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Acyl-acyl carrier protein (ACP) thioesterases (FAT) hydrolyze acyl-ACP complexes to release FA in plastids, which ultimately affects FA biosynthesis and profiles. Soybean GmFATA1 and GmFATA2 are homoeologous genes encoding oleoyl-ACP thioesterases whose role in seed oil accumulation and plant growth has not been defined. Using CRISPR/Cas9 gene editing mutation of Gmfata1 or 2 led to reduced leaf FA content and growth defect at the early seedling stage. In contrast, no homozygous double mutants were obtained. Combined this indicates that GmFATA1 and GmFATA2 display overlapping, but not complete functional redundancy. Combined transcriptomic and lipidomic analysis revealed a large number of genes involved in FA synthesis and FA chain elongation are expressed at reduced level in the Gmfata1 mutant, accompanied by a lower triacylglycerol abundance at the early seedling stage. Further analysis showed that the Gmfata1 or 2 mutants had increased composition of the beneficial FA, oleic acid. The growth defect of Gmfata1 could be at least partially attributed to reduced acetyl-CoA carboxylase activity, reduced abundance of five unsaturated monogalactosyldiacylglycerol lipids, and altered chloroplast morphology. On the other hand, overexpression of GmFATA in soybean led to significant increases in leaf FA content by 5.7%, vegetative growth, and seed yield by 26.9%, and seed FA content by 23.2%. Thus, overexpression of GmFATA is an effective strategy to enhance soybean oil content and yield.
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Ácidos Graxos , Glycine max , Proteínas de Plantas , Tioléster Hidrolases , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Glycine max/enzimologia , Ácidos Graxos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Sementes/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Sistemas CRISPR-Cas , Triglicerídeos/metabolismo , Edição de GenesRESUMO
The widespread application of zinc oxide nanoparticles (ZnO NPs) in our daily life has initiated an enhanced awareness of their biosafety concern. An incredible boom of evidence of organismal disorder has accumulated for ZnO NPs, yet there has been no relevant study at the single-cell level. Here, we profiled > 28,000 single-cell transcriptomes and assayed > 25,000 genes in testicular tissues from two healthy Sprague Dawley (SD) rats and two SD rats orally exposed to ZnO NPs. We identified 10 cell types in the rat testis. ZnO NPs had more deleterious effects on spermatogonia, Sertoli cells, and macrophages than on the other cell types. Cell-cell communication analysis indicated a sharp decrease of interaction intensity for all cell types except macrophages in the ZnO NPs group than in the control group. Interestingly, two distinct maturation states of spermatogonia were detected during pseudotime analysis, and ZnO NPs induced reservoir exhaustion of undifferentiated spermatogonia. Mechanically, ZnO NPs triggered fatty acid accumulation in GC-1 cells through protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling and peroxisome proliferator-activated receptor alpha (PPARα)/acyl-CoA oxidase 1 (Acox1) axis, contributing to cell apoptosis. In terms of Sertoli cells, downregulated genes were highly enriched for tight junction. In vitro and in vivo experiments verified that ZnO NPs disrupted blood-testis barrier formation and growth factors synthesis, which subsequently inhibited the proliferation and induced the apoptosis of spermatogonia. As for the macrophages, ZnO NPs activated oxidative stress of Raw264.7 cells through nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway and promoted cell apoptosis through extracellular signal-regulated kinase (ERK) 1/2 pathway. Collectively, our work reveals the cell type-specific and cellularly heterogenetic mechanism of ZnO NPs-induced testis damage and paves the path for identifying putative biomarkers and therapeutics against this disorder.
Assuntos
Nanopartículas , Óxido de Zinco , Masculino , Ratos , Animais , Óxido de Zinco/toxicidade , Testículo , Células de Sertoli , Espermatogônias , Ratos Sprague-Dawley , Estresse Oxidativo , Homeostase , MamíferosRESUMO
The objective of this research was to perform a genomics study of five cerium oxide particles, 4 nano and one micrometer-sized particles which have been studied previously by our group with respect to cytotoxicity, biochemistry and metabolomics. Human liver carcinoma HepG2 cells were exposed to between 0.3 to 300 ug/ml of CeO2 particles for 72 hours and then total RNA was harvested. Fatty acid accumulation was observed with W4, X5, Z7 and less with Q but not Y6. The gene expression changes in the fatty acid metabolism genes correlated the fatty acid accumulation we detected in the prior metabolomics study for the CeO2 particles named W4, Y6, Z7 and Q, but not for X5. In particular, the observed genomics effects on fatty acid uptake and fatty acid oxidation offer a possible explanation of why many CeO2 particles increase cellular free fatty acid concentrations in HepG2 cells. The major genomic changes observed in this study were sirtuin, ubiquitination signaling pathways, NRF2-mediated stress response and mitochondrial dysfunction. The sirtuin pathway was affected by many CeO2 particle treatments. Sirtuin signaling itself is sensitive to oxidative stress state of the cells and may be an important contributor in CeO2 particle induced fatty acid accumulation. Ubiquitination pathway regulates many protein functions in the cells, including sirtuin signaling, NRF2 mediated stress, and mitochondrial dysfunction pathways. NRF2-mediated stress response and mitochondrial were reported to be altered in many nanoparticles treated cells. All these pathways may contribute to the fatty acid accumulation in the CeO2 particle treated cells.
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Metabolic syndrome, characterized by centralobesity, hypertension, bycentralobesity, hypertension, and hyperlipidemia, increases the incidence and mortality of cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and other metabolic diseases. It is well known that insulin resistance, especially hepatic insulin resistance, is a risk factor for metabolic syndrome. Current research has shown that the accumulation of hepatic fatty acid can cause hepatic insulin resistance through increased gluconeogenesis, lipogenesis, chronic inflammation, oxidative stress and endoplasmic reticulum stress, and impaired insulin signal pathway. Mitochondria are the major sites of fatty acid β-oxidation, which is the major degradation mechanism of fatty acids. Mitochondrial dysfunction has been shown to be involved in the development of hepatic fatty acid-induced hepatic insulin resistance. Mitochondrial autophagy (mitophagy), a catabolic process, selectively degrades damaged mitochondria to reverse mitochondrial dysfunction and preserve mitochondrial dynamics and function. Therefore, mitophagy can promote mitochondrial fatty acid oxidation to inhibit hepatic fatty acid accumulation and improve hepatic insulin resistance. Here, we review advances in our understanding of the relationship between mitophagy and hepatic insulin resistance. Additionally, we also highlight the potential value of mitophagy in the treatment of hepatic insulin resistance and metabolic syndrome.
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Transmembrane 4 L six family member 5 (TM4SF5) is involved in nonalcoholic steatosis and further aggravation of liver disease. However, its mechanism for regulating FA accumulation is unknown. We investigated how TM4SF5 in hepatocytes affected FA accumulation during acute FA supply. TM4SF5-expressing hepatocytes and mouse livers accumulated less FAs, compared with those of TM4SF5 deficiency or inactivation. Binding of TM4SF5 to SLC27A2 increased gradually upon acute FA treatment, whereas TM4SF5 constitutively bound SLC27A5. Suppression of either SLC27A2 or SLC27A5 in hepatocytes expressing TM4SF5 differentially modulated initial and maximal FA uptake levels for a fast turnover of fatty acid. Altogether, TM4SF5 negatively modulates FA accumulation into hepatocytes via association with the transporters for an energy homeostasis, when FA are supplied acutely.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Metabolismo Energético , Proteínas de Transporte de Ácido Graxo/antagonistas & inibidores , Proteínas de Transporte de Ácido Graxo/genética , Células HEK293 , Células Hep G2 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Consumo de Oxigênio , RNA Interferente Pequeno/genéticaRESUMO
Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.
Assuntos
3-Isopropilmalato Desidrogenase/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipogênese/fisiologia , Mortierella/enzimologia , Mortierella/metabolismo , 3-Isopropilmalato Desidrogenase/genética , Acetilcoenzima A , Sequência de Aminoácidos , Aminoácidos/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Cetoácidos/metabolismo , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Metabolômica , Mortierella/genética , Mucor/metabolismo , Alinhamento de SequênciaRESUMO
Fatty acids in avocado fruit are crucial components influencing taste as well as fruit quality and nutritional value. Changes to fatty acid contents and concentrations in avocado fruit are important because of the associated effects on sensory properties. Hence, plant physiologists and molecular biologists interested in elucidating the influence of transcription factors on fatty acid accumulation in avocado fruit. In this study, APETALA2/ethylene-responsive factor (AP2/ERF) family members in avocado (Persea americana Mill.) were systematically and comprehensively analyze to identify potential PaAP2/ERF genes related to fatty acid accumulation. The results of bioinformatics analysis and the expression profiles of the AP2/ERF members suggested that 10 highly expressed PaAP2/ERF genes may encode transcription factors with functions related to the fatty acid accumulation in the avocado mesocarp. Furthermore, PaWRI1 and PaWRI2, two AP2/ERF transcription factor genes in avocado, were functionally characterized regarding their effects on fatty acid accumulation. The transcriptome and biochemical analyses of PaWRI1-2-overexpressing transgenic tomato plants revealed the up-regulated expression of 17 unigenes related to fatty acid synthesis and triacylglycerol assembly as well as increased fatty acid contents relative to the corresponding levels in the wild-type plants. In contrast, the overexpression of PaWRI2 in transgenic tomato plants up-regulated the expression of only six unigenes associated with fatty acid synthesis and triacylglycerol assembly and negligibly affected fatty acid accumulation when compared with wild-type plants. This systematic analysis provides a foundation for future studies regarding AP2/ERF functions associated with fatty acid accumulation.
