a pair of compounds of m/z 269.1288 in positive-mode liquid chromatography-mass spectrometry (LC-MS). The analytes fragmented in a manner typical of the stereoisomeric ergot alkaloids rugulovasine A and B, and the related rugulovasine producer Penicillium biforme accumulated the same isomeric pair of analytes. The P. camemberti eas genes were transcribed in culture, but comparison of the P. camemberti eas cluster with the functional cluster from P. biforme indicated 11 polymorphisms. Whereas other P. camembertieas genes functioned when expressed in N. fumigata, P. camembertieasC did not restore ergot alkaloids when expressed in an easC mutant. The data indicate that P. camemberti formerly had the capacity to produce the ergot alkaloids rugulovasine A and B.IMPORTANCE The presence of ergot alkaloid synthesis genes in the genome of Penicillium camemberti is significant, because the fungus is widely consumed in Brie and Camembert cheeses. Our results show that, although the fungus has several functional genes from the ergot alkaloid pathway, it produces only an early pathway intermediate in culture and does not produce ergot alkaloids in cheese. Penicillium biforme, a close relative of P. camemberti, contains a similar but fully functional set of ergot alkaloid synthesis genes and produces ergot alkaloids chanoclavine-I, chanoclavine-I aldehyde, and rugulovasine A and B. Our reconstruction of the P. camemberti pathway in the model fungus Neosartorya fumigata indicated that P. camemberti formerly had the capacity to produce these same ergot alkaloids. Neither P. camemberti nor P. biforme produced ergot alkaloids in cheese, indicating that nutritionally driven gene regulation prevents these fungi from producing ergot alkaloids in a dairy environment.

Molecular identification and characterization of endophytes from uncultivated barley.

Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with Hordeum species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum obtained from the US Department of Agriculture's (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to Epichloë species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable Hordeum species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.

Ergot Alkaloid Biosynthesis in the Maize (Zea mays) Ergot Fungus Claviceps gigantea.

Biosynthesis of the dihydrogenated forms of ergot alkaloids is of interest because many of the ergot alkaloids used as pharmaceuticals may be derived from dihydrolysergic acid (DHLA) or its precursor dihydrolysergol. The maize (Zea mays) ergot pathogen Claviceps gigantea has been reported to produce dihydrolysergol, a hydroxylated derivative of the common ergot alkaloid festuclavine. We hypothesized expression of C. gigantea cloA in a festuclavine-accumulating mutant of the fungus Neosartorya fumigata would yield dihydrolysergol because the P450 monooxygenase CloA from other fungi performs similar oxidation reactions. We engineered such a strain, and high performance liquid chromatography and liquid chromatography-mass spectrometry analyses demonstrated the modified strain produced DHLA, the fully oxidized product of dihydrolysergol. Accumulation of high concentrations of DHLA in field-collected C. gigantea sclerotia and discovery of a mutation in the gene lpsA, downstream from DHLA formation, supported our finding that DHLA rather than dihydrolysergol is the end product of the C. gigantea pathway.

Ergot alkaloids contribute to virulence in an insect model of invasive aspergillosis.

Neosartorya fumigata (Aspergillus fumigatus) is the most common cause of invasive aspergillosis, a frequently fatal lung disease primarily affecting immunocompromised individuals. This opportunistic fungal pathogen produces several classes of specialised metabolites including products of a branch of the ergot alkaloid pathway called fumigaclavines. The biosynthesis of the N. fumigata ergot alkaloids and their relation to those produced by alternate pathway branches in fungi from the plant-inhabiting Clavicipitaceae have been well-characterised, but the potential role of these alkaloids in animal pathogenesis has not been studied extensively. We investigated the contribution of ergot alkaloids to virulence of N. fumigata by measuring mortality in the model insect Galleria mellonella. Larvae were injected with conidia (asexual spores) of two different wild-type strains of N. fumigata and three different ergot alkaloid mutants derived by previous gene knockouts and differing in ergot alkaloid profiles. Elimination of all ergot alkaloids significantly reduced virulence of N. fumigata in G. mellonella (P < 0.0001). Mutants accumulating intermediates but not the pathway end product fumigaclavine C also were less virulent than the wild type (P < 0.0003). The data indicate that ergot alkaloids contribute to virulence of N. fumigata in this insect model and that fumigaclavine C is important for full virulence.

Biosynthesis of the Pharmaceutically Important Fungal Ergot Alkaloid Dihydrolysergic Acid Requires a Specialized Allele of cloA.

