RESUMO
The deep-sea fungus Phomopsis lithocarpus FS508 produces tenellone-macrolide conjugated hetero-dimer lithocarpins A-G with anti-tumor activities. The deficiency of new intermolecular Diels-Alder (DA) enzymes hindered the development of new bioactive hetero-dimers. A novel single-function intermolecular DA enzyme, g7882, was initially discovered in this study. The deletion of g7882 led to the disappearance of lithocarpin A and an increase in precursor level . the overexpression of g7882 significantly improved lithocarpin A yield. The in vitro function of g7882DA was also confirmed by biochemical reaction using tenellone B as a substrate. Additionally, the knockout of KS modules of PKS in cluster 41 and cluster 81 (lit cluster) eliminated the production of lithocarpins, which firstly explains the biosynthetic process of hetero-dimer lithocarpins mediated by DA enzyme in FS508. Furthermore, the removal of a novel acetyltransferase GPAT in cluster 41 and the oxidoreductase, prenyltransferase in cluster81 resulted in the reduction of lithocarpin A in P. lithocarpus. The overexpression of gpat in P. lithocarpus FS508 improved the yield of lithocarpin A significantly and produced a new tenellone derivative lithocarol G. This study offers a new DA enzyme tool for the biosynthesis of novel hetero-dimer and biochemical clues for the biosynthetic logic elucidation of lithocarpins.
Assuntos
Reação de Cicloadição , Ascomicetos/enzimologia , Família MultigênicaRESUMO
Gliotoxin can be developed as potent biopesticide. In this study, the positive transcriptional factor gliZ, glutathione-S transferase encoding gene gliG and gliN were firstly deleted by CRISPR/Cas9 system, which abolished the production of gliotoxin-like compounds in Dichotomomyces cejpii. CRISPR/dCas9 system targeting promoter of gliG was used to activate the biosynthetic genes in gli cluster. The overexpression of gliZ, gliN and gliG can significantly improve the yield of gliotoxin-like compunds. The gliotoxin yields was improved by 16.38 ± 1.36 fold, 18.98 ± 1.28 fold through gliZ overexpression and gliM deletion in D. cejpii FS110. In addtion, gliN was heterologously expressed in E. coli, the purified GliN can catalyze gliotoxin into methyl-gliotoxin. Furthermore, the binding sequences of GliZ in the promoters of gliG was determined by Dnase footprinting. This study firstly illustrated the transcriptional regulatory mechanism of DcGliZ for the gliotoxin biosynthesis in D. cejpii, and improved the yields of gliotoxins significantly in D. cejpii via biosynthetic approaches.
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Gliotoxina , Gliotoxina/química , Gliotoxina/metabolismo , Vias Biossintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Candida albicans is the main causal pathogen of fungal infections in human beings. Although diverse anti-C. albicans drugs have been explored, the drug resistance and side effects of these drugs are intensifying. Thus, it is urgent to explore new anti-C. albicans compounds from natural products. In this study, we identified trichoderma acid (TA), a compound from Trichoderma spirale with a strong inhibitory effect on C. albicans. Transcriptomic and iTRAQ-based proteomic analyses of TA-treated C. albicans in combination with scanning electronic microscopy and reactive oxygen species (ROS) detection were performed to investigate the potential targets of TA. The most significant differentially expressed genes and proteins after TA treatment were verified through Western blot analysis. Our results revealed that mitochondrial membrane potential, endoplasmic reticulum, ribosomes in the mitochondria, and cell walls were disrupted in TA-treated C. albicans, leading to the accumulation of ROS. The impaired enzymatic activities of superoxide dismutase further contributed to the increase in ROS concentration. The high concentration of ROS led to DNA damage and cell skeleton destruction. The expression levels of Rho-related GTP-binding protein RhoE (RND3), asparagine synthetase (ASNS), glutathione S-transferase, and heat shock protein 70 were significantly up-regulated in response to apoptosis and toxin stimulation. These findings suggest that RND3, ASNS, and supereoxide dismutase 5 are the potential targets of TA, as further demonstrated through Western blot analysis. The combination of transcriptomic, proteomic, and cellular analyses would provide clues for the anti-C. albicans mechanism of TA and the defensive response mechanism of C. albicans. TA is thus recognized as a promising new anti-C. albicans leading compound that alleviates the hazard of C. albicans infection in human beings.
