RESUMO
Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5' untranslated region (5'-UTR) from T7g10 gene (DC41 and DC51 plants), and 5' translation control region (5'-TCR), containing the 5'-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.
Assuntos
Proteínas Fúngicas , Oxigenases de Função Mista , Celulose/química , Proteínas Fúngicas/biossíntese , Oxigenases de Função Mista/biossíntese , Polissacarídeos/metabolismo , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , PlastídeosRESUMO
Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.
Assuntos
Abelhas/genética , Escherichia coli , Expressão Gênica , Proteínas de Insetos , Oxigenases de Função Mista , Animais , Abelhas/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Insetos/biossíntese , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.
Assuntos
Agrocybe/enzimologia , Magnaporthe/genética , Oxigenases de Função Mista/biossíntese , Agrocybe/genética , Fator de Iniciação 1 em Eucariotos/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Magnaporthe/metabolismo , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossínteseRESUMO
Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body's normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Bactérias/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oxigenases de Função Mista/farmacologia , Triptofano/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Modelos Moleculares , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estrutura Secundária de Proteína , Pseudomonas/química , Pseudomonas/enzimologia , Pseudomonas/genética , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The human aspartyl ß-hydroxylase (ASPH) is overexpressed in tumor tissues. Bronchoalveolar lavage (BAL) is a diagnostic procedure for infections and malignancies. The aim of this study was to investigate whether tumor exosomes carrying ASPH gene marker were present in bronchoalveolar fluid of patients with non-small cell lung cancer (NSCLC). A tissue microarray analysis was applied to explore the expression of ASPH in different histologic NSCLC. The human NSCLC cell lines and normal bronchial cell lines were used to study exosomal ASPH exprerssion. A total of 27 NSCLC, 21 benign tumor, and 15 healthy controls underwent BAL. Immunohistochemistry was performed to study the ASPH expression in malignant and normal lung tissues. The expression characteristics of ASPH in different NSCLC and normal bronchial cells and pneumocytes were confirmed by cell blocks. A reverse transcription-quantitative polymerase chain reaction was carried out to study the levels of exosomal ASPH expression. Immunohistochemical staining of tissue microarray demonstrated that overexpression of ASPH was found in NSCLC tissues including adenocarcinoma, large cell carcinoma, and squamous cell carcinoma, but absent in adjacent normal tissues. All NSCLC specimens exhibited high levels of ASPH immunoreactivity, while nonmalignant and normal lung tissues exhibited a very low level of expression. Overexpression of ASPH was found in exosomes from NSCLC cell lines but absent from the normal bronchial cell line NL-20. ASPH level from BAL exosomes was significantly increased in NSCLC patients compared with that from nonmalignant or health group. Our method of isolation of BAL exosomes was easily performed in the clinical laboratory. BAL exosomal ASPH can be a potential biomarker for NSCLC diagnosis.
Assuntos
Lavagem Broncoalveolar , Proteínas de Ligação ao Cálcio/biossíntese , Carcinoma Pulmonar de Células não Pequenas , Exossomos/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Proteínas de Membrana/biossíntese , Oxigenases de Função Mista/biossíntese , Proteínas Musculares/biossíntese , Proteínas de Neoplasias/biossíntese , Células A549 , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologiaRESUMO
Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.
Assuntos
Proteínas Fúngicas/biossíntese , Oxigenases de Função Mista/biossíntese , Pichia/enzimologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas/genética , Saccharomycetales/enzimologia , Estudos de Viabilidade , Proteínas Fúngicas/genética , Ensaios de Triagem em Larga Escala/métodos , Microrganismos Geneticamente Modificados , Oxigenases de Função Mista/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Saccharomycetales/genética , Sordariales/enzimologia , Sordariales/genéticaRESUMO
High-altitude (HA, >2500 m) hypoxic exposure evokes several physiological processes that may be abetted by differential genetic distribution in sojourners, who are susceptible to various HA disorders, such as high-altitude pulmonary edema (HAPE). The genetic variants in hypoxia-sensing genes influence the transcriptional output; however the functional role has not been investigated in HAPE. This study explored the two hypoxia-sensing genes, prolyl hydroxylase domain protein 2 (EGLN1) and factor inhibiting HIF-1α (HIF1AN) in HA adaptation and maladaptation in three well-characterized groups: highland natives, HAPE-free controls and HAPE-patients. The two genes were sequenced and subsequently validated through genotyping of significant single nucleotide polymorphisms (SNPs), haplotyping and multifactor dimensionality reduction. Three EGLN1 SNPs rs1538664, rs479200 and rs480902 and their haplotypes emerged significant in HAPE. Blood gene expression and protein levels also differed significantly (P < 0.05) and correlated with clinical parameters and respective alleles. The RegulomeDB annotation exercises of the loci corroborated regulatory role. Allele-specific differential expression was evidenced by luciferase assay followed by electrophoretic mobility shift assay, liquid chromatography with tandem mass spectrometry and supershift assays, which confirmed allele-specific transcription factor (TF) binding of FUS RNA-binding protein (FUS) with rs1538664A, Rho GDP dissociation inhibitor 1 (ARHDGIA) with rs479200T and hypoxia upregulated protein 1 (HYOU1) with rs480902C. Docking simulation studies were in sync for the DNA-TF structural variations. There was strong networking among the TFs that revealed physiological consequences through relevant pathways. The two hydroxylases appear crucial in the regulation of hypoxia-inducible responses.
