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Evidence for Bayer-Villiger monooxygenase activity in LAH biosynthesis. (A) Ratios of m/z intensities of ions obtained from extracts of M. brunneum ARSEF 9354 grown in unsupplemented G. mellonella larvae or larvae supplemented with l-alanine-2,3,3,3-D4 (D4-ala). Error bars show the standard error of the mean; n = 7 larvae per treatment. M, molecular ion, which for LAH is m/z 312. (B) l-alanine-2,3,3,3-D4-derived portion of Lps3-bound lysergyl-alanine. (C and D) Hypothetical oxygenated products of carbon 2 of the alanyl residue. (E) The side chain attached to the lysergyl group in LAH. Hydrolysis or reduction of the ester bond in C would yield LAH (E) while retaining all four deuteriums. Conversion of intermediate D to LAH requires a more complicated mechanism and would result in loss of one deuterium. Abbreviations: D, deuterium; R1, attachment to Lps3 via pantetheine; R2, attachment to carbonyl group of lysergic acid. The asterisk in panel C marks the carbon reduced by the reductase domain of Lps3.
Source publication
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. Man...
Citations
... The isomerase and reductase products of easA catalyze closure of the ergoline D ring before reduction via the product of easG occurs, generating either festuclavine or agroclavine, respectively (30,31). Production of lysergic acid amides, such as lysergic acid a-hydroxyethylamide (LAH), is then dependent upon the presence of other tailoring enzymes in an organism's eas cluster (32)(33)(34)(35)(36)(37)(38)(39). Ergopeptines consist of a tripeptide chain linked to d-lysergic acid and are synthesized by the combination of two nonribosomal peptide synthetases (LPS1 and LPS2) together with the Fe 21 /2-ketoglutarate-dependent dioxygenase encoded by easH found in Claviceps purpurea (40). ...
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.
... In contrast to A. fumigatus, many species of the genus Metarhizium are well-adapted insect pathogens that enter insects via appressoria and sporulate profusely on dead insects (23)(24)(25). Metarhizium species are aided in pathogenesis by several specialized metabolites (26,27), including the ergot alkaloid lysergic acid a-hydroxyethylamide (LAH) (28). LAH is the product of a different branch of the ergot alkaloid pathway relative to the branch producing fumigaclavine C in A. fumigatus ( Fig. 1) (29). ...
... To test the effects of lysergic acid amides on virulence of A. leporis, a CRISPR-Cas9-derived knockout was made in the easD gene of A. leporis. This fungus and gene were selected for mutation for several reasons: (i) we had developed a genetic transformation system for A. leporis but not for the other Aspergillus species; (ii) A. leporis was more virulent and produced significantly higher concentrations of ergot alkaloids in insects compared to the other Aspergillus species tested ( Fig. 2 and Table 1); (iii) easD is the only single-copy ergot alkaloid synthesis gene in the two LAH-associated gene clusters of A. leporis (31); and (iv) knockout of easD should block synthesis of LAH, which had been observed to contribute to virulence of the entomopathogen M. brunneum (28). ...
... Transformants in which easD was knocked out were found by a PCR screen spanning the target site for the mutagenic sgRNA. Two colonies contained target site amplicons of greater length than that obtained from wild-type A. leporis (see Fig. S3), indicating insertion of a DNA fragment into the target site during repair of the Cas9-cut locus, a phenomenon we had observed routinely when applying a similar mutagenesis strategy in M. brunneum (28,36,37). One particular easD mutant chosen for further study (easD ko 1) had a 2.1-kb increase in the length of the target site amplicon. ...
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.
... In addition to ergonovine and LAH, free lysergyl-alanine (liberated from the peptide synthetase without modification) and ergine (the simple amide of lysergic acid) accumulate in some fungi. Critical enzymes in the pathway from lysergic acid to lysergic acid amides include a combination of Lps2 and Lps3 for the synthesis of ergonovine from lysergic acid and alanine (Ortel & Keller, 2009) along with enzymes EasO and EasP to convert the lysergyl-alanine intermediate into LAH (Britton et al., 2022;Steen et al., 2021). All fungi that produce LAH have the capacity to produce ergonovine from the same lysergyl-alanine precursor; however, in all cases, the relative abundance of LAH compared to ergonovine is heavily shifted in favour of LAH (Table 2). ...
