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1.
J Agric Food Chem ; 68(6): 1684-1690, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31957431

RESUMO

The carotenoid-derived volatile ß-ionone plays an important role in the formation of green and black tea flavors due to its low odor threshold, but its formation and the gene(s) involved in its biosynthesis during the tea withering process is(are) still unknown. In this study, we found that the content of ß-ionone increased during the tea withering process catalyzed by an unknown enzyme(s). Correlation analysis of expression patterns of Camellia sinensis carotenoid cleavage dioxygenase genes (CsCCDs) and the ß-ionone content during the withering period revealed CsCCD4 as the most promising candidate. The full-length CsCCD4 gene was amplified from C. sinensis, and the biochemical function of the recombinant CsCCD4 protein was studied after coexpression in Escherichia coli strains engineered to accumulate ß-carotene. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' double bonds. Volatile ß-ionone was detected as the main product by gas and liquid chromatography-mass spectrometry. The accumulation of ß-ionone was consistent with the expression levels of CsCCD4 in different tissues and during the withering process. The CsCCD4 expression was induced by low temperature and mechanical damage stress but not by dehydration stress. The results demonstrate that CsCCD4 catalyzes the production of ß-ionone in the tea plant and provide insight into its formation mechanism during the withering process.


Assuntos
Camellia sinensis/enzimologia , Carotenoides/metabolismo , Dioxigenases/metabolismo , Norisoprenoides/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Biocatálise , Camellia sinensis/química , Camellia sinensis/genética , Camellia sinensis/metabolismo , Dioxigenases/genética , Manipulação de Alimentos , Espectrometria de Massas , Filogenia , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas/classificação , Plantas/enzimologia , Plantas/genética , Alinhamento de Sequência
2.
DNA Cell Biol ; 39(1): 37-49, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31750745

RESUMO

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH (p = 0.0495) and by the combination of SAH and procaine (p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH (p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine (p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine (p = 0.0007), SAH (p = 0.0060), and SAH/procaine (p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine (p = 0.0291), SAH (p = 0.0373), and procaine/SAH (p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.


Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Procaína/farmacologia , S-Adenosil-Homocisteína/farmacologia , Animais , Bovinos , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Dioxigenases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas/genética , Pele/citologia
3.
Food Chem ; 309: 125705, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31670122

RESUMO

Red-peeled huyou has a distinct red peel color due mainly to the presence of red apocarotenoid ß-citraurin as well as the increase in amount of total carotenoids. The expression level of carotenoid cleavage dioxygenase 4b1 (CCD4b1) accounted for 99.0% of total transcript abundance of CCD4s in red-peeled huyou peel and was nearly 100 times higher than that in ordinary huyou. ß-Citraurin accumulation and peel coloration was mostly favored at 15 °C but strongly inhibited at moderately high temperatures 20 °C and 25 °C. Exogenous ethylene application for 3 d had no obvious effect on ß-citraurin accumulation in red-peeled huyou but holding fruit at moderately higher temperatures (20 °C and 25 °C) for 3 d had a significant adverse effect on ß-citraurin accumulation. The expression of phytoene synthase 1 (PSY1) and CCD4b1 was higher at 10 °C and 15 °C and significantly lower at 20 °C and 25 °C. The mechanisms governing the accumulation of ß-citraurin are discussed.


Assuntos
Citrus/efeitos dos fármacos , Etilenos/farmacologia , Armazenamento de Alimentos/métodos , Carotenoides/análise , Carotenoides/isolamento & purificação , Citrus/química , Citrus/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Frutas/química , Frutas/efeitos dos fármacos , Frutas/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Extração Líquido-Líquido , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura Ambiente , beta Caroteno/análogos & derivados , beta Caroteno/análise , beta Caroteno/isolamento & purificação
4.
Int J Cancer ; 146(2): 373-387, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31211412

RESUMO

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.


