Several fungi in two different families––the Clavicipitaceae and the Trichocomaceae––produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.
Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii ؋ Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.
Aspergillus fumigatus is an opportunistic human pathogen that synthesizes a group of mycotoxins via a branch of the ergot alkaloid pathway. This fungus is globally distributed, and genetic data indicate that isolates recombine freely over that range; however, previous work on ergot alkaloids has focused on a limited number of isolates. We hypothesized that A. fumigatus harbors variation in the chemotype of ergot alkaloids and genotype of the ergot alkaloid gene cluster. Analysis of 13 isolates by high performance liquid chromatography revealed four distinct ergot alkaloid profiles or chemotypes. Five isolates completed the A. fumigatus branch of the ergot alkaloid pathway to fumigaclavine C. Six independent isolates accumulated fumigaclavine A, the pathway intermediate immediately before fumigaclavine C. One isolate accumulated only the early pathway intermediates chanoclavine-i and chanocla-vine-i aldehyde, and one isolate lacked ergot alkaloids altogether. A genetic basis for each of the observed chemotypes was obtained either by PCR analysis of the ergot alkaloid gene cluster or through sequencing of easL, the gene encoding the prenyl transferase that reverse prenylates fumigaclavine A to fumigaclavine C. Isolates also exhibited differences in pigmentation and sporulation. The ergot alkaloid chemotypes were widely distributed geographically and among substrate of origin.
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