... In the scenario illustrated in Figure 6, the reductase domain of Lps3 and the BVMO EasO are competing for the same Lps3-bound lysergyl-alanine substrate, and the great excess of LAH relative to ergonovine (Table 2) suggests that EasO outcompetes the reductase domain for the substrate. The Lps3bound carboxyl ester/thioester product of EasO could then be acted on by the reductase domain of Lps3 (via reduction of the same carbon as in ergonovine biosynthesis) or the α/β hydrolase fold protein EasP, a putative carboxyl esterase, to yield LAH (Britton et al., 2022;Steen et al., 2021). ...
... Knockout of easP in M. brunneum reduced LAH accumulation by >50% and the only homologue of easP in the M. brunneum genome did not contribute to LAH biosynthesis. Considering the activity of the reductase domain of Lps3 and a contributing (but not essential) role for EasP, three alternate and non-exclusive mechanisms have been proposed for the final steps in LAH biosynthesis: (1) The Lps3 reductase domain could reduce the carbonyl carbon of the carboxyl ester/thioester with a hydride ion (as it does the same carbon of lysergyl-alanine during ergonovine biosynthesis; Ortel & Keller, 2009) resulting in bond breakage on the carboxyl ester side to yield LAH directly (route 1 in Figure 6) ; (2) That same reduction (described in point a immediately above) could result in bond breakage on the thioester side, liberating a formate ester of LAH that might be acted on by the α/β hydrolase fold protein EasP to produce LAH (route 2 in Figure 6) (Britton et al., 2022;Steen et al., 2021); or, (3) EasP could act on the carboxyl ester (prior to reduction catalysed by Lps3 reductase domain) to produce LAH (route 3 in Figure 6) (Britton et al., 2022;Steen et al., 2021). This model provides an explanation for how knockout of easP reduces the concentration of LAH by more than 50%, but EasP is not essential (Britton et al., 2022). ...
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.
... Naturally occurring lysergic acid amides include ergonovine (called ergometrine in Europe) and lysergic acid α-hydroxyethylamide (LAH). Ergonovine is the primary lysergic acid amide in the rye ergot fungus Claviceps purpurea, which also produces abundant ergopeptines [2,4]; whereas, the plant root symbiont and insect pathogen Metarhizium brunneum produces ergonovine and LAH, with the concentration of LAH dwarfing that of ergonovine by approximately 200 to 1 [5,6]. In studies to date, all genes required for ergot alkaloid synthesis are clustered in the genomes of the producing fungi in ergot alkaloid synthesis (eas) clusters [7][8][9][10][11][12]. ...
... Both ergonovine and LAH are derived from the intermediate lysergyl-alanine [4,6] which is synthesized via a complex of two monomodular peptide synthetases, lysergyl peptide synthetase (Lps) 2 and Lps3. Lps2 recognizes lysergic acid, activates it by adenylation, binds it as a thioester, and condenses it with alanine that has been similarly recognized, activated, and thioesterified by Lps3. ...
... The reductase domain uses hydride ions obtained from its cofactor NADPH to reduce the carbonyl carbon of the alanyl moiety of lysergyl-alanine to an aldehyde and then a primary alcohol, liberating ergonovine from enzyme bound lysergyl-alanine [4] (Fig. 1). 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]. ...
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/β 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.
... 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). ...
... 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). ...
... A comparison of the EAS gene clusters of C. purpurea and C. paspali shows that both C. purpurea and C. paspali harbor The additional easO and easP are likely involved in the biosynthesis of LAH and LAA, in which LAH can spontaneously convert to LAA by a non-enzymatic process [10,24]. Recently, easO has been shown to control the biosynthesis of LAH in M. brunneum [25], suggesting the role of the enzyme in the biosynthesis of LAH in C. paspali. ...
