Generation of a Clean Host for Polyketide Production Using Agricultural Wastes in <i>Ascochyta Rabiei</i>

The plant pathogenic fungus <i>Ascochyta rabiei</i> produces solanapyrones, which are polyketide-derived secondary metabolites, during its saprobic growth on straws. Previously, we utilized <i>A. rabiei</i> as a heterologous host to produce a lichen-derived polyketide with anticancer activities. This study aimed to establish <i>A. rabiei</i> as a sustainable biorefinery for production of biologically active compounds through two objectives: optimizing culture conditions on agricultural waste substrates for polyketide production and generating a clean host using the Cre-loxP system for reusable antibiotic resistance markers. We found that soy hull is the most effective substrate among lignocellulosic materials. Neither light nor the addition of extra divalent cations were required for solanapyrone production in <i>A. rabiei</i>. Production of solanapyrones peaked 18 days after culture on soy hull pellets. To generate a clean host, we deleted the <i>pks1</i> gene, involved in melanin biosynthesis, and the <i>sol1</i> gene, responsible for solanapyrone production. Antibiotic resistance markers used in genetic transformation were recycled by establishing a Cre-loxP system in <i>A. rabiei</i>. In this system, Cre recombinase was expressed under the control of a promoter inducible during sporulation to mitigate the cytotoxicity of Cre. Here, we set the groundwork for developing <i>A. rabiei</i> as an environmentally-friendly biorefinery by generating a clean host with a Cre-loxP marker recycling system and optimizing growth conditions with soy hull pellets.