Assuntos
Doença da Altitude , Loci Gênicos , Hipertensão Pulmonar , Prolina Dioxigenases do Fator Induzível por Hipóxia , Oxigenases de Função Mista , Polimorfismo de Nucleotídeo Único , Edema Pulmonar , Proteínas Repressoras , Células A549 , Altitude , Doença da Altitude/enzimologia , Doença da Altitude/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Masculino , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Edema Pulmonar/enzimologia , Edema Pulmonar/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Fatores de RiscoRESUMO
Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.
Assuntos
Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genéticaRESUMO
N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.
Assuntos
Proteínas de Bactérias/metabolismo , Cadaverina/metabolismo , Desferroxamina/metabolismo , Oxigenases de Função Mista/biossíntese , Sideróforos/biossíntese , Streptomyces/enzimologia , Biocatálise , Domínio Catalítico , Dinitrocresóis/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Holoenzimas/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Ornitina/metabolismo , OxirreduçãoRESUMO
Pinocembrin derived flavones are the major bioactive compounds presented in the Lamiaceae plants that have long been of interest due to their great pharmaceutical and economical significance. Modifications on the central skeleton of the flavone moiety have a huge impact on their biological activities. However, the enzymes responsible for structure modification of most flavones are either inefficient or remain unidentified. By integrating omics analysis of Scutellaria barbata and synthetic biology tools in yeast chassis, we characterized a novel gene encoding flavone 7-O-methyltransferase (F7OMT) and discovered a new flavone 8-hydroxylase (F8H) with increased activity. We also identified a series of flavone 6-hydroxylases (F6Hs) and flavone 8-O-methyltransferases (F8OMTs) in this study. Subsequently, we constructed the biosynthetic pathway for chrysin production by assembling catalytic elements from different species and improved the titer to 10.06 mg/L. Using the established chrysin production platform, we achieved the de novo biosynthesis of baicalein, baicalin, norwogonin, wogonin, isowogonin, and moslosooflavone in yeast. Our results indicated that the combination of omics and synthetic biology can greatly speed up the efficiency of gene mining in plants and the engineered yeasts established an alternative way for the production of pinocembrin derivatives.
Assuntos
Flavanonas/biossíntese , Flavanonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas/fisiologia , Flavonas/biossíntese , Flavonas/metabolismo , Flavonoides/biossíntese , Flavonoides/metabolismo , Lamiaceae/metabolismo , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/metabolismoRESUMO
BACKGROUND: Recently, resveratrol (Res) has been suggested to suppress the migration and invasion of prostate cancer (PCa). In the present study, we aimed to investigate the effects of Res on genomic DNA methylation, as well as the migration and invasion of PCa cells. METHODS: The suppression by Res of the growth of PCa cells was verified through a cytotoxicity assay. In addition, the effects of Res on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and ten-eleven translocation 1 (TET1) levels were assessed, and the cell migration and invasion were also determined. The expressions of TET1, tissue inhibitor of metalloproteinases (TIMP) 2, TIMP3, MMP2, and MMP9 were detected through Western blot analysis. Afterward, TET1 was silenced using lentiviral short hairpin RNA to examine the effect of TET1 on the Res-triggered inhibition of migration and invasion of PCa cells. RESULTS: Our results showed that Res upregulated the 5hmC and TET1 levels and downregulated the 5mC level. Moreover, Res also inhibited the migration and invasion of PCa cells, promoted the demethylation of TIMP2 and TIMP3 to upregulate their expressions, and suppressed the expressions of MMP2 and MMP9. The silencing of TET1 in the presence of Res showed that Res could exert its effect through TET1. CONCLUSIONS: Our findings indicated that Res inhibited the migration and invasion of PCa cells via the TET1/TIMP2/TIMP3 pathway, which might potentially serve as a target for the treatment of PCa.
