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 × 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.

Keywords: P450 monooxygenase; dihydrolysergic acid; ergot alkaloids; lysergic acid.

FIG 1

Pathways to lysergic acid and dihydrolysergic acid and their derivatives. Enzymes responsible for catalysis are indicated between intermediates and products. Multiple arrows indicate the omission of intermediates to simplify the figure. Relevant customary ring and carbon labeling is indicated in the lysergic acid structure. DMAPP, dimethylallylpyrophosphate.

FIG 2

Analysis of ergot alkaloids from transformed strains. HPLC chromatograms of recipient strain (N. fumigata easM knockout), transformed strains containing E. typhina × E. festucae isolate Lp1 cloA or synthetic C. africana cloA, and DHLA standard. Fluorescence was detected at 372 nm after exciting at 272 nm. Mass spectra of fragmented parent ion of m/z 271.2 from DHLA standard and N. fumigata expressing synthetic C. africana cloA are inserted in relevant chromatograms.

FIG 3

Products derived from cDNA or genomic DNA isolated from N. fumigata expressing E. typhina × E. festucae isolate Lp1 or C. africana genomic alleles of cloA. Sizes of relevant fragments from BstEII-digested bacteriophage lambda DNA are indicated to the left. Gel was stained with ethidium bromide.

FIG 4

Graphic representation of C. africana cloA allele to demonstrate issues with intron processing in N. fumigata. Exons in genomic sequence are numbered and colored black. Introns are indicated by thinner lines. In the misspliced transcript, introns 2 and 4 and part of intron 7 are retained in the mRNA as indicated by the thicker, exonic lines. The stop codon resulting from the retention of intron 2 is indicated and exonic sequences downstream of that stop codon are shaded gray.