Assuntos
Neoplasias Encefálicas/genética , Carcinogênese/genética , Dioxigenases/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Biópsia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Glioblastoma/mortalidade , Glioblastoma/patologia , Código das Histonas/genética , Humanos , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Anticancer Res ; 39(11): 6007-6014, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704826

RESUMO

BACKGROUND/AIM: The histone demethylase NO66 regulates gene and protein expression. Epidermal growth factor receptor (EGFR) is a key oncogenic factor for glioblastoma. This study aimed to examine the role of NO66 in glioblastoma. MATERIALS AND METHODS: The prognostic value of NO66 expression in 263 human glioma tissues and 510 glioblastoma tissues was examined by Kaplan and Meier survival analysis. Immunoblot analysis of EGFR expression, cell proliferation assays and cell cycle analysis were performed in glioblastoma cells after NO66 knockdown. RESULTS: In 263 human glioma tissues, high levels of NO66 expression correlated with advanced disease stage and poor patient prognosis. In 510 glioblastoma tissues, high levels of NO66 expression also predicted poor patient prognosis. NO66 knockdown reduced EGFR expression and cell proliferation in glioblastoma cells. CONCLUSION: High levels of NO66 in glioma and glioblastoma tissues predict poor patient prognosis, and NO66 is required for EGFR expression and glioblastoma cell proliferation.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Dioxigenases/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Histona Desmetilases/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Dioxigenases/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Seguimentos , Glioma/genética , Glioma/metabolismo , Histona Desmetilases/genética , Humanos , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
6.
J Agric Food Chem ; 67(45): 12502-12510, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31623431

RESUMO

Succinic acid (SA) is applied in the food, chemical, and pharmaceutical industries. 5-Hydroxyleucine (5-HLeu) is a promising precursor for the biosynthesis of antituberculosis drugs. Here, we designed a promising synthetic route for the simultaneous production of SA and 5-HLeu by combining l-leucine dioxygenase (NpLDO), l-glutamate oxidase (LGOX), and catalase (CAT). Two bioconversion systems: "a multienzyme cascade catalysis in vitro" (MECCS) and "whole-cell catalysis system" (WCCS) were constructed. A high-activity NpLDO mutant was screened by error-prone polymerase chain reaction (PCR) and showed 6.1-fold improvement of catalytic activity. After optimization of reaction conditions, MECSS yielded 3.15 g/L SA and 3.92 g/L 5-HLeu, while the production of SA and 5-HLeu by the most effective WCSS reached 15.12 and 18.83 g/L, respectively. This is the first attempt to use ferrous iron/α-ketoglutarate-dependent dioxygenases for the simultaneous production of SA and hydroxy-amino-acid. This research provides a tool for industrial production of food of high-value products from low-cost raw materials.


Assuntos
Aminoácido Oxirredutases/química , Proteínas de Bactérias/química , Catalase/química , Dioxigenases/química , Leucina/química , Nostoc/metabolismo , Ácido Succínico/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Catalase/genética , Catalase/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Leucina/metabolismo , Nostoc/enzimologia , Nostoc/genética , Ácido Succínico/metabolismo
7.
Plant Sci ; 287: 110188, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481229

RESUMO

9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting enzyme for abscisic acid (ABA) biosynthesis. However, the molecular mechanisms of NCED5 that modulate plant development and abiotic stress tolerance are still unclear, particular in rice. Here, we demonstrate that a rice NCED gene, OsNCED5, was expressed in all tissues we tested, and was induced by exposure to salt stress, water stress, and darkness. Mutational analysis showed that nced5 mutants reduced ABA level and decreased tolerance to salt and water stress and delayed leaf senescence. However, OsNCED5 overexpression increased ABA level, enhanced tolerance to the stresses, and accelerated leaf senescence. Transcript analysis showed that OsNCED5 regulated ABA-dependent abiotic stress and senescence-related gene expression. Additionally, ectopic expression of OsNCED5 tested in Arabidopsis thaliana altered plant size and leaf morphology and delayed seed germination and flowering time. Thus, OsNCED5 may regulate plant development and stress resistance through control of ABA biosynthesis. These findings contribute to our understanding of the molecular mechanisms by which NCED regulates plant development and responses to abiotic stress in different crop species.