Ergometrine is widely used for the treatment of excessive postpartum uterine bleeding. Claviceps paspali is a common species for industrial production of ergometrine, which is often accompanied by lysergic acid α-hydroxyethylamide (LAH) and lysergic acid amide (LAA). Currently, direct evidence on the biosynthetic mechanism of LAH and LAA from lysergic acid in C. paspali is absent, except that LAH and LAA share the common precursor with ergometrine and LAA is spontaneously transformed from LAH. A comparison of the gene clusters between C. purpurea and C. paspali showed that the latter harbored the additional easO and easP genes. Thus, the knockout of easO and easP in the species should not only improve the ergometrine production but also elucidate the function. In this study, gene knockout of C. paspali by homologous recombination yielded two mutants ∆easOhetero-1 and ∆easPhetero-34 with ergometrine titers of 1559.36 mg∙L−1 and 837.57 mg∙L−1, which were four and two times higher than that of the wild-type control, respectively. While the total titer of LAH and LAA of ∆easOhetero-1 was lower than that of the wild-type control. The Aspergillus nidulans expression system was adopted to verify the function of easO and easP. Heterologous expression in A. nidulans further demonstrated that easO, but not easP, determines the formation of LAA.
... The Lps complex then forms thioesterified lysergyl-alanine as a precursor to the other lysergic acid amides (Fig. 1). Ergonovine may be released by the reduction of the carbonyl carbon of lysergyl-alanine to an alcohol by the reductase domain of Lps3 (20), or LAH may be released via a series of reactions initiated by the Baeyer-Villiger monooxygenase encoded by easO (21). Ergopeptines (ergotamine and ergocristine, etc.) result from the combination of lysergic acid-activating Lps2 with the trimodular NRPS Lps1 (encoded by lpsA), which incorporates lysergic acid and 3 amino acids into a lactam intermediate (18,19), which is subsequently cyclized by the dioxygenase EasH to an ergopeptine (22). ...
... The product of easO, which acts downstream from the Lps complex in the pathway to LAH (21), also varied in the Aspergillus species compared to the clavicipitaceous LAH producers. Whereas EasO in all of the LAH producers is a representative of the Baeyer-Villiger monooxygenase family, EasO of the Aspergillus species was derived from a different ancestral member of that family than was the version of EasO in the clavicipitaceous fungi FIG 4 Accumulation of lysergic acid (LA) and the lysergic acid amides lysergyl-alanine (LylAla), lysergic acid a-hydroxyethylamide (LAH), ergonovine, and ergine over time in 0.5-ml cultures of the indicated Aspergillus species. ...
... Since the volumes of the cultures were 0.5 ml, doubling the values for individual bars in the culture fluid samples yields values as nanomoles per milliliter or micromolar. (21) with each of the two A. leporis copies ( Fig. 6; Fig. S4). Strains of M. brunneum transformed with either version of easO from A. leporis accumulated LAH as opposed to ergonovine, the major product of the easO knockout of M. brunneum (Fig. 6) (21). ...
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.
Covering: 2000 to 2023The kingdom Fungi has become a remarkably valuable source of structurally complex natural products (NPs) with diverse bioactivities. Since the revolutionary discovery and application of the antibiotic penicillin from Penicillium, a number of fungi-derived NPs have been developed and approved into pharmaceuticals and pesticide agents using traditional "activity-guided" approaches. Although emerging genome mining algorithms and surrogate expression hosts have brought revolutionary approaches to NP discovery, the time and costs involved in developing these into new drugs can still be prohibitively high. Therefore, it is essential to maximize the utility of existing drugs by rational design and systematic production of new chemical structures based on these drugs by synthetic biology. To this purpose, there have been great advances in characterizing the diversified biosynthetic gene clusters associated with the well-known drugs and in understanding the biosynthesis logic mechanisms and enzymatic transformation processes involved in their production. We describe advances made in the heterogeneous reconstruction of complex NP scaffolds using fungal polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), PKS/NRPS hybrids, terpenoids, and indole alkaloids and also discuss mechanistic insights into metabolic engineering, pathway reprogramming, and cell factory development. Moreover, we suggest pathways for expanding access to the fungal chemical repertoire by biosynthesis of representative family members via common platform intermediates and through the rational manipulation of natural biosynthetic machineries for drug discovery.