Assuntos
Candida albicans , Trichoderma , Humanos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Trichoderma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteômica , Testes de Sensibilidade MicrobianaRESUMO
Epothilones are a kind of 16-member macrolides with strong anticancer activity, which was produced by Sorangium cellulosum. Epothlione D shows better drug resistance and safety than taxol in clinical trials. However, the low yield of epothilone D in Sorangium cellulosum and thereof toxicity limited the application of epothilone D. In this study, the epoK gene in gene cluster for epothilone was firstly inactivated by the employment of TALEN gene knockout system. The qRT-PCR analysis and sequencing were performed to confirm the gene deletion of epoK, resulting in the epothilone D yield improvement by 34.9±1.6% and the decrease of epothilone B yield by 34.2±2.5%, which was demonstrated by LC-MS analysis. This study would lay a foundation for the yield improvement of epothilones D, B and thereof derivatives in S. cellulosum by genetic engineering, thus promoting the applications of epothilones in the field of anticancer.
Assuntos
Antineoplásicos , Epotilonas , Epotilonas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Macrolídeos , Antineoplásicos/farmacologiaRESUMO
Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.
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Gliotoxina/biossíntese , Gliotoxina/toxicidade , Animais , Ascomicetos/efeitos dos fármacos , Humanos , Família Multigênica/genética , Pythium/efeitos dos fármacosRESUMO
Benzophenones are polyketides with diverse biological activities. Novel cytotoxic benzophenones cytosporaphenones A-C and cytorhizins A-D, which contain a new skeleton, were previously extracted from endophytic fungus Cytospora rhizophorae A761. However, the mechanism for the biosynthesis of these compounds remains unknown. Cytosporaphenone A was assumed to be the precursor for the biosynthesis of cytorhizins A-D. In this study, we sequenced the genome of C. rhizophorae A761 and characterized a benzoate 4-monooxygenase cytochrome P450(BAM). CRISPR/Cas9-mediated gene knockout and overexpression studies in C. rhizophorae confirmed the vital function of BAM in the biosynthesis of cytosporaphenones and cytorhizins. Overexpression of BAM also enhanced the yield of cytosporaphenone A by 1.868 folds. The in vitro function and enzymatic properties of BAM were also described. This study demonstrates the important role of BAM for the biosynthesis of cytosporaphenone A and cytorhizins and is also the first to provide approaches for the CRISPR-Cas9-mediated gene deletion and gene overexpression studies in C. rhizophoarae, thus laying a foundation for the elucidation of the biosynthetic mechanism of cytorhizins and the discovery of new benzophenones mediated by BAM.Key points⢠The novel bam gene encoding BAM protein in C. rhizophorae was firstly deleted using CRIPSR/Cas9 system.⢠The in vitro oxidation function of novel BAM protein and enzymatic properties was characterized.⢠The over expression of bam gene enhanced the yield of cytosporaphone A in C. rhizophorae significantly.
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Ascomicetos , Policetídeos , Ascomicetos/genética , Benzofenonas , Sistema Enzimático do Citocromo P-450/genéticaRESUMO
Trichothecene toxins cause serious hazard towards human health and economical crops. However, there are no sufficient molecular strategies to reduce the hazard of trichothecene toxins. Thus it is urgent to exploit novel approaches to control the hazard of trichothecenes. In this study, four trichothecene toxin-resistance genes including mfs1, GNAT1, TRP1 and tri12 in Paramyrothecium roridum were excavated based on genome sequencing results, and then expressed in toxin-sensitive Saccharomyces cerevisiae BJ5464, the toxin resistance genes pdr5, pdr10 and pdr15 of which were firstly knocked out simultaneously by the introduction of TAA stop codon employing CRISPR/Cas9 system. Therefore, three novel hazardous toxin-resistance genes mfs1, GNAT1, TRP1 in P. roridum were firstly excavated by the co-incubation of DON toxin and toxin resistant genes-containing BJ5464 strains. The in vitro function and properties of novel toxin-resistance genes coding proteins including GNAT1, MFS1 and TRP1 were identified by heterologous expression and cellular location analysis as well as in vitro biochemical reaction. The excavation of novel trichothecene toxin-resistance genes provide novel molecular clues for controlling the harm of trichothecenes, meanwhile, this study will also pave a new way for the yield improvement of trichothecenes by heterologous expression to facilitate the development of trichothecenes as anti-tumor lead compounds.
