Identification of a critical gene in the dihydroergot alkaloid pathway.

摘要

Ergot alkaloids, bioactive compounds produced by some species of fungi, have had significant impacts on agriculture and medicine. Claviceps purpurea and Epichloe spp. are ergot alkaloid producers associated with agricultural commodities and negatively affect humans and grazing animals, respectively. Another ergot alkaloid-producing fungus called Aspergillus fumigatus is ubiquitous in the environment; infections arising after inhaling the spores may kill immunocompromised persons. Ergot alkaloids and their derivatives are utilized by the pharmaceutical industry for the development of drugs. Some of these drugs are derived from lysergic acid, whereas others are derived from dihydrolysergic acid. Lysergic acid derivatives have vasoconstrictive properties and are used as treatment of acute headaches and in obstetrics. Dihydrolysergic acid derivatives are vasorelaxant and are the basis of drugs that help treat dementia, Alzheimer's, and Cushing's diseases. The ergot alkaloid pathway has largely been elucidated, but unknown steps remain, particularly in the dihydrolysergic acid branch. The gene encoding the enzyme that oxidizes festuclavine, a key intermediate in the dihydrolysergic acid branch, to fumigaclavine B is unknown. Based on DNA sequence analysis, we hypothesized that the A. fumigatus gene easM encodes this enzyme. To test this hypothesis we knocked out easM by inserting a hygromycin resistance gene in the middle of its coding sequence and transforming A. fumigatus with this construct. HPLC analysis showed that knock out of easM blocked the ergot pathway at festuclavine, and three downstream products (fumigaclavines B, A, and C) were eliminated. PCR results showed the hygromycin resistance fragment was present in the middle of easM -coding sequences in these transformants. Complementation, or reintroduction of the native non-disrupted easM gene restored the ability of the fungus to produce all downstream compounds. These results indicate that easM encodes the enzyme that oxidizes festuclavine to fumigaclavine B. The festuclavine-accumulating strain of A. fumigatus will be instrumental in future studies investigating the biosynthesis of dihydrolysergic acid derivatives, which are derived from festuclavine and are the basis for important vasorelaxant drugs.