RESUMEN
The biogenesis of peroxisomes in relation to the trafficking of proteins to peroxisomes has been extensively examined. However, the supply of phospholipids, which is needed to generate peroxisomal membranes in mammals, remains unclear. Therefore, we herein investigated metabolic alterations induced by clofibric acid, a peroxisome proliferator, in the synthesis of phospholipids, particularly phosphatidylethanolamine (PE) molecular species, and their relationship with the biogenesis of peroxisomal membranes. The subcutaneous administration of clofibric acid to rats at a relatively low dose (130 mg/kg) once a day time-dependently and gradually increased the integrated perimeter of peroxisomes per 100 µm2 hepatocyte cytoplasm (PA). A strong correlation was observed between the content (µmol/mg DNA) of PE containing arachidonic acid (20:4) and PA (r2 = 0.9168). Moreover, the content of PE containing octadecenoic acid (18:1) positively correlated with PA (r2 = 0.8094). The treatment with clofibric acid markedly accelerated the formation of 16:0-20:4 PE by increasing the production of 20:4 and the activity of acyl chain remodeling of pre-existing PE molecular species. Increases in the acyl chain remodeling of PE by clofibric acid were mainly linked to the up-regulated expression of the Lpcat3 gene. On the other hand, clofibric acid markedly increased the formation of palmitic acid (16:0)-18:1 PE through de novo synthesis. These results suggest that the enhanced formation of particular PE molecular species is related to increases in the mass of peroxisomal membranes in peroxisome proliferation in the liver.
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Ácido Araquidónico/biosíntesis , Ácido Araquidónico/química , Ácido Clofíbrico/farmacología , Membranas Intracelulares/efectos de los fármacos , Hígado/citología , Peroxisomas/efectos de los fármacos , Fosfatidiletanolaminas/química , Animales , Membranas Intracelulares/metabolismo , Masculino , Peroxisomas/metabolismo , Ratas , Ratas WistarRESUMEN
Arachidonic acid (AA), a major long-chain omega-6 polyunsaturated fatty acid, is associated with ischemic heart disease (IHD) and stroke. We assessed bi-directional associations of AA synthesis reflected by plasma phospholipid AA with CVD risk factors, and identified mediators of associations of AA with IHD and stroke using Mendelian randomization (MR). We used two-sample MR to assess bi-directional associations of AA synthesis with lipids, blood pressure, adiposity, and markers of inflammation and coagulation. We used multivariable MR to assess mediators of associations of AA with IHD and stroke. Genetically predicted AA (% of total fatty acids increase) was positively associated with apolipoprotein B (ApoB, 0.022 standard deviations (SD), 95% confidence interval (CI) 0.010, 0.034), high-density (0.030 SD, 95% CI 0.012, 0.049) and low-density lipoprotein cholesterol (LDL-C, 0.016 SD, 95% CI 0.004, 0.027) and lower triglycerides (-0.031 SD, 95% CI -0.049, -0.012) but not with other traits. Genetically predicted these traits gave no association with AA. The association of AA with IHD was attenuated adjusting for ApoB or LDL-C. Genetically predicted AA was associated with lipids but not other traits. Given ApoB is thought to be the key lipid in IHD, the association of AA with IHD is likely mediated by ApoB.
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Ácido Araquidónico/biosíntesis , Ácido Araquidónico/sangre , Factores de Riesgo de Enfermedad Cardiaca , Análisis de la Aleatorización Mendeliana/métodos , Isquemia Miocárdica/sangre , Accidente Cerebrovascular/sangre , Femenino , Finlandia , Humanos , Masculino , Reino UnidoRESUMEN
ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources.
Asunto(s)
Ácido Eicosapentaenoico/biosíntesis , Ácido Graso Desaturasas/genética , Ácidos Grasos Insaturados/biosíntesis , Mortierella/genética , Oomicetos/genética , Pythium/genética , Ácido Araquidónico/biosíntesis , Clonación Molecular , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/clasificación , Expresión Génica , Biblioteca de Genes , Ingeniería Metabólica/métodos , Mortierella/enzimología , Oomicetos/clasificación , Oomicetos/enzimología , Filogenia , Plásmidos/química , Plásmidos/metabolismo , Pythium/clasificación , Pythium/enzimología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Transformación Genética , TransgenesRESUMEN
Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.
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Ácido Araquidónico/biosíntesis , Ácido Araquidónico/genética , Fermentación , Edición Génica , Mortierella/genética , Mortierella/metabolismo , Reactores Biológicos , Vías Biosintéticas/genética , Medios de Cultivo , Humanos , Ingeniería Metabólica/métodos , Oxígeno/metabolismoRESUMEN
AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.
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Ácido Araquidónico/biosíntesis , Mortierella/metabolismo , Eliminación de Residuos/métodos , Solanum tuberosum , Ácido Araquidónico/economía , Biomasa , Carbono/metabolismo , Medios de Cultivo/economía , Medios de Cultivo/metabolismo , Fermentación , Lípidos/biosíntesis , Lípidos/economía , Mortierella/crecimiento & desarrollo , Nitrógeno/metabolismo , Solanum tuberosum/químicaRESUMEN
BACKGROUND AND OBJECTIVE: COVID-19 is a highly contagious viral disease. In this study, we tried to define and discuss all the findings on the potential association between arachidonic acid (AA) pathway and COVID-19 pathophysiology. METHODS: A literature search across PubMed, Scopus, Embase, and Cochrane database was conducted. A total of 25 studies were identified. RESULTS: The data elucidated that COX-2 and prostaglandins (PGs), particularly PGE2, have pro-inflammatory action in COVID-19 pathophysiology. Arachidonic acid can act as endogenous antiviral compound. A deficiency in AA can make humans more susceptible to COVID-19. Targeting these pro-inflammatory mediators may help in decreasing the mortality and morbidity rate in COVID-19 patients. CONCLUSIONS: PGE2 levels and other PGs levels should be measured in patients with COVID-19. Lowering the PGE2 levels through inhibition of human microsomal prostaglandin E synthase-1 (mPGES-1) can enhance the host immune response against COVID-19. In addition, the hybrid compounds, such as COX-2 inhibitors/TP antagonists, can be an innovative treatment to control the overall balance between AA mediators in patients with COVID-19.
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Ácido Araquidónico/biosíntesis , Infecciones por Coronavirus/fisiopatología , Ciclooxigenasa 2/biosíntesis , Inflamación/metabolismo , Neumonía Viral/fisiopatología , Prostaglandina-E Sintasas/biosíntesis , Antiinflamatorios no Esteroideos/farmacología , Betacoronavirus , COVID-19 , Ciclooxigenasa 2/sangre , Humanos , Pandemias , Fosfolipasas A2/biosíntesis , Prostaglandina-E Sintasas/sangre , Prostaglandinas/biosíntesis , Prostaglandinas/sangre , Proteína-Lisina 6-Oxidasa/biosíntesis , SARS-CoV-2 , Factores SexualesRESUMEN
Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.
Asunto(s)
Ácido Graso Desaturasas/genética , Ingeniería Genética/métodos , Mortierella/genética , Mortierella/metabolismo , Regiones Promotoras Genéticas/genética , Ácido Araquidónico/biosíntesis , Celulosa 1,4-beta-Celobiosidasa/genética , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/biosíntesis , Ácido Eicosapentaenoico/metabolismo , Ácidos Grasos Insaturados/metabolismo , Expresión Génica , Genes Reporteros/genética , Mortierella/crecimiento & desarrolloRESUMEN
The fungi in order Mortierellales are attractive producers for long-chain polyunsaturated fatty acids (PUFAs). Here, the genome sequencing and assembly of a novel strain of Mortierella sp. BCC40632 were done, yielding 65 contigs spanning of 49,964,116 total bases with predicted 12,149 protein-coding genes. We focused on the acetyl-CoA in relevant to its derived metabolic pathways for biosynthesis of macromolecules with biological functions, including PUFAs, eicosanoids and carotenoids. By comparative genome analysis between Mortierellales and Mucorales, the signature genetic characteristics of the arachidonic acid-producing strains, including Δ5-desaturase and GLELO-like elongase, were also identified in the strain BCC40632. Remarkably, this fungal strain contained only n-6 pathway of PUFA biosynthesis due to the absence of Δ15-desaturase or ω3-desaturase gene in contrast to other Mortierella species. Four putative enzyme sequences in the eicosanoid biosynthetic pathways were identified in the strain BCC40632 and others Mortierellale fungi, but were not detected in the Mucorales. Another unique metabolic trait of the Mortierellales was the inability in carotenoid synthesis as a result of the lack of phytoene synthase and phytoene desaturase genes. The findings provide a perspective in strain optimization for production of tailored-made products with industrial applications.
Asunto(s)
Acetilcoenzima A/biosíntesis , Ácido Araquidónico/genética , Genoma Fúngico/genética , Mortierella/metabolismo , Acetilcoenzima A/genética , Ácido Araquidónico/biosíntesis , Vías Biosintéticas/genética , Ácido Graso Desaturasas/genética , Elongasas de Ácidos Grasos/genética , Ácidos Grasos Insaturados/genética , Ácidos Grasos Insaturados/metabolismo , Mortierella/genética , Mucorales/genética , Mucorales/metabolismo , Ácido gammalinolénico/genética , Ácido gammalinolénico/metabolismoRESUMEN
Phytohormones are chemical messengers that have a positive effect at low concentrations on the biosynthesis of high-value compounds. Therefore, the effects of phytohormones on lipid and arachidonic acid (ARA) biosynthesis in Mortierella alpina were investigated in this study. At proper concentrations, the stimulatory effects of phytohormones on lipid production were determined to be as follows: 6-benzyl adenine (BA) > indole-3-acetic acid (IAA) > furfuryl adenine (KT) > gibberellin (GA) > indole-3-butyric acid (IBA) > abscisic acid (ABA). The results show that in the presence of 15 mg L-1 BA, the best positive effect was obtained, in which the lipid and ARA yields of M. alpina increased by 20.34% and 29.17%, respectively. Surprisingly, there was no synergy between the addition of two cytokinins (KT and BA), while adding cytokinins (KT or BA) and auxin (IAA) inhibited the growth of M. alpina and the ARA yield decreased by approximately 64%. Additional studies, such as those involving enzyme activity detection and quantitative real time polymerase chain reaction were carried out to check the fatty acid and lipid biosynthesis when the phytohormones were present. The activity of the main NADPH-supplying enzyme, 6-phosphoglucose dehydrogenase (G6PDH), increased by 19.52%. Moreover, the transcription levels of fatty acid synthase (FAS), Δ9-desaturase, and diacylglycerolacyltransferase (DGAT) increased by 9.3, 9.6 and 7.7 times, respectively, when only one type of phytohormone was present, indicating the enhancement of fatty acid and lipid biosynthesis in M. alpina. This study demonstrates the potential application of phytohormones for improving ARA yields of M. alpina.
Asunto(s)
Ácido Araquidónico/biosíntesis , Vías Biosintéticas/efectos de los fármacos , Mortierella/efectos de los fármacos , Mortierella/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacosRESUMEN
Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.
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Ácido Araquidónico/biosíntesis , Ingeniería Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos , Glucosa/metabolismo , Haptophyta/enzimologíaRESUMEN
Eicosanoids play crucial roles in mediating insect immune responses. In vertebrates, phospholipase A2 (PLA2) releases arachidonic acid (AA) from phospholipids (PLs) for biosynthesis of various eicosanoids. However, little AA is found in PLs of lepidopteran insects. Spodoptera exigua, a lepidopteran insect, is known to use eicosanoids to mediate immunity. Although AA was not detected in PLs of hemocytes and fat body (two immune tissues) of naïve larvae, it was detected at small but significant level after bacterial infection, suggesting induction of AA biosynthesis for immunity. Based on a mammalian AA biosynthetic pathway, this study hypothesizes that AA is synthesized from C18 polyunsaturated fatty acid (PUFA) precursor by subsequent desaturation and elongation reactions because PLs of S. exigua larvae are rich in linoleic acid. After inhibiting PLA2 activity to prevent release of free fatty acids, different PUFA precursors were injected to S. exigua larvae followed by assessment of eicosanoid-mediated cellular immune response. ω-6 PUFAs were effective in inducing immune response whereas α-linolenic acid (an ω-3 PUFA) was not. Several fatty acyl desaturases (SeDESs) have been predicted from S. exigua transcriptomes. Specific inhibitors against Δ5 or Δ6 DESs inhibited eicosanoid-mediated immune responses. Furthermore, RNA interference (RNAi) specific to Δ5 or Δ6 DES genes significantly suppressed eicosanoid-mediated immune responses. Four very long chain fatty acid elongase genes (SeEloV-Aâ¯â¼â¯SeEloV-D) were predicted. Among respective RNAi treatments of these genes, only one RNAi treatment specific to type 5 elongase (SeEloV-B) suppressed eicosanoid-mediated immune response. These results suggest that S. exigua larvae can synthesize AA from linoleic acid via Δ5- and Δ6-desaturations by SeDESs along with chain elongation by SeEloV-B. Finally, this study showed significant fitness cost of uncontrolled AA biosynthesis. AA injection alone without bacterial challenge significantly induced both cellular and humoral immune responses. This unnecessary energy expense due to free AA resulted in reduced pupal size and decreased adult egg production. The detrimental effect of free AA explains physiological significance of little AA content in lepidopteran insects except for life-or-death situation such as pathogen infection.
Asunto(s)
Ácido Araquidónico/biosíntesis , Spodoptera/metabolismo , Animales , Vías Biosintéticas , Eicosanoides/metabolismo , Escherichia coli , Ácidos Grasos Insaturados/metabolismo , Larva/genética , Larva/inmunología , Larva/metabolismo , Larva/microbiología , Interferencia de ARN , Spodoptera/genética , Spodoptera/inmunología , Spodoptera/microbiologíaRESUMEN
Temperature is a key factor for determining the lifespan of both poikilotherms and homeotherms. It is believed that animals live longer at lower body temperatures. However, the precise mechanism remains largely unknown. Here, we report that autophagy serves as a boost mechanism for longevity at low temperature in the nematode Caenorhabditis elegans. The adiponectin receptor AdipoR2 homolog PAQR-2 signaling detects temperature drop and augments the biosynthesis of two ω-6 polyunsaturated fatty acids, γ-linolenic acid and arachidonic acid. These two polyunsaturated fatty acids in turn initiate autophagy in the epidermis, delaying an age-dependent decline in collagen contents, and extending the lifespan. Our findings reveal that the adiponectin receptor PAQR-2 signaling acts as a regulator linking low temperature with autophagy to extend lifespan, and suggest that such a mechanism may be evolutionally conserved among diverse organisms.
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Autofagia/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Longevidad/fisiología , Proteínas de la Membrana/fisiología , Animales , Animales Modificados Genéticamente , Ácido Araquidónico/biosíntesis , Frío , Colágeno/metabolismo , Epidermis/metabolismo , Ácidos Grasos Omega-6/biosíntesis , Interferencia de ARN , Transducción de Señal/fisiologíaRESUMEN
Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.
Asunto(s)
Elongasas de Ácidos Grasos/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Salmo salar/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Ácido Araquidónico/biosíntesis , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Ácidos Docosahexaenoicos/biosíntesis , Ácido Eicosapentaenoico/biosíntesis , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos Omega-3/metabolismo , Técnicas de Inactivación de Genes , Metabolismo de los Lípidos/genética , Músculos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C18 PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase ("ωx2") mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase ("ωx1") catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C20 ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-14C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.
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Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Octopodiformes/metabolismo , Animales , Ácido Araquidónico/biosíntesis , Ácido Araquidónico/metabolismo , Ácidos Docosahexaenoicos/biosíntesis , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/biosíntesis , Ácido Graso Desaturasas/genética , Ácido Linoleico/biosíntesis , Octopodiformes/enzimología , Ácido alfa-Linolénico/biosíntesisRESUMEN
Biological significance of 18-carbon polyunsaturated fatty acids, γ-linolenic acid (GLA; C18:3 n-6) and dihomo-γ-linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6 PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized genes coding for Δ6-desaturase and Δ6-elongase of Pythium sp., and diacylglycerol acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result of a significant increase in triacylglycerol (TAG) content (52.0⯱â¯1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly, the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipid-accumulating stage of growth. Time-course expression analysis of the engineered strain revealed transcriptional control of TAG biosynthesis through a co-operation between the native DGAT2 of A. oryzae and the transformed mMaDGAT2.
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Aspergillus oryzae/metabolismo , Lípidos/biosíntesis , Ingeniería Metabólica/métodos , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidónico/biosíntesis , Aspergillus oryzae/genética , Aspergillus oryzae/fisiología , Vías Biosintéticas , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Proteínas Fúngicas/genética , Mortierella/genética , Triglicéridos/biosíntesis , Ácido gammalinolénico/biosíntesisRESUMEN
As the first marine teleost demonstrated to have the ability of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis from C18 PUFA precursors, the rabbitfish Siganus canaliculatus provides us a unique model for clarifying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts aiming at the replacement of dietary fish oil (rich in LC-PUFA) with vegetable oils (rich in C18 PUFA precursors but devoid of LC-PUFA). In the study of transcription regulation of gene encoding the Δ6Δ5 fatty acyl desaturase (Δ6Δ5 Fads), a rate-limiting enzyme catalyzing the first step of LC-PUFA biosynthesis in rabbitfish, a binding site for the transcription factor (TF), peroxisome proliferator-activated receptor γ (Pparγ), was predicted in Δ6Δ5 fads2 promoter by bioinformatics analysis, and thus the present study focused on the regulatory roles of Pparγ on Δ6Δ5 fads2. First, the activity of the Δ6Δ5 fads2 promoter was proved to be downregulated by pparγ overexpression and upregulated by treatment of Pparγ antagonist (GW9662), respectively, in HEK 293T cells with the dual luciferase reporter assay. Pparγ was further confirmed to interact with the promoter by electrophoretic mobility shift assay. Moreover, in S. canaliculatus hepatocyte line (SCHL) cells, GW9662 decreased the expression of pparγ together with increase of Δ6Δ5 fads2 mRNA. Besides, Δ6Δ5 fads2 expression was increased by pparγ RNAi knockdown and reduced by its mRNA overexpression. Furthermore, knockdown of pparγ induced a high conversion of 18:3n-3 to 18:4n-3 and 18:2n-6 to 18:3n-6, while pparγ mRNA overexpression led to a lower conversion of that, and finally a significant decrease of 20:4n-6(ARA), 20:5n-3(EPA), and 22:6n-3(DHA) production. The results indicate that Pparγ is involved in the transcriptional regulation of liver LC-PUFA biosynthesis by targeting Δ6Δ5 fads2 in rabbitfish, which is the first report of Pparγ involvement in the regulation of LC-PUFA biosynthesis in teleosts.
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Ácido Graso Desaturasas/genética , Proteínas de Peces/genética , Peces/genética , Hígado/metabolismo , PPAR gamma/genética , Regiones Promotoras Genéticas , Anilidas/farmacología , Animales , Organismos Acuáticos , Ácido Araquidónico/biosíntesis , Ácido Araquidónico/genética , Sitios de Unión , Línea Celular , Biología Computacional , Ácidos Docosahexaenoicos/biosíntesis , Ácidos Docosahexaenoicos/genética , Ácido Eicosapentaenoico/biosíntesis , Ácido Eicosapentaenoico/genética , Ácido Graso Desaturasas/metabolismo , Proteínas de Peces/metabolismo , Peces/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/genética , Hígado/citología , Luciferasas/genética , Luciferasas/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. RESULTS: The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L- 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. CONCLUSIONS: This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.
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Ácido Araquidónico/biosíntesis , Mortierella/efectos de los fármacos , Mortierella/efectos de la radiación , Reactores Biológicos , Ácido Graso Desaturasas/biosíntesis , Ácido Graso Sintasas/biosíntesis , Fermentación , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Iones Pesados , Mortierella/genética , Mortierella/metabolismo , Mutagénesis , Triclosán/farmacologíaRESUMEN
This study systematically examined the effect of nitrogen and phosphorous stress on the formation of linoleic acid (LA), arachidonic acid (ARA), and eicosapentaenoic acid (EPA) in Porphyridium cruentum gy-h56. P. cruentum was cultivated in six different media conferring different conditions of nitrogen (N) sufficiency/deprivation and phosphorous (P) sufficiency/limitation/deprivation. Over a 16-day cultivation process, the dry-weight content, proportion of total fatty acids (TFAs), and the concentration in the medium of linoleic acid (LA) were greatly improved by a maximum of 2.5-, 1.6-, and 1.1-fold, respectively, under conditions of N or P deprivation compared with N and P sufficiency. In contrast, levels of EPA or ARA were not enhanced under N or P stress conditions. Additionally, the results showed that N deprivation weakened the impact of P deficiency on the content and proportions of LA and EPA, while P deprivation enhanced the impact of N starvation on the content and proportions of LA and EPA. The conditions of N sufficiency and P deprivation (N+P-) were the optimal conditions for the production of LA, while the optimal conditions for EPA, ARA, and TFAs production were N sufficiency and P limitation (N+P-lim). This study suggests the potential application of combining N removal from saline wastewater with the production of LA, ARA, EPA, and biodiesel.
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Ácidos Grasos Insaturados/biosíntesis , Microbiología Industrial , Nitrógeno/metabolismo , Fósforo/metabolismo , Porphyridium/fisiología , Estrés Fisiológico , Aguas Residuales/química , Ácido Araquidónico/biosíntesis , Biocombustibles , Ácido Eicosapentaenoico/biosíntesis , Ácido Linoleico/biosíntesis , Nitrógeno/aislamiento & purificación , Nitrógeno/farmacología , Fósforo/farmacología , Porphyridium/efectos de los fármacosRESUMEN
Heavy and radioisotope labeling are commonly used methods to trace the pharmacological activity and metabolic fate of a biochemical or pharmaceutical in vivo. Recent years witnessed increased demand for molecules uniformly labeled with heavy carbon-13 (U-13C) or radioactive carbon-14 (U-14C) isotopes over singly labeled isotopic versions. Polyunsaturated fatty acids (PUFAs) represent one classic example where uniform 13C or 14C isotopic enrichment of the hydrocarbon backbone has numerous technical, metabolic and pharmacological advantages. PUFAs are usually produced in fungi or algae and uniform 13C or 14C enrichment of the hydrocarbon chain is achieved by feeding the microorganism a suitable U-13C or U-14C substrate. Previous literature methods describing the biosynthesis of U-13C or U-14C fatty acids reported variable isotopic enrichments that were less than anticipated and suffered from inconsistent growth of the microorganism due to radiotoxicity. In the present study, a single-tube method is described for the biosynthesis and extraction of U-13C and U-14C arachidonic acid (AA), a standard PUFA, from microcultures of the soil fungus Mortierella alpina. To produce U-13C-AA, a suspension of fungal spores and mycelial fragments was directly inoculated and grown into submerged cultures in a medium composed of U-13C-glucose and NaNO3 as the respective and only sources of carbon and nitrogen. The total 13C enrichment of AA was in excess of 95% and the percentage of U-13C-AA was in the range of 60-70%. These values have not been surpassed by previously reported methods. To produce U-14C-AA, the procedure was modified to limit the radiotoxic effects of 14C on fungal growth. Submerged cultures were initially grown on common 12C-glucose. Then, following glucose depletion, the biomass was collected and immediately cultured on U-14C-glucose. This approach is unprecedented in reported literature and has significantly limited the radiotoxic effects of 14C on the microorganism. Biomass transfer from 12C to 14C substrates was timed to keep an uninterrupted supply of carbon required to sustain the microorganism in the fatty acid synthesis mode and suppress ß-oxidation, a metabolic status that is prerequisite for enhanced isotopic purity of the 14C product. The specific activity of 14C enriched AA was estimated at 864â¯Ci/mol (range of 708-1020â¯Ci/mol) suggesting 69.2% (range of 56.7-81.7%) 14C enrichment along the AA hydrocarbon backbone. The present method used a single tube for microbial culture and lipid extraction to minimize manipulative losses and oxidative degradation of the labeled products. Production cost is comparatively cheaper to custom labeling and yields of U-13C and U-14C-AA are comparable to literature methods and sufficient for small scale in vitro and in vivo pharmacological studies.
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Ácido Araquidónico/biosíntesis , Isótopos de Carbono/química , Radioisótopos de Carbono/química , Mortierella/metabolismo , Ácido Araquidónico/aislamiento & purificación , Técnicas de Cultivo , Glucosa/químicaRESUMEN
Fatty acid (FA) composition of phospholipids in plasma and red blood cells (RBC) can influence calciuria, oxaluria and renal stone formation. In this regard, the ratio of arachidonic acid (AA) and its precursor linoleic acid (LA) appears to be important. Administration of γ-linolenic acid (GLA) has been shown to increase the concentration of dihomo-gamma linoleic acid (DGLA) relative to AA indicating that it may attenuate biosynthesis of the latter. Such effects have not been investigated in race groups having difference stone occurrence rates. Black (B) and white (W) healthy males ingested capsules containing linoleic acid (LA) and GLA, for 30 days. Plasma and RBC total phospholipid (TPL) FA profiles, serum and 24 h urine biomarkers of hypercalciuria and urinary stone risk factors were determined on days 0 and 30. Data were tested for statistical significance using GraphPadInstat version 3.02. Concentration and percentage content of DGLA in plasma TPL increased in W but not in B. Arachidonic acid (AA) did not change in either group. There was no change in calcium excretion in either group but oxalate and citrate excretion increased in W. We suggest that elongation of GLA to DGLA may occur more rapidly than desaturation of DGLA to AA in W and that depressed activity of the enzyme elongase may occur in B. Calciuric and citraturic effects may be dependent on the quantity of LA or on the mass ratio of LA/GLA in the FA supplement. Questions about the mooted DGLA-AA-oxaluria pathway arise. We speculate that there exists a potential for using GLA as a conservative treatment for hypocitraturia. The observation of different responses in B and W indicates that such differences may play a role in stone formation and prevention.