Assuntos
Antibiose , Proteínas Fúngicas/metabolismo , Hypocreales/metabolismo , Toxinas Biológicas/antagonistas & inibidores , Tricotecenos/antagonistas & inibidores , Antibiose/genética , Proteínas Fúngicas/genética , Expressão Gênica , Loci Gênicos , Hypocreales/genética , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tricotecenos/metabolismoRESUMO
Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.
Assuntos
Organismos Aquáticos , Proteínas Fúngicas , Genes Fúngicos , Gliotoxina/biossíntese , Hypocreales , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/enzimologia , Hypocreales/genéticaRESUMO
Marine toxins cause great harm to human health through seafood, therefore, it is urgent to exploit new marine toxins detection methods with the merits of high sensitivity and specificity, low detection limit, convenience, and high efficiency. Aptasensors have emerged to replace classical detection methods for marine toxins detection. The rapid development of molecular biological approaches, sequencing technology, material science, electronics and chemical science boost the preparation and application of aptasensors. Taken together, the aptamer-based biosensors would be the best candidate for detection of the marine toxins with the merits of high sensitivity and specificity, convenience, time-saving, relatively low cost, extremely low detection limit, and high throughput, which have reduced the detection limit of marine toxins from nM to fM. This article reviews the detection of marine toxins by aptamer-based biosensors, as well as the selection approach for the systematic evolution of ligands by exponential enrichment (SELEX), the aptamer sequences. Moreover, the newest aptasensors and the future prospective are also discussed, which would provide thereotical basis for the future development of marine toxins detection by aptasensors.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais/instrumentação , Toxinas Marinhas/análise , Animais , Técnicas Biossensoriais/métodos , Humanos , Ligantes , Limite de Detecção , Técnica de Seleção de AptâmerosRESUMO
Wheat yield is greatly reduced because of the occurrence of leaf spot diseases. Bipolaris sorokiniana is the main pathogenic fungus in leaf spot disease. In this study, B. sorokiniana from wheat leaf (W-B. sorokiniana) showed much stronger pathogenicity toward wheat than endophytic B. sorokiniana from Pogostemon cablin (P-B. sorokiniana). The transcriptomes and metabolomics of the two B. sorokiniana strains and transcriptomes of B. sorokiniana-infected wheat leaves were comparatively analyzed. In addition, the expression levels of unigenes related to pathogenicity, toxicity, and cell wall degradation were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Results indicated that pathogenicity-related genes, especially the gene encoding loss-of-pathogenicity B (LopB) protein, cell wall-degrading enzymes (particularly glycosyl hydrolase-related genes), and killer and Ptr necrosis toxin-producing related unigenes in the W-B. sorokiniana played important roles in the pathogenicity of W-B. sorokiniana toward wheat. The down-regulation of cell wall protein, photosystem peptide, and rubisco protein suggested impairment of the phytosynthetic system and cell wall of B. sorokiniana-infected wheat. The up-regulation of hydrolase inhibitor, NAC (including NAM, ATAF1 and CUC2) transcriptional factor, and peroxidase in infected wheat tissues suggests their important roles in the defensive response of wheat to W-B. sorokiniana. This is the first report providing a comparison of the transcriptome and metabolome between the pathogenic and endophytic B. sorokiniana strains, thus providing a molecular clue for the pathogenic mechanism of W-B. sorokiniana toward wheat and wheat's defensive response mechanism to W-B. sorokiniana. Our study could offer molecular clues for controlling the hazard of leaf spot and root rot diseases in wheat, thus improving wheat yield in the future.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Perfilação da Expressão Gênica , Metabolômica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Triticum/genética , Triticum/microbiologia , Ascomicetos/patogenicidade , Ascomicetos/ultraestrutura , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genoma de Planta , Anotação de Sequência Molecular , Micélio/ultraestrutura , Micotoxinas/metabolismo , Metabolismo Secundário/genética , TranscriptomaRESUMO
Epothilones are a kind of macrolides with strong cytotoxicity toward cancer cells and relatively lower side effects compared with taxol. Epothilone B derivate ixabepilone has been used for the clinical treatment of advanced breast cancer. However, the low yield of epothilones and the difficulty in the genetic manipulation of Sorangium cellulosum limited their wider application. Transcription activator-like effectors-Trancriptional factor (TALE-TF)-VP64 and clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-VP64 have been demonstrated as effective systems for the transcriptional improvement. In this study, a promoter for the epothilone biosynthesis cluster was obtained and the function has been verified. The TALE-TF-VP64 and CRISPR/dcas9-VP64 target P3 promoter were electroporated into S. cellulosum strain So ce M4, and the transcriptional levels of epothilone biosynthesis-related genes were significantly upregulated. The yield of epothilone B was improved by 2.89- and 1.53-fold by the introduction of recombinant TALE-TF-VP64-P3 and dCas9-VP64-P3 elements into So ce M4, respectively. The epothilone D yield was also improved by 1.12- and 2.18-fold in recombinant dCas9-So ce M4 and TALE-VP64 strains, respectively. The transcriptional regulation mechanism of TALE-TF-VP64 and the competition mechanism with endogenous transcriptional factor were investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), demonstrating the combination of the P3 promoter and TALE-TF element and the competition between TALE-TF and endogenous transcriptional protein. This is the first report on the transcriptional regulation of the epothilone biosynthetic gene cluster in S. cellulosum using the TALE-TF and dCas9-VP64 systems, and the regulatory mechanism of the TALE-TF system for epothilone biosynthesis in S. cellulosum was also firstly revealed, thus shedding light on the metabolic engineering of S. cellulosum to improve epothilone yields substantially and promoting the application of epothilones in the biomedical industry.
RESUMO
Agarwood is a precious traditional Chinese medicine with a variety of pharmacological effects. Although efforts have been made in elucidating the mechanism of agarwood formation, little progress is obtained till now. Therefore, the molecular mechanism of agarwood formation needs to be further explored using different biological approaches. In this study, the quantitative proteomic analysis using iTRAQ technology combined with transcriptomic and metabolomic analyses on chemically induced Aquilaria sinensis is performed to elucidate the agarwood formation mechanism by formic acid stimulus. Data are available via ProteomeXchange with identifier PXD007586; 1884 proteins are detected, 504 differential proteins that show at least twofold differences in their expression levels are selected based on GO annotations, KEGG, STRING analysis, and quantitative RT-PCR analysis. The results indicate that sesquiterpene synthase, germin-like protein, pathogenesis-related protein, 6-phosphogluconate dehydrogenase, lipoyl synthase, and superoxide dismutase play important roles in the agarwood formation, suggesting that the proteins related to the plant defensive response, the removal of peroxide, the disease-resistance, the biosythesis of glycan, fatty acids, and sesquiterpene are crucial for agarwood formation.