Assuntos
Dioxigenases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Desidratação , Dioxigenases/genética , Oryza/genética , Oryza/fisiologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico , Água/metabolismo
8.
Planta ; 250(5): 1613-1620, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31388830

RESUMO

MAIN CONCLUSION: A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA9to GA20, but not GA4to GA1. Gibberellins (GAs) play essential roles in plant growth and development. Previous characterization of GA20- and GA3-oxidases yielded a large number of genetic elements that can interconvert different GAs. However, enzymes that catalyze the 13-hydroxylation step are rarely identified. Here, we report that the GA13-oxidase of Tripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase instead of reported cytochrome P450 oxygenases, among 376 differential proteins in comparative proteomics. Phylogenetic analysis showed that the enzyme resides in its own independent branch in the DOXC class. Unexpectedly, it specifically catalyzes the conversion of GA9 to GA20, but not GA4 to GA1. Contrary to the previous research, TwGA13ox transcriptional expression was upregulated ~ 146 times by exogenous application of methyl jasmonate (MeJA). RNAi targeting of TwGA13ox in T. wilfordii led to an 89.9% decrease of triptolide, a diterpenoid epoxide with extensive anti-inflammatory and anti-tumor properties. In subsequent MeJA supplementation experiments, triptolide production increased 13.4-times. TwGA13ox displayed root-specific expression. Our results provide a new GA13-oxidase from plants and elucidate the metabolic associations within the diterpenoid biosynthetic pathway (GAs, triptolide) at the genetic level.


Assuntos
Acetatos/farmacologia , Ciclopentanos/farmacologia , Dioxigenases/metabolismo , Regulação Enzimológica da Expressão Gênica , Giberelinas/metabolismo , Oxirredutases/metabolismo , Oxilipinas/farmacologia , Tripterygium/enzimologia , Vias Biossintéticas , Dioxigenases/genética , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oxirredutases/genética , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tripterygium/genética
9.
J Biotechnol ; 305: 11-17, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31430497

RESUMO

Fatty aldehydes are among the most important flavor and fragrance compounds. Most biotechnological production approaches make use of the one step conversion of fatty acids from renewable sources by the enzymes α-dioxygenase (αDox) or carboxylic acid reductase (CAR). Their reaction mechanisms and cofactor dependencies are very different. In contrast to heme-containing αDox which requires only oxygen as cosubstrate, CAR needs NADPH and ATP, which is a clear argument for the application of a whole cell catalyst. Therefore we compared fatty acid biotransformations with growing Escherichia coli cells expressing αDox or CAR to investigate their suitability for fatty aldehyde and also fatty alcohol production. Our results show the main product of fatty acid conversions with αDox-expressing cells to be the expected Cn-1 aldehyde. However, 14% of the products consist of the corresponding alcohol, but in addition, 17% of the products consist of further shortened aldehydes, alcohols and acids that result from the consecutive activity of αDox and a putative endogenous fatty aldehyde dehydrogenase activity in E. coli. Conversely, CAR-expressing cells produced only the unshortened fatty aldehyde and alcohol, whereby the latter surprisingly accounts for at least 80% of the products. The considerably higher extend of aldehyde reduction of CAR-expressing cells was shown to be causally connected to the CAR-mediated fatty acid conversion. Our study provides an overview about the applicability of αDox- or CAR-based whole cell catalysts and gives a detailed description of side products as well as suggestions for tailored strain engineering.


Assuntos
Dioxigenases/metabolismo , Escherichia coli/crescimento & desenvolvimento , Ácidos Graxos/biossíntese , Álcoois Graxos/metabolismo , Oxirredutases/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeídos , Catálise , Dioxigenases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Engenharia Genética , NADP/metabolismo , Oryza/enzimologia , Oryza/genética , Oxirredução , Oxirredutases/genética
10.
Oncol Rep ; 42(5): 2139-2148, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31436299

