| Summary: | Ergot alkaloids are fungal natural products with important roles in agriculture and medicine. We used heterologous expression and gene knockout approaches to investigate potential roles for the product of a major facilitator superfamily transporter gene (<i>easT</i>) recently found in an ergot alkaloid biosynthetic gene cluster in <i>Aspergillus leporis</i>. A strain of <i>Aspergillus fumigatus</i> previously engineered to accumulate lysergic acid, but which did not convert the precursor agroclavine to lysergic acid efficiently or secrete lysergic acid well, was chosen as an expression host for <i>easT</i>. Expression of <i>easT</i> in this strain resulted in accumulation of significantly more pathway intermediates but no detectable lysergic acid. Secretion of ergot alkaloids was reduced in the <i>easT</i>-expressing strain. EasT localized to discrete vesicle-like structures in the cytosol of <i>A. fumigatus</i>, with no localization detected in the plasma membrane. When <i>easT</i> was knocked out in <i>A. leporis</i>, accumulation of lysergic acid amides was reduced relative to the wild type. There was no negative effect on secretion of ergot alkaloids in the knockout mutant. The data indicate that <i>easT</i> encodes a product that contributes to accumulation of ergot alkaloids, perhaps by transporting intermediates between cellular compartments, but does not have a significant role in secreting ergot alkaloids.
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