Assuntos
Formiatos/farmacologia , Metaboloma , Proteínas de Plantas/análise , Proteoma , Thymelaeaceae/metabolismo , Transcriptoma , Madeira/metabolismo , Regulação da Expressão Gênica de Plantas , Thymelaeaceae/efeitos dos fármacos , Thymelaeaceae/genética , Thymelaeaceae/crescimento & desenvolvimento , Madeira/químicaRESUMO
Gliotoxin, produced by fungi, is an epipolythiodioxopiperazine (ETP) toxin with bioactivities such as anti-liver fibrosis, antitumor, antifungus, antivirus, antioxidation, and immunoregulation. Recently, cytotoxic gliotoxins were isolated from a deep-sea-derived fungus, Dichotomomyces cejpii. However, the biosynthetic pathway for gliotoxins in D. cejpii remains unclear. In this study, the transcriptome of D. cejpii was sequenced using an Illumina Hiseq 2000. A total of 19,125 unigenes for D. cejpii were obtained from 9.73 GB of clean reads. Ten genes related to gliotoxin biosynthesis were annotated. The expression levels of gliotoxin-related genes were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The GliG gene, encoding a glutathione S-transferase (DC-GST); GliI, encoding an aminotransferase (DC-AI); and GliO, encoding an aldehyde reductase (DC-AR), were cloned and expressed, purified, and characterized. The results suggested the important roles of DC-GST, DC-AT, and DC-AR in the biosynthesis of gliotoxins. Our study on the genes related to gliotoxin biosynthesis establishes a molecular foundation for the wider application of gliotoxins from D. cejpii in the biomedical industry in the future.
Assuntos
Fungos/genética , Gliotoxina/biossíntese , Transcriptoma/genética , Aldeído Redutase/genética , Fungos/metabolismo , Perfilação da Expressão Gênica/métodos , Glutationa Transferase/genéticaRESUMO
Sesquiterpene synthases are key enzymes for biosynthesis of sesquiterpene compounds and are important for agarwood formation in Aquilaria sinensis.The As-sesTPS gene encoding a novel sesquiterpene synthase was expressed in Escherichia coli strain BL21 (DE3) as an inclusion body and purified by Ni affinity chromatography. The molecular weight of the protein was lower than the theoretical value. Amino acid sequencing results indicated that the 27.2kDa-recombinant protein was a truncated sesquiterpene synthase from chemically induced A. sinensis. After refolding, the truncated As-SesTPS protein catalyzed the conversion of farnesyl pyrophosphate (FPP) to nerolidol which is a characteristic component of agarwood. The optimal reaction pH for the As-SesTPS protein was 8.0, and the optimal temperature was 30°C. The values of Km and Vmax of As-SesTPS protein towards FPP were 0.0548mM, 42.83µmol/mg.min, respectively. The results of qPCR and iTRAQ demonstrated the much higher expression level of As-SesTPS gene in agarwood than that in whitewood. This study provides a foundation for elucidating the mechanism of agarwood formation in A. sinensis and the potential of the novel gene for improving the quality of artificial agarwood.
Assuntos
Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Thymelaeaceae/enzimologia , Sequência de Aminoácidos , Carbono-Carbono Liases/química , Carbono-Carbono Liases/isolamento & purificação , Catálise , Clonagem Molecular , Ativação Enzimática , Expressão Gênica , Genes de Plantas , Filogenia , Análise de Sequência de DNA , Sesquiterpenos/metabolismo , Thymelaeaceae/genéticaRESUMO
Myrothecium roridum is a plant pathogenic fungus that infects different crops and decreases the yield of economical crops, including soybean, cotton, corn, pepper, and tomato. Until now, the pathogenic mechanism of M. roridum has remained unclear. Different types of trichothecene mycotoxins were isolated from M. roridum, and trichothecene was considered as a plant pathogenic factor of M. roridum. In this study, the transcriptome of M. roridum in different incubation durations was sequenced using an Illumina Hiseq 2000. A total of 35,485 transcripts and 25,996 unigenes for M. roridum were obtained from 8.0 Gb clean reads. The protein-protein network of the M. roridum transcriptome indicated that the mitogen-activated protein kinases signal pathway also played an important role in the pathogenicity of M. roridum. The genes related to trichothecene biosynthesis were annotated. The expression levels of these genes were also predicted and validated through quantitative real-time polymerase chain reaction. Tri5 gene encoding trichodiene synthase was cloned and expressed, and the purified trichodiene synthase was able to catalyze farnesyl pyrophosphate into different kinds of sesquiterpenoids.Tri4 and Tri11 genes were expressed in Escherichia coli, and their corresponding enzymatic properties were characterized. The phylogenetic tree of trichodiene synthase showed a great discrepancy between the trichodiene synthase from M. roridum and other species. Our study on the genes related to trichothecene biosynthesis establishes a foundation for the M. roridum hazard prevention, thus improving the yields of economical crops.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Micotoxinas/biossíntese , Transcriptoma , Tricotecenos/biossíntese , Vias Biossintéticas , Catálise , Biologia Computacional/métodos , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anotação de Sequência Molecular , Filogenia , Doenças das Plantas/microbiologia , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2 (+) As2.4). LC-MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2 (+) As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2 (+) As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering.