Ergot alkaloids are specialized fungal metabolites that are important as the bases of several pharmaceuticals. Many ergot alkaloids are derivatives of lysergic acid (LA) and have vasoconstrictive activity, whereas several dihydrolysergic acid (DHLA) derivatives are vasorelaxant. The pathway to LA is established, with the P450 monooxygenase CloA playing a key role in oxidizing its substrate agroclavine to LA. We analyzed the activities of products of cloA alleles from different fungi relative to DHLA biosynthesis by expressing them in a mutant of the fungus Neosartorya fumigata that accumulates festuclavine, the precursor to DHLA. Transformants expressing CloA from Epichloë typhina × Epichloë festucae, which oxidizes agroclavine to LA, failed to oxidize festuclavine to DHLA. In substrate feeding experiments, these same transformants oxidized exogenously supplied agroclavine to LA, indicating that a functional CloA was produced. A genomic clone of cloA from Claviceps africana, a sorghum ergot fungus that produces a DHLA derivative, was cloned and expressed in the festuclavine-accumulating mutant of N. fumigata, but several introns in this genomic clone were not processed properly. Expression of a synthetic intron-free version of C. africanacloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In substrate feeding experiments, the C. africana CloA also accepted agroclavine as the substrate, oxidizing it to LA. The data indicate that a specialized allele of cloA is required for DHLA biosynthesis and that the pharmaceutically important compound DHLA can be produced in engineered N. fumigataIMPORTANCE Ergot alkaloids are fungal metabolites that have impacted humankind historically as poisons and more recently as pharmaceuticals used to treat dementia, migraines, and other disorders. Much is known about the biosynthesis of ergot alkaloids that are derived from lysergic acid (LA), but important questions remain about a parallel pathway to ergot alkaloids derived from dihydrolysergic acid (DHLA). DHLA-derived alkaloids have minor structural differences compared to LA-derived alkaloids but can have very different activities. To understand how DHLA is made, we analyzed activities of a key enzyme in the DHLA pathway and found that it differed from its counterpart in the LA pathway. Our data indicate a critical difference between the two pathways and provide a strategy for producing DHLA by modifying a model fungus. The ability to produce DHLA in a model fungus may facilitate synthesis of DHLA-derived pharmaceuticals.

Ergot Alkaloids of the Family Clavicipitaceae.

Ergot alkaloids are highly diverse in structure, exhibit diverse effects on animals, and are produced by diverse fungi in the phylum Ascomycota, including pathogens and mutualistic symbionts of plants. These mycotoxins are best known from the fungal family Clavicipitaceae and are named for the ergot fungi that, through millennia, have contaminated grains and caused mass poisonings, with effects ranging from dry gangrene to convulsions and death. However, they are also useful sources of pharmaceuticals for a variety of medical purposes. More than a half-century of research has brought us extensive knowledge of ergot-alkaloid biosynthetic pathways from common early steps to several taxon-specific branches. Furthermore, a recent flurry of genome sequencing has revealed the genomic processes underlying ergot-alkaloid diversification. In this review, we discuss the evolution of ergot-alkaloid biosynthesis genes and gene clusters, including roles of gene recruitment, duplication and neofunctionalization, as well as gene loss, in diversifying structures of clavines, lysergic acid amides, and complex ergopeptines. Also reviewed are prospects for manipulating ergot-alkaloid profiles to enhance suitability of endophytes for forage grasses.

Toxin-producing Epichloë bromicola strains symbiotic with the forage grass Elymus dahuricus in China.

Cool-season grasses (Poaceae subfamily Poöideae) are an important forage component for livestock in western China, and many have seed-transmitted symbionts of the genus Epichloë, fungal endophytes that are broadly distributed geographically and in many tribes of the Poöideae. Epichloë strains can produce any of several classes of alkaloids, of which ergot alkaloids and indole-diterpenes can be toxic to mammalian and invertebrate herbivores, whereas lolines and peramine are more selective against invertebrates. The authors characterized genotypes and alkaloid profiles of Epichloë bromicola isolates symbiotic with Elymus dahuricus, an important forage grass in rangelands of China. The endophyte was seed-transmitted and occasionally produced fruiting bodies (stromata), but its sexual state was not observed on this host. The genome sequence of E. bromicola isolate E7626 from El. dahuricus in Xinjiang Province revealed gene sets for peramine, ergot alkaloids, and indole-diterpenes. In multiplex polymerase chain reaction (PCR) screens of El. dahuricus-endophyte isolates from Beijing and two locations in Shanxi Province, most were also positive for these genes. Ergovaline and other ergot alkaloids, terpendoles and other indole-diterpenes, and peramine were confirmed in El. dahuricus plants with E. bromicola. The presence of ergot alkaloids and indole-diterpenes in this grass is a potential concern for managers of grazing livestock.

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte.

Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected "knockoff" endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production.