RESUMO

Curcumin is a natural antioxidant polyphenol, which decreases epithelial­mesenchymal transition (EMT) and cell migration in cervical cancer cells. However, the mechanism by which such a decrease occurs is unclear. It is well established that cervical cancer can be caused by high­risk human papillomavirus (HPV), which overexpresses E6 and E7 oncoproteins. Recent findings have suggested that viral oncoproteins regulate the expression of Pirin, which is an oxidative stress sensor involved in EMT and cell migration. Molecular markers associated with EMT, pirin and HPV were evaluated using reverse transcription­reverse quantitative PCR and western blotting. In addition, the migratory ability of cells was evaluated using a Transwell assay. In order to evaluate the role of Pirin in curcumin­mediated inhibition of EMT, SiHa cervical carcinoma cells, which contain two integrated copies of HPV16, were exposed to curcumin. Cell migration, and the expression levels of EMT biomarkers and the pirin protein, which is a product of the PIR gene, were subsequently evaluated. The results demonstrated a significant decrease in EMT following exposure to 20 µM curcumin for 72 h. This finding was supported by a decrease in the protein expression levels of N­cadherin, Vimentin and Slug. Furthermore, it was observed that PIR expression and Pirin protein levels were significantly decreased when SiHa cells were exposed to curcumin. Subsequently, to analyze the effects of Pirin on EMT, SiHa cells were transfected with a small interfering RNA (siRNA) to knockdown PIR. A significant increase in E­cadherin mRNA expression and a decrease in N­cadherin protein expression were observed. In addition, a similar decrease was observed when SiHa cells were exposed to both PIR siRNA and curcumin. Finally, a significant decrease in SiHa cell migration was observed in the presence of 20 µM curcumin compared with in the control group. These findings suggested that curcumin may decrease EMT, at least in part by a Pirin­dependent mechanism. Therefore, Pirin protein may be an important pharmacological target for cervical cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Dioxigenases/genética , Dioxigenases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
11.
Microb Cell Fact ; 18(1): 120, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277660

RESUMO

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.


Assuntos
Vias Biossintéticas , Carotenoides/biossíntese , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Crocus/enzimologia , Crocus/genética , Dioxigenases/genética , Escherichia coli/genética , Gardenia/enzimologia , Gardenia/genética , Genes de Plantas , Glicosiltransferases/genética , Proteínas de Plantas/genética
12.
Food Chem ; 299: 125089, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31319343

RESUMO

Synthesis of ß-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a ß-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by ß-ionone quantification via RP-HPLC. Two single-point mutants increased ß-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed ß-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of ß-ionone synthesized by metabolically engineered yeast.


Assuntos
Carotenoides/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Norisoprenoides/biossíntese , Engenharia de Proteínas , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética
13.
BMC Plant Biol ; 19(1): 317, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307384

RESUMO

BACKGROUND: Anthocyanins, which are colored pigments, have long been used as food and pharmaceutical ingredients due to their potential health benefits, but the intermediate signals through which environmental or developmental cues regulate anthocyanin biosynthesis remains poorly understood. Fleshy fruits have become a good system for studying the regulation of anthocyanin biosynthesis, and exploring the mechanism underlying pigment metabolism is valuable for controlling fruit ripening. RESULTS: The present study revealed that ABA accumulated during Lycium fruit ripening, and this accumulation was positively correlated with the anthocyanin contents and the LbNCED1 transcript levels. The application of exogenous ABA and of the ABA biosynthesis inhibitor fluridon increased and decreased the content of anthocyanins in Lycium fruit, respectively. This is the first report to show that ABA promotes the accumulation of anthocyanins in Lycium fruits. The variations in the anthocyanin content were consistent with the variations in the expression of the genes encoding the MYB-bHLH-WD40 transcription factor complex or anthocyanin biosynthesis-related enzymes. Virus-induced LbNCED1 gene silencing significantly slowed fruit coloration and decreased both anthocyanin and ABA accumulation during Lycium fruit ripening. An qRT-PCR analysis showed that LbNCED1 gene silencing clearly reduced the transcript levels of both structural and regulatory genes in the flavonoid biosynthetic pathway. CONCLUSIONS: Based on the results, a model of ABA-mediated development-dependent anthocyanin biosynthesis and fruit coloration during Lycium fruit maturation was proposed. In this model, the developmental cues transcriptionally activates LbNCED1 and thus enhances accumulation of the phytohormone ABA, and the accumulated ABA stimulates transcription of the MYB-bHLH-WD40 transcription factor complex to upregulate the expression of structural genes in the flavonoid biosynthetic pathway and thereby promoting anthocyanin production and fruit coloration. Our results provide a valuable strategy that could be used in practice to regulate the ripening and quality of fresh fruit in medicinal and edible plants by modifying the phytohormone ABA.