Assuntos
Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Resveratrol/farmacologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Invasividade Neoplásica , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Resveratrol/farmacocinética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genética , Regulação para CimaRESUMO
Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT. To investigate this issue, we constructed phzM-lacZ fusion on a vector and on the chromosome. By quantifying ß-galactosidase activities, we confirmed that expression of the phzM was up-regulated when the rpoS gene was inactivated. However, no changes occurred in the expression of phzS and phzH when the rpoS was knocked out. Taken together, overproduction of the SAM-dependent methyltransferase (PhzM) might contribute to the increased pyocyanin in the absence of RpoS in P. aeruginosa.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Metiltransferases/biossíntese , Oxigenases de Função Mista/biossíntese , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Fator sigma/genética , Humanos , Metiltransferases/genética , Oxigenases de Função Mista/genética , Fenazinas/metabolismo , Pseudomonas aeruginosa/genética , Fatores de Virulência/metabolismoRESUMO
A bacterial strain designated Lysinibacillus fusiformis 15-4 was isolated from oil-free soil on the Qinghai-Tibet Plateau, which can grow well utilizing petroleum hydrocarbons as a carbon source at a lower temperature. To deeply characterize the molecular adaptations and metabolic processes of this strain when grown in a petroleum-containing environment, transcriptome analysis was performed. A total of 4664 genes and the expression of 3969 genes were observed in strain 15-4. When the strain was grown in petroleum-containing medium, 2192 genes were significantly regulated, of which 1312 (60%) were upregulated and 880 (40%) were downregulated. This strain degraded and adapted to petroleum via modulation of diverse molecular processes, including improvements in transporter activity, oxidoreductase/dehydrogenase activity, two-component system/signal transduction, transcriptional regulation, fatty acid catabolism, amino acid metabolism, and environmental stress responses. Many strain-specific genes were involved in the oxidation of hydrocarbon compounds, such as several luciferase family alkane monooxygenase genes, flavin-utilizing monooxygenase family genes, and flavoprotein-like family alkanesulfonate monooxygenase genes. Several cold shock protein genes were also induced suggesting adaptation to cold environments and the potential for petroleum degradation at low temperatures. The results obtained in this study may broaden our understanding of molecular adaptation of bacteria to hydrocarbon-containing environments and may provide valuable data for further study of L. fusiformis.
Assuntos
Bacillaceae/genética , Bacillaceae/metabolismo , Petróleo/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adaptação Fisiológica , Bacillaceae/isolamento & purificação , Biodegradação Ambiental , Proteínas e Peptídeos de Choque Frio/biossíntese , Proteínas e Peptídeos de Choque Frio/genética , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hidrocarbonetos/metabolismo , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Microbiologia do Solo , TibetRESUMO
Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.
Assuntos
Arabidopsis , Flavanonas/biossíntese , Flavonóis/biossíntese , Proteínas de Plantas/biossíntese , Flavanonas/isolamento & purificação , Flavonoides/biossíntese , Flavonoides/isolamento & purificação , Flavonóis/isolamento & purificação , Oxigenases de Função Mista/biossíntese , Oxirredutases/biossíntese , Extratos Vegetais/biossíntese , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/isolamento & purificaçãoRESUMO
The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 ).
Assuntos
Caproatos/metabolismo , Cumarínicos/metabolismo , Escherichia coli/metabolismo , Lactonas/metabolismo , Engenharia Metabólica , Catálise , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genéticaRESUMO
BACKGROUND: While aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types. RESULTS: Here, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched. CONCLUSIONS: Our finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/biossíntese , Regulação Leucêmica da Expressão Gênica , Oxigenases de Função Mista/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Humanos , Camundongos , Camundongos Transgênicos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Increasing evidence revealed that ten-eleven translocation 1 (TET1) plays an important role in tumorigenesis and chemoresistance, but its functions in gemcitabine resistance in cholangiocarcinoma (CCA) remain unknown. This study aims to investigate the effect of TET1 on gemcitabine resistance in CCA and the possible effect on P-glycoprotein (P-gp) expression encoded by multidrug resistance (MDR) genes. We established two kinds of gemcitabine-resistant CCA cell lines and confirmed its specific features. The expression of TET1 and P-gp was evaluated in gemcitabine-resistant CCA cells and their parental cells at mRNA and protein level by quantitative RT-PCR and western blot analysis. After transfecting the gemcitabine-resistant CCA cell lines with TET1 gene or siRNA, the cell viability test was obtained to verify the effect of TET1 on the sensitivity of CCA cells to gemcitabine. And then, the possible