Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi

Abstract

The PKS-NRPS-derived tetramic acid equisetin and its <i>N</i>-desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., <i>Staphylococcus aureus</i>. The equisetin biosynthetic gene cluster was first described in <i>Fusarium heterosporum</i>, a species distantly related to the notorious rice pathogen <i>Fusarium fujikuroi</i>. Here we present the activation and characterization of a homologous, but silent, gene cluster in <i>F. fujikuroi</i>. Bioinformatic analysis revealed that this cluster does not contain the equisetin <i>N</i>-methyltransferase gene <i>eqxD</i> and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from <i>Aspergillus niger</i> achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in <i>F. fujikuroi</i>. Overexpression of one of the two cluster-specific transcription factor (TF) genes, <i>TF22</i>, led to an activation of the three biosynthetic cluster genes, including the <i>PKS-NRPS</i> key gene. In contrast, overexpression of <i>TF23</i>, encoding a second Zn(II)₂Cys₆ TF, did not activate adjacent cluster genes. Instead, <i>TF23</i> was induced by the final product trichosetin and was required for expression of the transporter-encoding gene <i>MFS-T</i>. TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus.