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.
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Acknowledgments
We gratefully acknowledge the support of Grants R15GM114774 from the National Institutes of Health, National Institute of General Medical Sciences and 2012-67013-19384 from the United States Department of Agriculture, National Institute of Food and Agriculture. This work is published with the approval of the West Virginia Agriculture and Forestry Experiment Station as article number 3269. Technology described in this article is embodied in U.S. Provisional Patent Application No. PCT/US2015/035784. We thank Paige Bragg for technical assistance.
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Communicated by M. Kupiec.
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Bilovol, Y., Panaccione, D.G. Functional analysis of the gene controlling hydroxylation of festuclavine in the ergot alkaloid pathway of Neosartorya fumigata . Curr Genet 62, 853–860 (2016). https://doi.org/10.1007/s00294-016-0591-5
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DOI: https://doi.org/10.1007/s00294-016-0591-5