title: Functional analysis of the gene controlling hydroxylation of festuclavine in the ergot alkaloid pathway of Neosartorya fumigata.
abstract: Bioactive ergot alkaloids produced by several species of fungi are important molecules in agriculture and medicine. Much of the ergot alkaloid pathway has been elucidated, but a few steps, including the gene controlling hydroxylation of festuclavine to fumigaclavine B, remain unsolved. Festuclavine is a key intermediate in the fumigaclavine branch of the ergot alkaloid pathway of the opportunistic pathogen Neosartorya fumigata and also in the dihydrolysergic acid-based ergot alkaloid pathway of certain Claviceps species. Based on several lines of evidence, the N. fumigata gene easM is a logical candidate to encode the festuclavine-hydroxylating enzyme. To test this hypothesis we disrupted easM function by replacing part of its coding sequences with a hygromycin resistance gene and transforming N. fumigata with this construct. High-pressure liquid chromatography analysis demonstrated that easM deletion mutants were blocked in the ergot alkaloid pathway at festuclavine, and downstream products were eliminated. An additional alkaloid, proposed to be a prenylated form of festuclavine on the basis of mass spectral data, also accumulated to higher concentrations in the easM knockout. Complementation with the wild-type allele of easM gene restored the ability of the fungus to produce downstream compounds. These results indicate that easM encodes an enzyme required for fumigaclavine B synthesis likely by hydroxylating festuclavine. The festuclavine-accumulating strain of N. fumigata may facilitate future investigations of the biosynthesis of dihydrolysergic acid derivatives, which are derived from festuclavine and are the basis for several important drugs.
authors.affiliation: Genetics and Developmental Biology Program, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, 26506, USA.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Bilovol
authors.affiliation: Genetics and Developmental Biology Program, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, 26506, USA. danpan@wvu.edu.
authors.forename: Daniel G
authors.identifier: http://orcid.org/0000-0002-4159-164X
authors.initials: DG
authors.lastname: Panaccione
mesh_terms: D004873:Ergolines / Q000378:metabolism*; D004876:Ergot Alkaloids / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006900:Hydroxylation; D053858:Metabolic Networks and Pathways; D005810:Multigene Family; D055326:Neosartorya / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004873:Ergolines; D004876:Ergot Alkaloids; C033546:costaclavin
keywords: Aspergillus fumigatus; Clavines; Gene cluster; Mycotoxin; P450 monooxygenase