Assuntos
Ácidos Graxos/metabolismo , Persea , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Persea/genética , Persea/metabolismo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
Rapid whole-genome sequencing (rWGS) allows for a diagnosis to be made quickly and impact medical management, particularly in critically ill children. Variants identified by this approach are often not identified using other testing methodologies, such as carrier screening or gene sequencing panels, targeted panels, or chromosomal microarrays. However, rWGS can identify variants of uncertain significance (VUSs), which challenges clinicians in the rapid return of information to families. Here we present a case of the metabolic condition D-bifunctional protein deficiency in a neonate with epilepsy and hypotonia born to consanguineous parents. Sequencing revealed a homozygous VUS in HSD17B4, c.1619A > G (p.His540Arg). Preliminary results were delivered within 3 d of sample receipt. Previous parental carrier screening included the HSD17B4 gene but was reported as negative. The molecular finding directed the clinical team to assess phenotypic overlap and investigate next steps in terms of confirmation of the findings and potential medical management of the patient. Clinical metabolic testing of fatty acids confirmed the diagnosis. Computational analysis of HSD17B4 His540Arg showed the change to likely impact dimerization based on structural insights, with the histidine conserved and selected throughout all 223 species assessed for this amino acid. This variant clusters around several pathogenic and likely pathogenic variants in HSD17B4 This case demonstrates the utility of rWGS, the potential for receiving uncertain results, and the downstream implications for confirmation or rejection of a molecular diagnosis by the clinical team.
Assuntos
Homozigoto , Proteína Multifuncional do Peroxissomo-2/deficiência , Proteína Multifuncional do Peroxissomo-2/genética , Deficiência de Proteína/genética , Sequenciamento Completo do Genoma , Ácidos Graxos , Feminino , Predisposição Genética para Doença/genética , Humanos , Lactente , Modelos Moleculares , Hipotonia Muscular , Patologia Molecular/métodos , Proteína Multifuncional do Peroxissomo-2/química , Conformação ProteicaRESUMO
n-3 Polyunsaturated fatty acid binding phospholipids (n-3 PUFA-PLs) are known to be potent carriers of n-3 PUFAs and provide health benefits. We previously prepared n-3 PUFA binding phosphatidylglycerol (n-3 PUFA-PG) by phospholipase D-mediated transphosphatidylation. Because PG has excellent emulsifiability, n-3 PUFA-PG is expected to work as a functional molecule with properties of both PG and n-3 PUFAs. In the present study, the health benefits and tissue accretion of dietary n-3 PUFA-PG were examined in diabetic/obese KK-Ay mice. After a feeding duration over 30 days, n-3 PUFA-PG significantly reduced the total and non-HDL cholesterols in the serum of diabetic/obese KK-Ay mice. In the mice fed n-3 PUFA-PG, but not n-3 PUFA-TAG, hepatic lipid content was markedly alleviated depending on the neutral lipid reduction compared with the SoyPC-fed mice. Further, the n-3 PUFA-PG diet increased eicosapentaenoic acid and docosahexaenoic acid (DHA) and reduced arachidonic acid in the small intestine, liver, perirenal white adipose tissue, and brain, and the ratio of the n-6 PUFAs to n-3 PUFAs in those tissues became lower compared to the SoyPC-fed mice. Especially, the DHA level was more significantly elevated in the brains of n-3 PUFA-PG-fed mice compared to the SoyPC-fed mice, whereas n-3 PUFA-TAG did not significantly alter DHA in the brain. The present results indicate that n-3 PUFA-PG is a functional lipid for reducing serum and liver lipids and is able to supply n-3 PUFAs to KK-Ay mice.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Síndrome Metabólica/metabolismo , Fosfatidilgliceróis/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Ácido Araquidônico , Encéfalo/metabolismo , Colesterol/metabolismo , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Ácidos Graxos Ômega-3/administração & dosagem , Intestino Delgado/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos ObesosRESUMO
Metabolic syndrome, characterized by central obesity, hypertension, and hyperlipidemia, increases the morbidity and mortality of cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and other metabolic diseases. It is well known that insulin resistance, especially hepatic insulin resistance, is a risk factor for metabolic syndrome. Current research has shown that hepatic fatty acid accumulation can cause hepatic insulin resistance through increased gluconeogenesis, lipogenesis, chronic inflammation, oxidative stress and endoplasmic reticulum stress, and impaired insulin signal pathway. Mitochondria are the major sites of fatty acid ß-oxidation, which is the major degradation mechanism of fatty acids. Mitochondrial dysfunction has been shown to be involved in the development of hepatic fatty acid-induced hepatic insulin resistance. Mitochondrial autophagy (mitophagy), a catabolic process, selectively degrades damaged mitochondria to reverse mitochondrial dysfunction and preserve mitochondrial dynamics and function. Therefore, mitophagy can promote mitochondrial fatty acid oxidation to inhibit hepatic fatty acid accumulation and improve hepatic insulin resistance. Here, we review advances in our understanding of the relationship between mitophagy and hepatic insulin resistance. Additionally, we also highlight the potential value of mitophagy in the treatment of hepatic insulin resistance and metabolic syndrome.
RESUMO
In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.
Assuntos
AMP Desaminase/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia , AMP Desaminase/genética , Monofosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Proteínas Fúngicas/genética , Metabolismo dos Lipídeos , Mortierella/genética , Mortierella/crescimento & desenvolvimento , Mortierella/metabolismoRESUMO
Tea oil camellia (Camellia oleifera) is an important woody oil tree in southern China. However, little is known regarding the molecular mechanisms that contribute to high oleic acid accumulation in tea oil camellia. Herein, we measured the oil content and fatty acid compositions of high- and low-oil tea oil camellia seeds and investigated the global gene expression profiles by RNA-seq. The results showed that at the early, second and third seed developmental stages, a total of 64, 253, and 124 genes, respectively, were significantly differentially expressed between the high- and low-oil cultivars. Gene ontology (GO) enrichment analysis of the identified differentially expressed transcription factors (TFs; ABI3, FUS3, LEC1, WRI1, TTG2 and DOF4.6) revealed some critical GO terms associated with oil biosynthesis and fatty acid accumulation, including glycolysis, zinc ion binding, positive regulation of fatty acid biosynthetic process, triglyceride biosynthetic process, seed coat development, abscisic acid-mediated signaling pathway and embryo development. Comprehensive comparisons of transcriptomic profiles and expression analysis of multigenes based on qRT-PCR showed that coordinated high expression of the upstream genes HAD, EAR and KASI directly increased the relative levels of C16:0-ACP, which provided enough precursor resources for oleic acid biosynthesis. Continuous high expression of the SAD gene accelerated oleic acid synthesis and accumulation, and coordinated low expression of the downstream genes FAD2, FAD3, FAD7, FAD8 and FAE1 decreased the consumption of oleic acid for conversion. The coordinated regulation of these multigenes ensures the high accumulation of oleic acid in the seeds of tea oil camellia. Our data represent a comprehensive transcriptomic study of high- and low-oil tea oil camellia, not only increasing the number of sequences associated with lipid biosynthesis and fatty acid accumulation in public resource databases but also providing a scientific basis for genetic improvement of the oleic acid content in woody oil trees.
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Using clinical exome sequencing (ES), we identified an autosomal recessive missense variant, c.153C>A (p.F51L), in the peroxisome biogenesis factor 26 gene (PEX26) in a 19-yr-old female of Ashkenazi Jewish descent who was referred for moderate to severe hearing loss. The proband and three affected siblings are all homozygous for the c.153C>A variant. Skin fibroblasts from this patient show normal morphology in immunostaining of matrix proteins, although the level of catalase was elevated. Import rate of matrix proteins was significantly decreased in the patient-derived fibroblasts. Binding of Pex26-F51L to the AAA ATPase peroxins, Pex1 and Pex6, is severely impaired and affects peroxisome assembly. Moreover, Pex26 in the patient's fibroblasts is reduced to â¼30% of the control, suggesting that Pex26-F51L is unstable in cells. In the patient's fibroblasts, peroxisome-targeting signal 1 (PTS1) proteins, PTS2 protein 3-ketoacyl-CoA thiolase, and catalase are present in a punctate staining pattern at 37°C and in a diffuse pattern at 42°C, suggesting that these matrix proteins are not imported to peroxisomes in a temperature-sensitive manner. Analysis of peroxisomal metabolism in the patient's fibroblasts showed that the level of docosahexaenoic acid (DHA) (C22:6n-3) in ether phospholipids is decreased, whereas other lipid metabolism, including peroxisomal fatty acid ß-oxidation, is normal. Collectively, the functional data support the mild phenotype of nonsyndromic hearing loss in patients harboring the F51L variant in PEX26.