effect of TET1 on the expression of P-gp was examined by western blot analysis. Xenograft tumor experiment was conducted to confirm the association between TET1 and P-gp expression under gemcitabine chemoresistance. The associations between clinical outcomes of CCA patients with chemotherapy and TET1 expression were analyzed in 82 patients. The results showed that TET1 expression was significantly decreased, and P-gp expression was increased in gemcitabine-resistant CCA cells. Additionally, overexpression of TET1 augmented the sensitivity of CCA cells to gemcitabine and induced the decreased expression of P-gp in gemcitabine-resistant CCA cells. Furthermore, multivariate Cox regression analysis indicated that TET1 expression and TNM stage were independent risk factors (P < 0.001) for the clinical outcomes of CCA patients with chemotherapy. Additionally, Kaplan-Meier survival and the log-rank test showed that decreased expression of TET1 was associated with poorer prognosis of CCA patients with chemotherapy. These findings suggest that TET1 expression reverses gemcitabine resistance in CCA accompanied by a reduction in P-gp expression. Thus, TET1 may be a promising target to overcome chemoresistance in CCA.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Desoxicitidina/análogos & derivados , Oxigenases de Função Mista/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
DNA hypermethylation is a driving force in carcinogenesis. However, the role of active DNA hypomethylation in cancer remains largely unknown. This process, facilitated by ten-eleven translocation methylcytosine dioxygenase 1 (TET1), which oxidizes 5-methylcytosine (5â¯mC) to 5-hydroxymethylcytosine (5hmC), has never been studied in cervical cancer. Here, we found that TET1 and 5hmC correlative increases from normal cervix to Low-grade squamous intraepithelial lesion (LSIL), maximizing in High-grade squamous intraepithelial lesion (HSIL), and decreasing in invasive cancer. Full-length HPV-immortalized HSIL cells demonstrated higher TET1/5hmC levels, and stemness properties, compared to invasive cancer cells. TET1 silencing promoted the epithelial-mesenchymal transition (EMT), to transform precancerous cells in vivo. TET1 increased 5hmC in the ZEB1 and VIM promoters, surprisingly, silencing both genes. TET1 interaction with the histone modifiers, LSD1 and EZH2, on the ZEB1 promoter, resulted in gene silencing, via loss of histone H3K4 trimethylation, and gain of histone H3K27 trimethylation. Taken together, TET1 promotes stemness properties, and inhibits EMT, in HSIL cells, through 5hmC-dependent and -independent mechanisms.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Células HeLa , Xenoenxertos , Humanos , Camundongos , Oxigenases de Função Mista/biossíntese , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas/biossíntese , Lesões Intraepiteliais Escamosas Cervicais/metabolismo , Lesões Intraepiteliais Escamosas Cervicais/patologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Vimentina , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/patologiaRESUMO
Cytochrome P450 monooxygenases (P450s) are a large class of enzymes that play essential roles in metabolic processes such as hormone synthesis and the catabolism of toxins and other chemicals in insects. In the present study, we identified 82 P450 genes using comprehensive RNA sequencing in the flea beetle Agasicles hygrophila, and all of the sequences were validated by cloning and sequencing. Phylogenetic analysis showed that the P450 genes in A. hygrophila fell into the mitochondrial clan, CYP2 clan, CYP3 clan and CYP4 clan and were classified into 20 families and 48 subfamilies. Most A. hygrophila P450 genes had high sequence homology with those from other coleopteran insects. To understand the effects of high temperatures on the metabolic processes of female and male adults, we studied the effects of two temperature regimes (constant temperature of 28 °C for 20 h with a 4-h period of high temperatures of 30 °C and 39 °C) on the expression levels of P450 genes in A. hygrophila using RT-PCR and qRT-PCR. The results showed that there were no differences in expression in 30 P450 genes between the control and high-temperature-treated A. hygrophila adults, while 22 P450 genes showed up-regulated expression and 19 P450 genes were down-regulated in A. hygrophila female adults after high-temperature treatment. For A. hygrophila male adults exposed to high temperatures, we found that 8 P450 genes had higher expression levels and 12 P450 genes had lower expression levels under the same conditions. The P450 genes are candidates that showed significantly different expression levels after high-temperature treatments in A. hygrophila adults, and further studies are needed to determine their possible roles in metabolic processes during the response to elevated temperatures.
Assuntos
Besouros/enzimologia , Besouros/efeitos da radiação , Sistema Enzimático do Citocromo P-450/biossíntese , Perfilação da Expressão Gênica , Temperatura Alta , Oxigenases de Função Mista/biossíntese , Animais , Clonagem Molecular , Besouros/genética , Sistema Enzimático do Citocromo P-450/genética , Feminino , Masculino , Oxigenases de Função Mista/genética , Filogenia , Análise de Sequência de DNA , Análise de Sequência de RNA , Estresse FisiológicoRESUMO
Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC-MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality.