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1.
J Agric Food Chem ; 68(1): 128-137, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31825613

RESUMO

Salmonellosis is a world-wide epidemic, and n-3 long chain polyunsaturated fatty acids (LCPUFAs) possess various health benefits. This study is aimed to investigate the preventive effects of n-3 LCPUFAs against Salmonella infection. By pretreatment with n-3 LCPUFAs, but not n-6 LCPUFAs, the survival rate of the infected mice was increased. Further studies showed that n-3 LCPUFAs significantly increased the fecal contents of short-chain fatty acids (SCFAs). The cytokine expression in the liver and production in serum were both modulated by n-3 LCPUFAs into an anti-inflammatory profile against infection. Moreover, the changes in gut microbiota by n-3 LCPUFAs favored the host against pathogens, closely related to the modified SCFA production and immune responses. In conclusion, n-3 LCPUFAs prevented Salmonella infection through multiple mechanisms, especially by the interaction with gut microbiota and host immunology. Our results suggested great perspectives for n-3 LCPUFAs and their related products to control the prevalence of Salmonella, a most predominant food-borne pathogen.


Assuntos
Suplementos Nutricionais/análise , Ácidos Graxos Ômega-3/administração & dosagem , Infecções por Salmonella/prevenção & controle , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Modelos Animais de Doenças , Ácidos Graxos Voláteis/metabolismo , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Salmonella/efeitos dos fármacos , Salmonella/fisiologia , Infecções por Salmonella/microbiologia
2.
mSystems ; 4(5)2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594827

RESUMO

Gut microbiota play important roles in host metabolism, especially in diabetes. However, why different diets lead to similar diabetic states despite being associated with different microbiota is not clear. Mice were fed two high-energy diets (HED) with the same energy density but different fat-to-sugar ratios to determine the associations between the microbiota and early-stage metabolic syndrome. The two diets resulted in different microbiota but similar diabetic states. Interestingly, the microbial gene profiles were not significantly different, and many common metabolites were identified, including l-aspartic acid, cholestan-3-ol (5ß, 3α), and campesterol, which have been associated with lipogenesis and inflammation. Our study suggests that different metabolic-syndrome-inducing diets may result in different microbiota but similar microbiomes and metabolomes. This suggests that the metagenome and metabolome are crucial for the prognosis and pathogenesis of obesity and metabolic syndrome.IMPORTANCE Various types of diet can lead to type 2 diabetes. The gut microbiota in type 2 diabetic patients are also different. So, two questions arise: whether there are any commonalities between gut microbiota induced by different pro-obese diets and whether these commonalities lead to disease. Here we found that high-energy diets with two different fat-to-sugar ratios can both cause obesity and prediabetes but enrich different gut microbiota. Still, these different gut microbiota have similar genetic and metabolite compositions. The microbial metabolites in common between the diets modulate lipid accumulation and macrophage inflammation in vivo and in vitro This work suggests that studies that only use 16S rRNA amplicon sequencing to determine how the microbes respond to diet and associate with diabetic state are missing vital information.

3.
Int J Mol Sci ; 20(7)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30935072

RESUMO

Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from Mortierella alpina and oRiFADS17 from Rhizophagus irregularis, which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in Saccharomyces cerevisiae INVSc1 indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.


Assuntos
Ácidos Graxos Dessaturases/química , Proteínas Fúngicas/química , Glomeromycota/enzimologia , Simulação de Acoplamento Molecular , Mortierella/enzimologia , Ácido Araquidônico/química , Sítios de Ligação , Ácidos Graxos Dessaturases/metabolismo , Proteínas Fúngicas/metabolismo , Ácido Linoleico/metabolismo , Ligação Proteica , Especificidade por Substrato
4.
Front Microbiol ; 10: 299, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30842764

RESUMO

Pathogen-induced infectious diseases pose great threats to public health. Accordingly, many studies have investigated effective strategies targeting pathogenic infections. We previously reported the preventive effects of Lactobacillus plantarum ZS2058 (ZS2058) and L. rhamnosus GG (LGG) against Salmonella spp. in a murine model. Here, we compared the mechanisms underlying the preventive effects of these Lactobacillus strains in vivo. Notably, reduced C-reactive protein levels were observed with both ZS2058 and LGG, which suggests abrogated anti-infection and inflammatory responses. ZS2058 more efficiently reduced the pathogenicity of Salmonella by increasing the level of propionic acid in feces and production of mucin 2 in the mouse colon and activity through the interleukin (IL)-23/IL-22 and IL-23/IL-17 pathways. Meanwhile, LGG more strongly alleviated gut inflammation, as indicated by changes in the levels of tissue necrosis factor (TNF)-α, IL-10 and myeloperoxidase (MPO) in infected mice. Moreover, both ZS2058 and LGG restored the levels of interferon (INF)-γ, a cytokine suppressed by Salmonella, albeit through different pathways. Our results demonstrate that ZS2058 and LGG prevent Salmonella infection via different mechanisms.

5.
J Microbiol Methods ; 158: 80-85, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30708087

RESUMO

Screening oleaginous microorganisms capable of accumulating considerable lipids is essential for industrial lipid production. Here we demonstrated forty-seven filamentous fungal isolates were obtained from eight soil samples using a new screening strategy with both triphenyltetrazolium chloride (TTC), a redox indicator used for testing oil presence, and cerulenin, an inhibitor of fatty acid synthase (FAS), supplemented in screening medium. Among these fungal isolates, nineteen have high lipid content (>20% dry biomass weight) and were affiliated with the genus Mortierella by morphology identification and phylogenetic analysis based on ITS gene sequences. Notably, one strain designated as SL-4 reached 32% of its biomass weight as lipid, displaying the highest potential. Two candidates with high lipid content as well as biomass production were selected for exploring the effect of different carbon and nitrogen sources on morphology, biomass and lipid accumulation.

6.
Appl Microbiol Biotechnol ; 102(22): 9679-9689, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30255230

RESUMO

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Mortierella/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Análise Mutacional de DNA , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/química , Simulação de Acoplamento Molecular , Mortierella/química , Mortierella/genética , Mutação de Sentido Incorreto , Alinhamento de Sequência , Especificidade por Substrato
7.
Front Microbiol ; 9: 1878, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154780

RESUMO

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have important therapeutic and nutritional benefits in humans. In the biosynthesis pathways of these LC-PUFAs, omega-3 desaturase plays a critical role. In this study, we report a new omega-3 desaturase (PPD17) from Phytophthora parasitica. This desaturase shares high similarities with the known omega-3 desaturases and was expressed in Saccharomyces cerevisiae for the activity and substrate specificity research. The desaturase has a wide omega-6 fatty acid substrate, containing both 18C and 20C fatty acids, and exhibits a strong activity of delta-17 desaturase but a weak activity of delta-15 desaturase. The new desaturase converted the omega-6 arachidonic acid (AA, C20:4) to EPA (an omega-3 LC-PUFA, C20:5) with a substrate conversion rate of 70%. To obtain a high EPA-producing strain, we transformed PPD17 into Mortierella alpina, an AA-producing filamentous fungus. The EPA content of the total fatty acids in reconstruction strains reached 31.5% and was followed by the fermentation optimization of the EPA yield of up to 1.9 g/L. This research characterized a new omega-3 desaturase and provides a possibility of industrially producing EPA using M. alpina.

8.
Food Funct ; 9(7): 3673-3682, 2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-29956713

RESUMO

Salmonella is a common food-borne pathogen; since lactobacilli show great potential for protecting against Salmonella infections, they are used as dietary supplements in functional foods. The aim of this study is to investigate the strain-specific properties and the involved mechanisms of action of Lactobacillus plantarum towards prevention of Salmonella infection. Mice were pretreated with mixed strains or single strain of Lactobacillus plantarum for 10 d prior to infection with Salmonella typhimurium SL1344, and the survival rates showed that lactobacilli exhibited strain-specific properties for preventing Salmonella infection. Then, in vitro and in vivo studies were carried out to investigate the involved mechanism of the strain-specific properties. The results showed that different Lactobacillus plantarum strains had different effects on inhibiting Salmonella growth, thus preventing adhesion to and invasion of epithelial cells by pathogens and enhancing immune responses. The present study demonstrated strain-specific properties of probiotics to prevent Salmonella infection and elucidated their underlying mechanisms.


Assuntos
Lactobacillus plantarum/fisiologia , Probióticos/administração & dosagem , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/efeitos dos fármacos , Animais , Aderência Bacteriana/efeitos dos fármacos , Células Epiteliais/microbiologia , Feminino , Humanos , Lactobacillus plantarum/classificação , Camundongos , Probióticos/classificação , Infecções por Salmonella/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologia , Especificidade da Espécie
9.
Food Funct ; 9(5): 2787-2795, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29691525

RESUMO

Probiotics are now prevalent world-wide, as functional food supplements with many benefits for humans and animals, such as protective effects against pathogenic infection. We showed that oral supplementation of Lactobacillus pentosus AT6 (AT6) decreased the mortality rate of mice with Salmonella infection. A series of experiments showed that the protective effects of AT6 on mice involved multiple mechanisms, including (1) the inhibition of Salmonella Typhimurium growth by AT6 or its cell-free culture supernatants (CFCSs); (2) the reduction of the bacterial loads of Salmonella Typhimurium in intestinal contents and internal organs, such as the liver and spleen; (3) the inhibition of adhesion and invasion of Salmonella Typhimurium into intestinal epithelial cells; and (4) the regulation of host immunities by modifying the production of a chain of cytokines. In conclusion, AT6 inhibited Salmonella infection via multiple mechanisms and therefore has great potential for the development of functional foods with anti-Salmonella activities.


Assuntos
Lactobacillus pentosus/fisiologia , Probióticos/administração & dosagem , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Salmonella typhimurium/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Baço/microbiologia
10.
Lipids Health Dis ; 16(1): 136, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28697730

RESUMO

BACKGROUND: α-linolenic acid (ALA) is an n-3 polyunsaturated fatty acid (PUFA) and the substrate for long-chain n-3 PUFAs. The beneficial effects of ALA on chronic diseases are still in dispute, unlike those of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). METHODS: The primary objective of this investigation was to evaluate the efficiency of ALA uptake from a vegetable oil source and its subsequent conversion to n-3 long-chain PUFAs (LCPUFAs) in the tissues of growing mice, and to investigate its protective role in a prostate cancer animal model. We carried out the investigation in prostate-specific Pten-knockout mice with specified low-ALA (L-ALA, 2.5%) and high-ALA (H-ALA, 7.5%) diets. Total fatty acids in blood, liver, epididymal fat pad, prostate were detected and prostate weight were adjusted for body weight (mg/25 g). RESULTS: We found that dietary ALA triggered significant increases in ALA, EPA, docosapentaenoic acid (DPA) and DHA levels and a significant decrease in arachidonic acid levels during the mice's growth stage. A dose-dependent effect was observed for ALA, EPA and DPA, but not DHA. Furthermore, the average prostate weights in the L-ALA and H-ALA groups were lower than those in the control and n-6 groups, and similar to those in the EPA and n-3 groups. CONCLUSIONS: Our data suggest that dietary supplementation with ALA is an efficient means of improving n-3 LCPUFAs in vivo, and it has a biologically effective role to play in prostate cancer, similar to that of fish oils.


Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-3/metabolismo , Neoplasias da Próstata/sangue , Neoplasias da Próstata/metabolismo , Ácido alfa-Linoleico/sangue , Ácido alfa-Linoleico/metabolismo , Animais , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/sangue , Ácido Eicosapentaenoico/metabolismo , Masculino , Camundongos , Camundongos Knockout
11.
Food Funct ; 8(8): 2847-2856, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28726934

RESUMO

Numerous medicinal plants have been reported to prevent various chronic diseases. In this study, we screened a new FASN inhibitor-alcohol extract of clove (AEC) using a fast microplate method developed in our laboratory. The major components of AEC were: eugenol (42.27%), acetyl eugenol (29.12%), caryophyllene (15.40%), and humulene (3.22%). Fatty acid synthase (FASN) is a key enzyme for de novo lipogenesis, and it has been suggested as a potential therapeutic target in cancer and obesity. We have tested the ability of AEC to inhibit FASN in mammalian cells and tissues. Furthermore, we found that AEC as a FASN inhibitor could inhibit the S-phase DNA replication of HepG2 cells and adipocyte differentiation of OP9 cells. AEC also limited the development of high fat diet (HFD) induced obesity. AEC supplementation significantly reduced body weight and abdominal adipose tissue weight, lowered lipid accumulation in the liver and epididymal adipose tissue compared with the HFD control group. The serum lipid profiles showed that AEC could regulate the content of total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C). Collectively, our data suggest that FASN inhibitor AEC is a potential therapeutic agent for obesity.


Assuntos
Fármacos Antiobesidade/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Ácido Graxo Sintases/antagonistas & inibidores , Obesidade/prevenção & controle , Extratos Vegetais/administração & dosagem , Syzygium/química , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Animais , Modelos Animais de Doenças , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Humanos , Lipoproteínas LDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Triglicerídeos/metabolismo
12.
J Agric Food Chem ; 65(24): 5074-5082, 2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28557429

RESUMO

Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.


Assuntos
Proteínas Fúngicas/metabolismo , Lipídeos/biossíntese , Mucor/metabolismo , Proteoma/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mucor/química , Mucor/classificação , Mucor/genética , Proteoma/química , Proteoma/genética , Ácido gama-Linolênico/análise , Ácido gama-Linolênico/metabolismo
13.
Sci Rep ; 8: 45840, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28374781

RESUMO

As a long-standing biomedical model, rats have been frequently used in studies exploring the correlations between gastrointestinal (GI) bacterial biota and diseases. In the present study, luminal and mucosal samples taken along the longitudinal axis of the rat digestive tract were subjected to 16S rRNA gene sequencing-based analysis to determine the baseline microbial composition. Results showed that the community diversity increased from the upper to lower GI segments and that the stratification of microbial communities as well as shift of microbial metabolites were driven by biogeographic location. A greater proportion of lactate-producing bacteria (such as Lactobacillus, Turicibacter and Streptococcus) were found in the stomach and small intestine, while anaerobic Lachnospiraceae and Ruminococcaceae, fermenting carbohydrates and plant aromatic compounds, constituted the bulk of the large-intestinal core microbiota where topologically distinct co-occurrence networks were constructed for the adjacent luminal and mucosal compartments. When comparing the GI microbiota from different hosts, we found that the rat microbial biogeography might represent a new reference, distinct from other murine animals. Our study provides the first comprehensive characterization of the rat GI microbiota landscape for the research community, laying the foundation for better understanding and predicting the disease-related alterations in microbial communities.


Assuntos
Bactérias/genética , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/microbiologia , RNA Ribossômico 16S/genética , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Firmicutes/genética , Firmicutes/isolamento & purificação , Intestino Delgado/microbiologia , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Filogenia , Ratos , Análise de Sequência de DNA , Streptococcus
14.
J Agric Food Chem ; 65(17): 3474-3480, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28391699

