Characterization of novel gliotoxin biosynthesis-related genes from deep-sea-derived fungus Geosmithia pallida FS140
Graphical abstract
Introduction
Gliotoxins belong to diketopiperazines and are characterized by a disulfide bridge across a piperazine ring, which were mainly produced by Aspergillus sp. Gliotoxin [[1], [2], [3]], and gliotoxin was reported to inhibit the proliferation of cancer cells and the replication of virus RNAs, thus inducing the apoptosis of hepatic stellate cells [[1], [2], [3]], and suppressing the expression of transcription factor NF-κB to reduce inflammation [[4], [5], [6]]. Gliotoxins also have a bright future in the field of biomedicine and agriculture. A synthetic analog of the natural gliotoxin-like compound derivative produced by marine-derived A. ustus CNC-139, named as plinabulin, has entered phase III clinical study to treat non-small cell lung cancer [7]. Therefore, gliotoxins show the great potential to be developed as leading compounds for anticancer drugs. Gliotoxin from Trichoderma viride also exhibited antifungal acitivty and has the latent capacity to be developed as fungicide [8]. It also reported that the presence of gliotoxin can promote the survival rate of A. fumigatus being exposed to high levels of H2O2 [9], suggesting the possibility to be developed as antioxidant.
The mechanism of gliotoxin biosynthesis in A. fumigatus has been elucidated, the gli cluster related to the gliotoxin biosynthesis in A. fumigatus was identified [[10], [11], [12]], which consists of 13 genes. It has been demonstrated that gliG encodes a glutathione S-transferase (GST), which conjugates two glutathione (GSH) molecules to form a bis-glutathionylated biosynthetic intermediate, which is responsible for the sulfurization of gliotoxin, demonstrating that GSTs play an important biosynthetic role in the fungus [10].
gliT is an essential functional gene for the process of gliotoxin biosynthesis, which encodes GliT protein, a thioredoxin reductase. GliT protein is reported to be responsible for the formation of the disulfide bond in gliotoxins, which is vital for the bioactivity of gliotoxins from A. fumigatus [13]. Thioredoxin reductase (TrxR) is a NADPH-dependent dimeric protein that surrounds the FAD domain, which is the only selenozyme that catalyzes the formation of thioredoxin. Thioredoxin reductase is vital for the antioxidant response and cell growth regulation [13,14]. Studies on thioredoxin reductase in mammals revealed that these enzymes are important pathogenic factors for tumorigenesis and cardiovascular disease [15,16]. The thioredoxin reductases reported in Arabidopsis thaliana [17] and rice [18] play an important role in resisting abiotic stress such as low temperature and drought, and may also be involved in the synthesis of plant starch and shikimic acid. More importantly, as an electron donor, it is of great significance for redox signal transmission [19]. Moreover, the practice of clinical treatment of human diseases demonstrated that some microbial thioredoxin reductases showed great application foreground. For example, thioredoxin reductase in Vibrio harveyi can also be used as a potent subunit vaccine for the immunological prevention of vibriosis [20]; thioredoxin reductase in A. fumigatus displayed a strong immune response with the serum of patients towards invasive aspergillosis, which lays a foundation for the serological diagnosis of the disease [21].
gliM, encoding GliM, a kind of O-methyltransferase, is also a key gene for the biosynthesis of gliotoxins and their derivatives. Methyltransferase catalyzing the methylation reaction utilizes S-adenosine-methionine (SAM) as the methyl donor. Methylation is distributed in most organisms, from eukaryote to prokaryote. methyltransferase is necessary for the biosynthesis of child protection hormone. At the same time, methyltransferase is also associated with the biosynthesis of many secondary metabolites such as lignin and parenting phenol [22]. And it is assumed that GliM protein is responsible for the methylation of hydroxyl group at the side chain of gliotoxins to produce diverse gliotoxin derivatives.
gliK encoding GliK protein, was annotated as a kind of gamma-glutamyl cyclotransferase. gliK belongs to gene cluster involved in the biosynthesis of gliotoxins. GliK was served as a glutamyl cyclotransferase to remove both gamma-glutamyl moieties to yield an intermediate for gliotoxins in A. fumigatus [10]. However, the function of GliK for the biosynthesis of novel gliotoxin-like compounds in D. cejpii from deep sea remains obscure. As acetyl-gliotoxin was discovered in Dichotomomyces cejpii and G. pallida, it was proposed that GliK also exert the function of acetylation.
