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

Set1 and Kdm5 are antagonists for H3K4 methylation and regulators of the major conidiation‐specific transcription factor gene ABA1 in Fusarium fujikuroi

“…In the performed microarray, PKS‐NRPS1 , encoding the key enzyme in trichosetin biosynthesis, was upregulated in Δ set1 under nitrogen limitation. The tetramic acid trichosetin is a potent mycotoxin, which was shown to inhibit vegetative growth when overproduced by F. fujikuroi (Janevska et al ., ). Noteworthy, PKS‐NRPS1 expression was similarly induced in F. fujikuroi KMT6 knock‐down mutants, also exhibiting poor vegetative growth (Studt et al ., ).…”

“…japonica cv. Nipponbare (kindly provided by the USDA ARS Dale Bumpers National Rice Research Center, Arkansas, USA) with mycelial plugs of the WT, Δ set1 , Δ kdm5 and OE:: KDM5 mutants was performed for 7 days as described elsewhere (Janevska et al ., ). Addition of 100 ppm GA 3 and H 2 O served as positive and negative control respectively.…”

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

“…The glnA and alcA promoters are both strong promoters. Besides these, a Tet-on system has been developed for F. fujikuroi (Janevska et al, 2017). This Teton system was established based on the adaptation of a Tet-on system of Aspergillus niger (Meyer et al, 2011).…”

“…In F. fujikuroi, the choices of selection markers are very limited. Drug resistance markers, as the representatives the nourseothricin resistance marker nat1 (Teichert et al, 2006;Bomke et al, 2008;Janevska et al, 2017), hygromycin FIGURE 2 | The construct of Tet-on promoter for conditional expression in F. fujikuroi. The promoter region is composed of a tetracycline-dependent transactivator rtTA2 S -M2 (on the left of the construct, encodes rtTA protein) and an rtTA protein driven operator tetO7.…”

An Interpreted Atlas of Biosynthetic Gene Clusters from 1000 Fungal Genomes

“…Prospect, we identified two closely related GCFs (HYBRIDS_11/HYBRIDS_610) containing known BGCs responsible for biosynthesis of equisetin (35), trichosetin (36), and phomasetin (37) as well as BGCs from Alternaria likely responsible for the biosynthesis of altersetin found in multiple Alternaria species (32,38).…”