RESUMO
The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with inâ vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500â mg L(-1) , thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.
Assuntos
Enzimas/metabolismo , Alcaloides de Claviceps/biossíntese , Proteínas Fúngicas/metabolismo , Alcaloides Indólicos/metabolismo , Aspergillus fumigatus/genética , Ciclopropanos/química , Enzimas/genética , Alcaloides de Claviceps/química , Proteínas Fúngicas/genética , Alcaloides Indólicos/química , Família Multigênica , Saccharomyces cerevisiae/metabolismoRESUMO
The ergots are a structurally diverse group of alkaloids derived from tryptophan and dimethylallyl pyrophosphate (DMAPP) . The potent bioactivity of ergot alkaloids have resulted in their use in many applications throughout human history. In this highlight, we recap some of the history of the ergot alkaloids, along with a brief description of the classifications of the different ergot structures and producing organisms. Finally we describe what the advancements that have been made in understanding the biosynthetic pathways, both at the genomic and the biochemical levels. We note that several excellent review on the ergot alkaloids, including one by Wallwey and Li in Nat. Prod. Rep., have been published recently. We provide a brief overview of the ergot alkaloids, and highlight the advances in biosynthetic pathway elucidation that have been made since 2011 in Section 4.
Assuntos
Alcaloides de Claviceps , Fungos , Vias Biossintéticas , Alcaloides de Claviceps/biossíntese , Alcaloides de Claviceps/química , Alcaloides de Claviceps/classificação , Fungos/química , Fungos/genética , Fungos/metabolismo , Hemiterpenos/metabolismo , Humanos , Estrutura Molecular , Compostos Organofosforados/metabolismo , Triptofano/metabolismoRESUMO
Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I.
Assuntos
Aspergillus fumigatus/metabolismo , Catalase , Ergolinas/metabolismo , Alcaloides de Claviceps/biossíntese , Proteínas Fúngicas/metabolismo , Oxirredutases/metabolismo , Proteínas Recombinantes/metabolismo , Compostos Alílicos/metabolismo , Aspergillus fumigatus/genética , Catalase/genética , Catalase/metabolismo , Claviceps/genética , Claviceps/metabolismo , Clonagem Molecular , Ergonovina/metabolismo , Alcaloides de Claviceps/metabolismo , Escherichia coli , Proteínas Fúngicas/genética , Peróxido de Hidrogênio/metabolismo , Alcaloides Indólicos/metabolismo , Família Multigênica , Oxirredutases/genética , Proteínas Recombinantes/genética , Recombinação Genética , Deleção de Sequência , Triptofano/análogos & derivados , Triptofano/metabolismoRESUMO
Ergot alkaloids, secondary metabolites produced by filamentous fungi, elicit a diverse array of pharmacological effects. The biosynthesis of this class of natural products has not been fully elucidated. Here we demonstrate that a homologue of Old Yellow Enzyme encoded in the Aspergillus fumigatus ergot gene cluster catalyzes reduction of the alpha,beta unsaturated alkene of chanoclavine-I aldehyde 3. This reduction, which yields dihydrochanoclavine aldehyde, facilitates an intramolecular reaction between a secondary amine and aldehyde to form the D ring of the ergot alkaloid structural framework.
Assuntos
Alcaloides de Claviceps/biossíntese , NADPH Desidrogenase/metabolismo , Aldeídos/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Biocatálise , Produtos Biológicos/biossíntese , Ergolinas/metabolismo , Alcaloides de Claviceps/metabolismo , Família Multigênica , NADPH Desidrogenase/química , Oxirredução , Homologia de Sequência de AminoácidosRESUMO
The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The biosynthetic intermediate chanoclavine-I aldehyde 1 represents a branch point in ergot biosynthesis. Ergot alkaloids festuclavine 2 and agroclavine 3 derive from alternate enzymatic pathways originating from the common biosynthetic precursor chanoclavine-I aldehyde 1. Here we show that while the Old Yellow Enzyme homologue EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii, in contrast, produces agroclavine 3. Mutational analysis suggests a mechanistic rationale for the switch in activity that controls this critical branch point of ergot alkaloid biosynthesis.
Assuntos
Alcaloides de Claviceps/biossíntese , Alcaloides de Claviceps/química , Aspergillus fumigatus/enzimologia , Família Multigênica , NADPH Desidrogenase/química , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Neotyphodium/genética , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus. However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA, a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA-encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.
Assuntos
Aspergillus fumigatus/metabolismo , Vias Biossintéticas , Claviceps/metabolismo , Alcaloides de Claviceps/biossíntese , Proteínas Fúngicas/metabolismo , NADPH Desidrogenase/metabolismo , Aspergillus fumigatus/genética , Claviceps/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Modelos Biológicos , Estrutura Molecular , NADPH Desidrogenase/genéticaRESUMO
The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with inâ vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500â mg L-1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.