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

Slavica Janevska,Birgit Arndt,Hans-Ulrich Humpf,Lisa Apken,Lucas Mauriz Marques,Bettina Tudzynski,Leonie Baumann

doi:10.3390/toxins9040126

Slavica Janevska, Birgit Arndt + Show 5 more

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https://doi.org/10.3390/toxins9040126

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Journal: Toxins	Publication Date: Apr 5, 2017
Citations: 37	License type: CC BY 4.0

Affiliation: University of Münster

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.

Highlights

Fusarium fujikuroi belongs to the Fusarium (Gibberella) fujikuroi species complex (FFC) [1,2], which causes the bakanae (“foolish seedling”) disease of rice plants [3]
F. fujikuroi that is highly homologous to the F. heterosporum equisetin gene cluster [26]
We activated a heretofore silent gene cluster in the rice pathogen F. fujikuroi by two different approaches and found that it is responsible for the synthesis of trichosetin, the penultimate intermediate in the synthesis of equisetin

Summary

Introduction

Fusarium fujikuroi belongs to the Fusarium (Gibberella) fujikuroi species complex (FFC) [1,2], which causes the bakanae (“foolish seedling”) disease of rice plants [3]. The chlorosis and hyper-elongation of plant internodes characteristic of this disease occur due to the production of the phytohormone gibberellic acid by F. fujikuroi [4,5]. The tetramic acid (pyrrolidine-2,4-dione) ring system is a characteristic feature of natural products. Occurring tetramic acid derivatives are of great interest because they show a large spectrum of bioactivities, including antibacterial, antiviral, fungicidal, phytotoxic, and cytotoxic effects [20,21]. The tetramic acid equisetin was initially isolated as an antibiotic from cultures of Fusarium equiseti that effectively inhibited growth of Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus [22]. Equisetin has been reported to be toxic to various mono- and dicotyledonous plants [23] and to inhibit the human immunodeficiency virus type 1 integrase [24,25]. FN080326 [26,27]

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