RESUMO

Artocarpus heterophyllus is an evergreen tree distributed in tropical regions, and its fruit (jackfruit) is well-known as the world's largest tree-borne fruit. Although A. heterophyllus has been widely used in folk medicines against inflammation, its potential in cancer chemoprevention remains unclear. Herein we identified artocarpin from A. heterophyllus as a promising colorectal cancer chemopreventive agent by targeting Akt kinase. Phenotypically, artocarpin exhibited selective cytotoxicity against human colon cancer cells. Artocarpin impaired the anchorage-independent growth capability, suppressed colon cancer cell growth, and induced a G1 phase cell cycle arrest which was followed by apoptotic as well as autophagic cell death. Mechanistic studies revealed that artocarpin directly targeted Akt 1 and 2 kinase activity evidenced by in vitro kinase assay, ex vivo binding assay as well as Akt downstream cellular signal transduction. Importantly, oral administration of artocarpin attenuated colitis-associated colorectal tumorigenesis in mice. Taken together, artocarpin, a bioactive component of A. heterophyllus, might merit investigation as a potential colorectal cancer chemopreventive agent.


Assuntos
Artocarpus/química , Neoplasias Colorretais/prevenção & controle , Lectinas de Ligação a Manose/administração & dosagem , Compostos Fitoquímicos/administração & dosagem , Extratos Vegetais/administração & dosagem , Lectinas de Plantas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/fisiopatologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
15.
Lipids Health Dis ; 16(1): 10, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28095863

RESUMO

BACKGROUND: Dietary polyunsaturated fatty acids (PUFAs), especially n-3 PUFAs, are important for human health. The intestinal tract, a location that is heavily colonized by microorganisms, is the main organ for absorbing fatty acids. METHODS: The purpose of this study was to analyze the effects of dietary n-3 and n-6 PUFAs on the distribution of different types of fatty acids and their bioavailability along the gut. Mice were fed for a week with experimental diets containing high n-3 or high n-6 fatty acid levels. Blood was collected at different time points, and after 7 days the mice were euthanized and their digestive tract was divided into 17 segments for fatty acids analyses. RESULTS: We found that supplementing n-3 fatty acids significantly changed the ratio of n-6/n-3 PUFAs, increased the bioavailability of n-3 PUFAs, and altered fatty acid distribution. In addition, in the n-3 diet group, the absorption of saturated fatty acids (SFAs) along the gut was found to be inhibited, which was confirmed by feeding the mice with a diet containing deuterium-labeled palmitic acid and stearic acid. CONCLUSION: These results show that a diet rich in n-3 PUFAs can significantly modify the distribution and bioavailability of fatty acids, and particularly, may block the absorption of SFAs in the mouse gastrointestinal (GI) tract.


Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3 , Ácidos Graxos/metabolismo , Trato Gastrointestinal/metabolismo , Animais , Disponibilidade Biológica , Masculino , Camundongos
16.
Artigo em Inglês | MEDLINE | ID: mdl-29404322

RESUMO

In the industrial oleaginous fungus Mortierella alpina, the arachidonic acid (AA; C20:4; ω-6) fraction can reach 50% of the total fatty acids (TFAs) in vivo. However, the eicosapentaenoic acid (EPA; C20:5; ω-3) fraction is less than 3% when this fungus is cultivated at a low temperature (12°C). Omega-3 fatty acid desaturase is a key enzyme in ω-3 long-chain polyunsaturated fatty acids biosynthesis pathways. To enhance EPA production, we transformed the ω-3 fatty acid desaturase (PaD17), which exhibits strong Δ-17 desaturase activity, into M. alpina, thus increasing the AA to EPA conversion rate to 49.8%. This PaD17-harboring M. alpina reconstruction strain produced 617 mg L-1 of EPA at room temperature in broth medium, this yield was increased to 1.73 g L-1 after culture medium optimization (i.e., about threefold higher than that under original culture conditions), with concomitant respective increases in dry cell weight and TFA content to 16.55 and 6.46 g L-1. These findings suggest a new platform for the future industrial production of EPA.

17.
Microbiology ; 162(9): 1544-1553, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27488762

RESUMO

Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH4), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH4salvage pathway in a fungus.