Assuntos
Perda Auditiva/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Peroxissomos/metabolismo , Síndrome de Zellweger/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Sequência de Aminoácidos , Feminino , Perda Auditiva/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Linhagem , Ligação Proteica , Estabilidade Proteica , Análise de Sequência de DNA , Síndrome , Adulto Jovem , Síndrome de Zellweger/metabolismoRESUMO
As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.
Assuntos
Arabidopsis/genética , Biocombustíveis , Ácidos Graxos/genética , Microbiologia Industrial/métodos , Óleos Industriais , Microalgas/genética , Saccharomyces cerevisiae/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Biocombustíveis/análise , Biocombustíveis/microbiologia , Diacilglicerol Colinofosfotransferase/genética , Diacilglicerol Colinofosfotransferase/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Graxos/metabolismo , Óleos Industriais/análise , Óleos Industriais/microbiologia , Engenharia Metabólica/métodos , Microalgas/enzimologia , Microalgas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
The purpose of this study was to clarify the interrelation between the mitochondrial alternative oxidase (AOX) pathway and fatty acid accumulation in marine microalga Isochrysis galbana. Under normal conditions, the activity of the AOX pathway was maintained at a low level in I. galbana. Compared with the normal condition, nitrogen deprivation significantly increased the AOX pathway activity and fatty acid accumulation. Under nitrogen deprivation, the inhibition of the AOX pathway by salicylhydroxamic acid caused the accumulation of reducing equivalents and the over-reduction of chloroplasts in I. galbana cells, leading to a decrease in the photosynthetic O2 evolution rate. The over-production of reducing equivalents due to the inhibition of the AOX pathway under nitrogen deprivation further enhanced the accumulation of fatty acids in I. galbana cells.
Assuntos
Ácidos Graxos/metabolismo , Haptófitas/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Nitrogênio/química , Nitrogênio/metabolismoRESUMO
In order to live, cells need to import different molecules, such as sugars, amino acids or lipids, using transporters. In Saccharomyces cerevisiae, the ScFAT1 gene encodes the long-chain fatty acid transporter; however, the transport of fatty acids (FAs) in the oleaginous yeast Yarrowia lipolytica has not yet been studied. In contrast to what has previously been found for ΔScfat1 strains, ΔYlfat1 yeast was still able to grow on substrates containing short-, medium- or long-chain FAs. We observed a notable difference in cell lipid content between wild-type (WT) and deletion mutant strains after 24 h of culture in minimal oleate medium: in the WT strain, lipids represented 24% of cell dry weight (CDW), while they accounted for 37% of CDW in the ΔYlfat1 strain. This result indicates that YlFat1p is not involved in cell lipid uptake. Moreover, we also observed that fatty acid remobilisation was decreased in the ΔYlfat1 strain and that fluorescence-tagged YlFat1p proteins localised to the interfaces between lipid bodies, which suggests that YlFat1p may play a role in the export of FAs from lipid bodies.
Assuntos
Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Sequência Conservada , Meios de Cultura/química , Proteínas de Transporte de Ácido Graxo/química , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Ácido Oleico/química , Ácido Oleico/metabolismo , Transporte Proteico , Alinhamento de Sequência , Yarrowia/crescimento & desenvolvimentoRESUMO
In this study, the Saccharomyces cerevisiae wild type strain and engineered strain with an overexpressed heterologous ATP-citrate lyase (acl) were cultured in medium with different carbon and nitrogen concentrations, and their fatty acid production levels were investigated. The results showed that when the S. cerevisiae engineered strain was cultivated under nitrogen limited culture condition, the yield of mono-unsaturated fatty acids showed higher than that under non-nitrogen limited condition; with the carbon concentration increased, the accumulation become more apparent, whereas in the wild type strain, no such correlation was found. Besides, the citrate level in the S. cerevisiae under nitrogen limited condition was found to be much higher than that under non-nitrogen limited condition, which indicated a relationship between the diminution of nitrogen and accumulation of citrate in the S. cerevisiae. The accumulated citrate could be further cleaved by acl to provide substrate for fatty acid synthesis.