Assuntos
Ácido Abscísico/metabolismo , Antocianinas/biossíntese , Frutas/metabolismo , Lycium/metabolismo , Pigmentação , Reguladores de Crescimento de Planta/metabolismo , Dioxigenases/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Lycium/genética , Lycium/crescimento & desenvolvimento , Proteínas de Plantas/genética , Transdução de Sinais
14.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31308081

RESUMO

The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.


Assuntos
Hidrolases de Éster Carboxílico/genética , Dioxigenases/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium abscessus/genética , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Células A549 , Animais , Antibiose/genética , Caenorhabditis elegans/microbiologia , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Dioxigenases/metabolismo , Dioxigenases/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mycobacterium abscessus/enzimologia , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/genética , Piocianina/metabolismo , Quinolonas/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
15.
PLoS Genet ; 15(7): e1008252, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283754

RESUMO

The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Reparo do DNA , Replicação do DNA , Dioxigenases/genética , Oxirredutases N-Desmetilantes/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Dioxigenases/metabolismo , Masculino , Meiose , Mutação , Oxirredutases N-Desmetilantes/metabolismo , Análise de Sequência de RNA
16.
Mar Pollut Bull ; 142: 419-427, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31232319

RESUMO

This study investigated the network of polycyclic aromatic hydrocarbon (PAH) degraders in the Yangtze estuarine and coastal areas. Along the estuarine gradients, Proteobacteria and Bacteroidetes were the dominant bacterial phyla, and forty-six potential PAH degraders were identified. The abundance of genes encoding the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDα) of gram-negative bacteria ranged from 5.5 × 105 to 5.8 × 107 copies g-1, while that of gram-positive bacteria ranged from 1.3 × 105 to 2.0 × 107 copies g-1. The PAH-degraders could represent up to 0.2% of the total bacterial community and mainly respond to PAHs and Cu concentrations, which indicate anthropogenic activities. Salinity and pH showed negative regulating effects on the PAH-degrading potential and the tolerance of bacteria to pollutants. PAH degraders such as Novosphingobium and Mycobacterium exhibit heavy-metal tolerance and core roles in the network of PAH degraders. These outcomes have important implications for bioremediation.


Assuntos
Bactérias/metabolismo , Sedimentos Geológicos/microbiologia , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Microbiologia da Água , Poluentes Químicos da Água/metabolismo , Bactérias/genética , Biodegradação Ambiental , Biodiversidade , China , Dioxigenases/genética , Dioxigenases/metabolismo , Estuários , Sedimentos Geológicos/análise , Sedimentos Geológicos/química , Concentração de Íons de Hidrogênio , Metais/análise , Metais/toxicidade , Mycobacterium/genética , Mycobacterium/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/análise , Proteobactérias/genética , Proteobactérias/metabolismo , Água do Mar/química , Água do Mar/microbiologia , Poluentes Químicos da Água/análise
17.
J Agric Food Chem ; 67(26): 7399-7409, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244203

RESUMO

Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice ( Oryza sativa) cultivar "Ilmi" OsFLS includes highly conserved 2-ODD-specific motifs and FLS-specific regions. Recombinant OsFLS exhibited both FLS and flavanone 3ß-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco ( Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3- O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2-ODD enzyme and functions in flavonol production in planta.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Oryza/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antocianinas/biossíntese , Cor , Flavonóis/biossíntese , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oryza/genética , Tabaco/genética , Tabaco/metabolismo
18.
Yonsei Med J ; 60(7): 659-666, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31250580