Assuntos
Biopterina/análogos & derivados , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Mortierella/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Biopterina/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Proteínas Fúngicas/genética , Mortierella/genética , Mortierella/metabolismo , Tetra-Hidrofolato Desidrogenase/genética
18.
Microb Cell Fact ; 15(1): 117, 2016 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27364006

RESUMO

BACKGROUND: Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels. RESULT: To enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate. CONCLUSION: An ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.


Assuntos
Ácido Eicosapentaenoico/biossíntese , Proteínas Fúngicas/metabolismo , Linoleoil-CoA Desaturase/metabolismo , Mortierella/enzimologia , Ácido alfa-Linoleico/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cinética , Linoleoil-CoA Desaturase/química , Linoleoil-CoA Desaturase/genética , Mortierella/química , Mortierella/genética , Mortierella/metabolismo
19.
Biotechnol Lett ; 38(10): 1761-8, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27395065

RESUMO

OBJECTIVE: To clarify the molecular mechanism of GDP-L-fucose biosynthesis in Mortierella alpina. RESULTS: Analysis of the M. alpina genome suggests that there were two isofunctional GDP-D-mannose-4,6-dehydratase genes (GMD1 and GMD2) that have never been found in a microorganism before. GMD2 was expressed heterologously in Escherichia coli and purified to homogeneity. The addition of exogenous NAD(+) or NADP(+) was not essential for GMD2 activity. GMD2 may have considerable importance for GDP-L-fucose biosynthesis under nitrogen starvation. The transcriptional regulation of GMD1 may be more susceptible to GDP and GTP than that of GMD2. Significant changes were observed in the concentration of GDP-L-fucose (30 and 36 % inhibition respectively) and total fatty acids (18 and 12 % inhibition respectively) in M. alpina grown on GMD inhibitors medium, which suggests that GDP-L-fucose is functionally significant in lipid metabolism. CONCLUSIONS: This is the first time that an isofunctional GDP-D-mannose-4,6-dehydratase has been characterized in a microorganism.


Assuntos
Clonagem Molecular/métodos , Guanosina Difosfato Fucose/biossíntese , Hidroliases/genética , Hidroliases/metabolismo , Mortierella/enzimologia , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Hidroliases/isolamento & purificação , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Metabolismo dos Lipídeos , Mortierella/genética , Nitrogênio/metabolismo
20.
Glycobiology ; 26(8): 880-887, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26957583

RESUMO

GDP-l-fucose functions as a biological donor for fucosyltransferases, which are required for the catalysis of l-fucose to various acceptor molecules including oligosaccharides, glycoproteins and glycolipids. Mortierella alpina is one of the highest lipid-producing fungi and can biosynthesis GDP-l-fucose in the de novo pathway. Analysis of the M. alpina genome suggests that there is a gene encoding l-fucokinase (FUK) for the conversion of fucose to l-fucose-1-phosphate in the GDP-l-fucose salvage pathway, which has never been found in fungi before. This gene was characterized to explore its role in GDP-l-fucose synthesis. The yield of GDP-l-fucose is relatively higher in lipid accumulation phase (0.096 mg per g cell) than that in cell multiplication phase (0.074 mg per g cell) of M. alpina Additionally, the transcript level of FUK is up regulated by nitrogen exhaustion when M. alpina starts to accumulate lipid, highlights the functional significance of FUK in the GDP-l-fucose biosynthesis in M. alpina Gene encoding FUK was expressed heterologously in Escherichia coli and the resulting protein was purified to homogeneity. The product of FUK reaction was analyzed by liquid chromatography and mass spectrometry. Kinetic parameters and other properties of FUK were investigated. Comparative analyses between the FUK protein and other homologous proteins were performed. To our knowledge, this study is the first to report a comprehensive characterization of FUK in a fungus. Mortierella alpina could be used as an alternative source for the production of GDP-l-fucose.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Guanosina Difosfato Fucose/biossíntese , Mortierella/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fucose/metabolismo , Proteínas Fúngicas/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Redes e Vias Metabólicas/genética , Mortierella/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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