RESUMO
Trichothecene mycotoxins are a type of sesquiterpenoid produced by various kinds of plantpathogenic fungi. In this study, two trichothecene toxins, namely, a novel cytotoxic epiroridin acid and a known trichothecene, mytoxin B, were isolated from the endophytic fungus Myrothecium roridum derived from the medicinal plant Pogostemon cablin. The two trichothecene mytoxins were confirmed to induce the apoptosis of HepG-2 cells by cytomorphology inspection, DNA fragmentation detection, and flow cytometry assay. The cytotoxic mechanisms of the two mycotoxins were investigated by quantitative real time polymerase chain reaction, western blot, and detection of mitochondrial membrane potential. The results showed that the two trichothecene mycotoxins induced the apoptosis of cancer cell HepG-2 via activation of caspase-9 and caspase-3, up-regulation of bax gene expression, down-regulation of bcl-2 gene expression, and disruption of the mitochondrial membrane potential of the HepG-2 cell. This study is the first to report on the cytotoxic mechanism of trichothecene mycotoxins from M. roridum. This study provides new clues for the development of attenuated trichothecene toxins in future treatment of liver cancer.
Assuntos
Apoptose/efeitos dos fármacos , Hypocreales/química , Micotoxinas/administração & dosagem , Tricotecenos/administração & dosagem , Caspases/biossíntese , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Micotoxinas/química , Neoplasias/tratamento farmacológico , Pogostemon/microbiologia , Sesquiterpenos/administração & dosagem , Sesquiterpenos/química , Tricotecenos/químicaRESUMO
BACKGROUND: Agarwood is a traditional Chinese medicine used as a clinical sedative, carminative, and antiemetic drug. Agarwood is formed in Aquilaria sinensis when A. sinensis trees are threatened by external physical, chemical injury or endophytic fungal irritation. However, the mechanism of agarwood formation via chemical induction remains unclear. In this study, we characterized the transcriptome of different parts of a chemically induced A. sinensis trunk sample with agarwood. The Illumina sequencing platform was used to identify the genes involved in agarwood formation. METHODOLOGY/PRINCIPAL FINDINGS: A five-year-old Aquilaria sinensis treated by formic acid was selected. The white wood part (B1 sample), the transition part between agarwood and white wood (W2 sample), the agarwood part (J3 sample), and the rotten wood part (F5 sample) were collected for transcriptome sequencing. Accordingly, 54,685,634 clean reads, which were assembled into 83,467 unigenes, were obtained with a Q20 value of 97.5%. A total of 50,565 unigenes were annotated using the Nr, Nt, SWISS-PROT, KEGG, COG, and GO databases. In particular, 171,331,352 unigenes were annotated by various pathways, including the sesquiterpenoid (ko00909) and plant-pathogen interaction (ko03040) pathways. These pathways were related to sesquiterpenoid biosynthesis and defensive responses to chemical stimulation. CONCLUSIONS/SIGNIFICANCE: The transcriptome data of the different parts of the chemically induced A. sinensis trunk provide a rich source of materials for discovering and identifying the genes involved in sesquiterpenoid production and in defensive responses to chemical stimulation. This study is the first to use de novo sequencing and transcriptome assembly for different parts of chemically induced A. sinensis. Results demonstrate that the sesquiterpenoid biosynthesis pathway and WRKY transcription factor play important roles in agarwood formation via chemical induction. The comparative analysis of the transcriptome data of agarwood and A. sinensis lays the foundation for elucidating the mechanism of agarwood formation via chemical induction, and thus, enables future improvements in agarwood quality while protecting endangered wild A. sinensis.