Interaction of a Novel Zn2Cys6 Transcription Factor DcGliZ with Promoters in the Gliotoxin Biosynthetic Gene Cluster of the Deep-Sea-Derived Fungus Dichotomomyces cejpii

Abstract

Gliotoxin is an important epipolythiodioxopiperazine, which was biosynthesized by the gli gene cluster in Aspergillus genus. However, the regulatory mechanism of gliotoxin biosynthesis remains unclear. In this study, a novel Zn2Cys6 transcription factor DcGliZ that is responsible for the regulation of gliotoxin biosynthesis from the deep-sea-derived fungus Dichotomomyces cejpii was identified. DcGliZ was expressed in Escherichia coli and effectively purified from inclusion bodies by refolding. Using electrophoretic mobility shift assay, we demonstrated that purified DcGliZ can bind to gliG, gliM, and gliN promoter regions in the gli cluster. Furthermore, the binding kinetics and affinity of DcGliZ protein with different promoters were measured by surface plasmon resonance assays, and the results demonstrated the significant interaction of DcGliZ with the gliG, gliM, and gliN promoters. These new findings would lay the foundation for the elucidation of future gliotoxin biosynthetic regulation mechanisms in D. cejpii.The putative gene cluster (gli) of gliotoxin is composed of 13 genes that encode crucial enzymes responsible for the biosynthesis of various gliotoxins and their derivatives in A. fumigatus [9]. The non-ribosomal peptide synthetase encoded by gliP gene catalyzes diketopiperazine scaffold formation, the first biosynthetic reaction in a gliotoxin biosynthesis pathway [10]. Subsequently, cytochrome P450 monooxygenase encoded by gliC catalyzes the hydroxylation at the α-carbon of L-Phe [11]. Then, glutathione S-transferase (GST) encoded by the gliG promotes the sulfurization of gliotoxin biosynthetic imtermediates; GST is known for its ability in the catalyzation of carbon-sulfur bond formation, as opposed to detoxification [12,13]. After the process by the enzymes of GliK and GliJ, the γ-glutamyl moieties are removed [14,15]. The carbon-sulfur lyase expressed by the gliI gene then catalyzes the intermediate to generate a notorious epidithiol moiety [16]. After the catalysis of cytochrome P450 monooxygenases (GliF or GliC) and GliH (function remains elusive), the N-methyltransferase encoded by gliN or gliM functions as a freestanding amide to promote amide methylation and confer stability on ETP [17,18]. Finally, the oxidoreductase GliT-mediated disulfide bridge closure might be a prerequisite for the formation of gliotoxins, and the major facilitator superfamily transporter GliA also plays an important role in exporting the toxins to prevent the harmful effect of gliotoxin on hosts [9,19].Many regulators involved in gliotoxin biosynthesis pathway have been identified in A. fumigatus, regardless of whether they deploy a non-gli cluster or a gli cluster. GtmA (or termed TtmA) is encoded outside the gli cluster and functions as a bis-thiomethyl transferase for the conversion of dithiogliotoxin to bisdethiobis (methylthio) gliotoxin (BmGT), which mainly attenuates the formation of disulfide bridge closure [20]. Other non-gli clusters encoding transcription factors, including the global regulator laeA, C 2 H 2...

Fungi.guru: Comparative genomic and transcriptomic resource for the fungi kingdom

“…1 C). 11 out of 13 gliotoxin biosynthesis genes are found in the gliotoxin gli coexpression cluster, with the exception of GliA , responsible for the exportation of extracellular gliotoxin essential for gliotoxin tolerance [47] and GliH whose function is still unknown [48] . Interestingly, the gliotoxin biosynthesis genes found in the coexpression cluster are also part of the genomic gli cluster, where the gli biosynthetic genes are found on the same chromosomal locus [49] .…”

Cmcrf1, a Putative Zn2Cys6 Fungal Transcription Factor, Is Involved in Conidiation, Carotenoid Production, and Fruiting Body Development in Cordyceps militaris

“…The Zn 2 Cys 6 zinc cluster proteins are a family of transcription factors found exclusively in fungi [ 33 ]. They function as transcriptional regulators and are involved in a wide variety of biological processes, including asexual and sexual development, secondary metabolite biosynthesis, carbon and nitrogen metabolism, stress responses, pleiotropic drug resistance, and fungal pathogenesis [ 34 , 35 , 36 , 37 , 38 ]. However, the functions of Zn 2 Cys 6 TFs in different fungi are diverse.…”

Deacetylation by sirtuins is important forAspergillus fumigatuspathogenesis and virulence

“…Several putative cytochrome p450 monooxygenase enzymes (CYP450), which contribute to the production and diversity of various SMs 43 were also upregulated in the ΔAfSirE strain. Among downregulated genes in this strain, six are determinants of virulence, such as sensor histidine kinase/response regulator (Fos-1/TcsA; Afu6g10240), also found downregulated in response to itraconazole 44 , conidial hydrophobin (Afu5g09580), putative transporter (Afu3g12900), and the C6 transcription factor hasA (Afu3g12890) which controls the production of hexadehydroastechrome 45 . On the SIRTKO strain, 1697 DEGs were identified, with 845 up-and 848 downregulated (Figure 8B).…”