RESUMO

PURPOSE: To investigate associations for polymorphisms in ß-carotene 9',10'-oxygenase (BCO2, rs10431036 and rs11214109), proprotein convertase subtilisin kexin type 9 (PCSK9, rs11583680), and tribbles pseudokinase 1 (TRIB1, rs17321515 and rs2954029), as well as lifestyle factors, with ischemic stroke (IS). MATERIALS AND METHODS: This nested case-control study included 161 patients with IS and 483 matched control individuals. We collected medical reports, lifestyle details, and blood samples from individuals and used the PCR-ligase detection reaction method to genotype single nucleotide polymorphisms (SNPs). RESULTS: The GA+AA genotype of rs10431036 (p<0.001) and rs17321515 (p=0.003), the CT+TT genotype of rs11214109 (p=0.005), and the TA+AA genotype of rs2954029 (p=0.006) in dominant models increased the risk of IS. In additive models, the GG genotype of rs17321515 (p=0.005) and the TT genotype of rs2954029 (p=0.008) increased the risk of IS. Adequate intake of fruits/vegetables reduced the risk of IS (p=0.005). Although there was no interaction between genes and fruits/vegetables, people with inadequate intake of fruits/vegetables who carried a risk genotype had a higher risk of IS than those only having inadequate fruits/vegetables intake or those only carrying a risk genotype. Also, the haplotypes AC, AT, and GT (comprising rs10431036 and rs11214109) and GT (comprising rs2954029 and rs17321515) were found to be associated with an increased risk of IS (p<0.05). CONCLUSION: Polymorphisms in BCO2 and TRIB1 and fruits/vegetables intake were associated with IS. These results provide the theoretical basis for gene screening to prevent chronic cerebrovascular diseases.


Assuntos
Isquemia Encefálica/complicações , Dioxigenases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estilo de Vida , Polimorfismo de Nucleotídeo Único , Pró-Proteína Convertase 9/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Acidente Vascular Cerebral/genética , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Humanos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Acidente Vascular Cerebral/complicações
19.
Genetics ; 212(4): 1445-1452, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31160321

RESUMO

Domestic animals are adapted to conditions vastly different from those of their wild ancestors, and this is particularly true for their diets. The most numerous of all domestic species, the chicken, originated from the Red Junglefowl (RJF), a native of subtropical forests in Southeast Asia. Surprisingly however, in domestic chicken breeds, a common haplotype of the ß-carotene oxygenase 2 (BCO2) gene, which is involved in carotenoid metabolism, is introgressed from a related species, the Gray Junglefowl, and has been under strong selective pressure during domestication. This suggests that a hybridization event may have conferred a fitness advantage on chickens carrying the derived allele. To investigate the possible biological function of the introgressed BCO2 allele in chicken, we introgressed the ancestral BCO2 allele into domestic White Leghorn chickens. We measured gene expression as well as carotenoid accumulation in skin and eggs of chickens carrying either the ancestral or the derived BCO2 allele. The derived haplotype was associated with down-regulation of BCO2 in skin, muscle, and adipose tissue, but not in liver or duodenum, indicating that carotenoid accumulation occurred in the tissues with reduced gene expression. Most importantly, we found that hens with the derived BCO2 genotype were capable of allocating stored carotenoids to their eggs, suggesting a functional benefit through buffering any shortage in the diet during egg production. Nevertheless, it is of interest that loss of function mutations in BCO2 gene are prevalent in other domesticates including cows, rabbits, and sheep, and, given the importance of carotenoids in development, reproduction, and immunity, it is possible that derived BCO2 alleles may provide a general mechanism in multiple domestic species to deal with higher demand for carotenoids in an environment with carotenoid shortage in the diet.


Assuntos
Alelos , Proteínas Aviárias/genética , Carotenoides/metabolismo , Galinhas/genética , Dioxigenases/genética , Aptidão Genética , Animais , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Dieta , Dioxigenases/metabolismo , Domesticação , Duodeno/metabolismo , Evolução Molecular , Hibridização Genética , Fígado/metabolismo , Músculo Esquelético/metabolismo
20.
Nucleic Acids Res ; 47(14): 7333-7347, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31165872

RESUMO

Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.


Assuntos
Sistemas CRISPR-Cas , Dioxigenases/genética , Infecções por HIV/genética , HIV-1/genética , Histona Desmetilases/genética , Proteínas Nucleares/genética , Latência Viral/genética , Aminopiridinas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , Células Cultivadas , Desmetilação/efeitos dos fármacos , Dioxigenases/antagonistas & inibidores , Dioxigenases/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Hidrazonas/farmacologia , Metilação/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Interferência de RNA
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