| Summary: | Ergometrine is widely used for the treatment of excessive postpartum uterine bleeding. <i>Claviceps paspali</i> is a common species for industrial production of ergometrine, which is often accompanied by lysergic acid <i>α</i>-hydroxyethylamide (LAH) and lysergic acid amide (LAA). Currently, direct evidence on the biosynthetic mechanism of LAH and LAA from lysergic acid in <i>C. paspali</i> is absent, except that LAH and LAA share the common precursor with ergometrine and LAA is spontaneously transformed from LAH. A comparison of the gene clusters between <i>C. purpurea</i> and <i>C. paspali</i> showed that the latter harbored the additional <i>easO</i> and <i>easP</i> genes. Thus, the knockout of <i>easO</i> and <i>easP</i> in the species should not only improve the ergometrine production but also elucidate the function. In this study, gene knockout of <i>C. paspali</i> by homologous recombination yielded two mutants ∆<i>easO<sub>hetero</sub></i>-1 and ∆<i>easP<sub>hetero</sub></i>-34 with ergometrine titers of 1559.36 mg∙L<sup>−1</sup> and 837.57 mg∙L<sup>−1</sup>, which were four and two times higher than that of the wild-type control, respectively. While the total titer of LAH and LAA of ∆<i>easO<sub>hetero</sub></i>-1 was lower than that of the wild-type control. The <i>Aspergillus nidulans</i> expression system was adopted to verify the function of <i>easO</i> and <i>easP</i>. Heterologous expression in <i>A. nidulans</i> further demonstrated that <i>easO</i>, but not <i>easP</i>, determines the formation of LAA.
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