Marine-derived fungi have been proved as a promising source to exploit bioactive metabolites, and a growing number of marine fungi have been reported to produce bioactive secondary metabolites [23,24]. In recent years, gliotoxin and its derivatives were isolated from marine fungi such as D. cejpii (Aspergillus cejpii), and genes related to gliotoxins biosynthesis were identified [25]. Different kinds of gliotoxins and their derivatives including rare gliotoxin dimers were isolated from D. cejpii [21]. Known gliotoxins with cytotoxicity and rare gliotoxins like compounds with a bioactivity of inhibiting angiotensin-converting enzyme (ACE) were isolated from G. pallida [26]. Novel gliotoxin like compounds including gliotoxin dimers were excavated from G. pallida, which is different from the gliotoxins from other species including A. fumigatus and D. cejpii, and the genes involved in the biosynthesis of GT showed differences with that in other species, suggesting the specialty of gliotoxin biosynthetic gene clusters in G. pallida. Therefore, it is necessary to characterize the genes associated with the biosynthesis of gliotoxins in G. pallida, thus providing the molecular foundation for the mechanism elucidation of gliotoxin biosynthesis in G. pallida, as well as paving a way for the exploiting of novel gliotoxin like compounds via biosynthetic approaches and in vitro biochemical reaction. In this study, the functions of gliotoxin biosynthesis-related genes including gliT, gliM and gliK from the deep-sea-derived fungus G. pallida were firstly characterized in E. coli. The three genes were obtained by PCR using cDNA of G. pallida, cloned and expressed in E. coli BL21 (DE3), then purified by Ni affinity chromatography, which was demonstrated by SDS-PAGE, Western blot and MALDI-TOF analysis. The enzymatic properties of the recombinant enzymes including GliT, GliM, and GliK from deep-sea fungi were also characterized. Besides the glutamyl cyclotransferase activity, the acetyl-transferase activity of GliK from G. pallida was firstly demonstrated.
Section snippets
Materials and reagents
The strain G. pallida FS140 (Accession No. MK047400) was isolated from a deep-sea sedimental sample in the South China Sea (19°28.5810′ N, 115°27.2510’ E, depth 2403 m) [29]. The strain FS140 was inoculated on a PDA (Potato Dextrose Agar) medium and incubated at 28 °Cfor 7 days. The total RNAs of the strain were extracted using a RNA extracting kit (Magen, Guangzhou, China). Then the cDNA was synthesized through the reverse transcription kit (Abm, Vancouver, BC, Canada). The genes gliT and gliM
The clone of gliT, gliM, and gliK
The total RNA of the deep-sea-derived fungus G. pallida was extracted to obtained cDNA, the corresponding genes of gliT, gliM and gliK were amplified by gliT-F/gliT-R, gliM-F/gliM-R, and gliK-F/gliK-R primers. Agarose gel electrophores analysis showed that the 624 bp, 264 bp and 825 bp bands were corresponding to gliT, gliM and gliK, respectively (Fig. S1). The phylogenetic trees of gliT, gliM, and gliK based on their nucleotide sequences and similar genes were constructed using MEGA 7.0 (Fig. 1
Discussion
The function of GliT, GliM and GliK proteins related to the biosynthesis of gliotoxins in deep sea fungus G. pallida were investigated in this study. It has been demonstrated that thioredoxin reductase encoded by GliT is of great importance to maintain the environment of intracellular oxidation, and the activity of thioredoxin reductase is often closely related to the oxidative stress level [27]. For instance, the apoptosis of cerebellar granular neurons is significantly affected by the
Conclusion
In this study, gliT, gliM and gliK genes related to the biosynthesis of gliotxins in the deep-sea fungus G. pallida were cloned, expressed and characterized for the first time. The target genes including gliT, giM and gliK were cloned and then expressed in E. coli BL21 (DE3). GliT, GliM and GliK proteins were purified by Ni affinity chromatography, which was confirmed by SDS-PAGE, Western-blot analyses and sequencing. And the functions of GliT and GliM as thioredoxin reductase and
Author contributions
Conception of the work, W.Y and W.-M.Z.; collection of data, S.L., W.Y., S.-N.L. Y.-L.K., L.-T.L. and S.L.; analysis of data, W.Y., S.L., S.-N.L. and W.-M.Z.; writing of manuscript, W.Y. and S.L; any other relevant contribution, W.Y., W.-M.Z., W.-Y.Z.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
Financial support for this research was provided by the Guangdong Provincial Special Fund for Marine Economic Development Project (Yue Natural Resources Contract No. [2020]042), the National Natural Science Foundation of Guangdong province (2019A1515011829, 2019A1515011702), the Natural Science Foundation of Guangdong Province (2019A1515011829), the Team Project of Natural Science Foundation of Guangdong Province (2016A030312014).
References (31)
Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: exacerbation by caspase and serine protease inhibition
J. Hepatol.
(2007)Gliotoxin-mediated apoptosis of activated human hepatic stellate cells
J. Hepatol.
(2003)Discovery of gliotoxin as a new small molecule targeting thioredoxin redox system
Biochem. Biophys. Res. Commun.
(2007)The effect of gliotoxin upon macrophage function
Int. J. Immunopharm.
(1986)Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties
J. Biol. Chem.
(1988)Gliotoxin effects on fungal growth: mechanisms and exploitation
Fungal Genet. Biol.
(2012)Resistance is not futile: gliotoxin biosynthesis
functionality and utility
(2015)Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes
Free Radic. Biol. Med.
(2011)Involvement of thioredoxin reductase 1 in the regulation of redox balance and viability of rheumatoid synovial cells
Biochem. Biophys. Res. Commun.
(2008)A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana
J. Biol. Chem.
(2004)
Aspergilones A and B, two benzylazaphilones with an unprecedented carbon skeleton from the gorgonian-derived fungus Aspergillus sp
Bioorg. Med. Chem. Lett
Geospallins A-C: new thiodiketopiperazines with inhibitory activity against angiotensin-converting enzyme from a deep-sea-derived fungus Geosmithia pallida FS140
Mar. Drugs
Purification, cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis Protein Expr
Purif
Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells
Toxicol. Sci.
Biological activity and interaction mechanism of the diketopiperazine derivatives as tubulin polymerization inhibitors
RSC Adv.
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indicate contributed equally to this article.