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

Abstract

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 ϫ 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. africana cloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatographymass 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. fumigata. IMPORTANCE 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.KEYWORDS ergot alkaloids, lysergic acid, dihydrolysergic acid, P450 monooxygenase E rgot alkaloids are specialized fungal metabolites that have impacted humankind historically as toxins and more recently as pharmaceuticals used to treat dementia, migraines, hyperprolactinemia, and Parkinson's disease (1-4). Many natural ergot alka-

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

“…In previous work with the C. africana allele of cloA , no fully processed mRNA was observed when the genomic clone of cloA was expressed in the N. fumigata easM knockout. 21 The most abundant transcript observed in that study contained two retained introns and a third misprocessed intron.…”

“…Peaks with these same retention times were observed when the C. africana allele of cloA was expressed in the N. fumigata easM knockout. 21 Extracts of the recipient strain, N. fumigata easM knockout, contained large quantities of 3 and nothing that eluted near 5 (Figure 2). …”

“…20 Similarly, when a synthetic version of C. africana cloA was expressed in N. fumigata easM knockout, 5 but not 4 was detected. 21 …”

“…21 The ability of CloA from C. gigantea to oxidize 7 was tested by feeding 7 to a C. gigantea cloA -transformed N. fumigata strain. Twelve cultures each of the C. gigantea cloA -transformed strain and N. fumigata easM knockout strain, as a negative control, were grown from 60 000 conidia in 200 µL of malt extract broth.…”

“…21 In contrast, the dihydroergot alkaloid producer C. africana has a version of CloA that accepts 3 as substrate, oxidizing it to dihydrolysergic acid, 5 (DHLA). 21 Most 9 producers incorporate 9 into ergopeptines or lysergic acid amides using a combination of the enzymes Lps1, Lps2, and EasH. In the 9 producer C. purpurea , the relevant downstream enzymes Lps2 and EasH accept dihydrogenated substrates as well as lysergic acid-based substrates.…”