RESUMO
The malate shuttle is traditionally understood to maintain NAD+/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8+ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD+/NADH ratio and limited antiviral CD8+ T cell responses to chronic infection; however, increasing the NAD+/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8+ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8+ T cells is not to maintain the NAD+/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8+ T cells during chronic infection.
Assuntos
Ácidos Cetoglutáricos , NAD , Humanos , Oxirredução , NAD/metabolismo , Ácidos Cetoglutáricos/metabolismo , Amônia , Malatos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Infecção Persistente , AntiviraisRESUMO
Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.
Assuntos
Malatos , NAD , Ácido Aspártico/metabolismo , Glucose/metabolismo , Glicólise , Ácido Láctico , Malatos/metabolismo , NAD/metabolismoRESUMO
Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Ácido Cítrico/metabolismo , Microscopia Crioeletrônica , Malatos/farmacologia , Fenilbutiratos/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/química , Sítios de Ligação , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Ácido Cítrico/química , Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ácidos Dicarboxílicos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Malatos/química , Modelos Moleculares , Mutação , Fenilbutiratos/química , Multimerização Proteica , Sódio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Simportadores/genética , Simportadores/ultraestruturaRESUMO
Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood; however, molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2201 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals might be generated.
Assuntos
Proteínas de Bactérias , Malatos , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas de Bactérias/metabolismo , Ligantes , Escherichia coli/metabolismo , Quimiotaxia/fisiologia , Bactérias/metabolismo , CitratosRESUMO
Metabolites derived from the intestinal microbiota play an important role in maintaining skeletal muscle growth, function, and metabolism. Here, we found that D-malate (DMA) is produced by mouse intestinal microorganisms and its levels increase during aging. Moreover, we observed that dietary supplementation of 2% DMA inhibits metabolism in mice, resulting in reduced muscle mass, strength, and the number of blood vessels, as well as the skeletal muscle fiber type I/IIb ratio. In vitro assays demonstrate that DMA decreases the proliferation of vascular endothelial cells and suppresses the formation of blood vessels. In vivo, we further demonstrated that boosting angiogenesis by muscular VEGFB injection rescues the inhibitory effects of D-malate on muscle mass and fiber area. By transcriptomics analysis, we identified that the mechanism underlying the effects of DMA depends on the elevated intracellular acetyl-CoA content and increased Cyclin A acetylation rather than redox balance. This study reveals a novel mechanism by which gut microbes impair muscle angiogenesis and may provide a therapeutic target for skeletal muscle dysfunction in cancer or aging.
Assuntos
Células Endoteliais , Microbiota , Camundongos , Animais , Células Endoteliais/metabolismo , Acetilação , Ciclina A/metabolismo , Angiogênese , Malatos/metabolismo , Músculo Esquelético/metabolismo , EnvelhecimentoRESUMO
Poly(ß-L-malic acid) (PMLA) is a natural polyester produced by numerous microorganisms. Regarding its biosynthetic machinery, a nonribosomal peptide synthetase (NRPS) is proposed to direct polymerization of L-malic acid in vivo. Chemically versatile and biologically compatible, PMLA can be used as an ideal carrier for several molecules, including nucleotides, proteins, chemotherapeutic drugs, and imaging agents, and can deliver multimodal theranostics through biological barriers such as the blood-brain barrier. We focus on PMLA biosynthesis in microorganisms, summarize the physicochemical and physiochemical characteristics of PMLA as a naturally derived polymeric delivery platform at nanoscale, and highlight the attachment of functional groups to enhance cancer detection and treatment.
Assuntos
Neoplasias , Polímeros , Humanos , Malatos , Neoplasias/tratamento farmacológicoRESUMO
In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.
Assuntos
Compostos de Amônio , Brassica napus , Compostos de Amônio/metabolismo , Nitratos/metabolismo , Brassica napus/genética , Rizosfera , Malatos/metabolismo , Nitrogênio/metabolismo , Concentração de Íons de HidrogênioRESUMO
Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.
Assuntos
Citrulinemia , Hiperamonemia , Humanos , Citrulinemia/genética , Citrulinemia/metabolismo , NAD/metabolismo , Malatos , Ácido Aspártico/metabolismo , Hiperamonemia/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Hepatócitos/metabolismo , Glicólise , Ureia/metabolismo , Ácidos GraxosRESUMO
Ear length (EL) is a key trait that contributes greatly to grain yield in maize (Zea mays). While numerous quantitative trait loci for EL have been identified, few causal genes have been studied in detail. Here we report the characterization of ear apical degeneration1 (ead1) exhibiting strikingly shorter ears and the map-based cloning of the casual gene EAD1. EAD1 is preferentially expressed in the xylem of immature ears and encodes an aluminum-activated malate transporter localizing to the plasma membrane. We show that EAD1 is a malate efflux transporter and loss of EAD1 leads to lower malate contents in the apical part of developing inflorescences. Exogenous injections of malate rescued the shortened ears of ead1. These results demonstrate that EAD1 plays essential roles in regulating maize ear development by delivering malate through xylem vessels to the apical part of the immature ear. Overexpression of EAD1 led to greater EL and kernel number per row and the EAD1 genotype showed a positive association with EL in two different genetic segregating populations. Our work elucidates the critical role of EAD1 in malate-mediated female inflorescence development and provides a promising genetic resource for enhancing maize grain yield.
Assuntos
Inflorescência , Zea mays , Mapeamento Cromossômico/métodos , Grão Comestível/genética , Inflorescência/genética , Malatos/metabolismo , Fenótipo , Locos de Características Quantitativas , Zea mays/metabolismoRESUMO
Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation1. In CD4 T cells, T-cell-receptor ligation-along with co-stimulatory and cytokine signals-induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (TH1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of TH1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses TH1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state.
Assuntos
Diferenciação Celular , Mitocôndrias/metabolismo , Células Th1/citologia , Células Th1/imunologia , Acetilação , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Transporte de Elétrons , Feminino , Histonas/metabolismo , Humanos , Ativação Linfocitária/genética , Malatos/metabolismo , Masculino , Camundongos , Succinato Desidrogenase/metabolismo , Células Th1/metabolismo , Transcrição GênicaRESUMO
Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with d-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.
Assuntos
Malatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Malatos/metabolismo , Oxirredução , Catálise , Flavoproteínas/metabolismo , Flavinas/metabolismo , Zinco/metabolismo , Cinética , Especificidade por SubstratoRESUMO
Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with â¼40 s-1kcat for d-malate. Treatment with 1 mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin ε450 at pH 7.4 from 13.5 to 11.8 mM-1 cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity.
Assuntos
Malatos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Ácido Edético , Oxirredução , Flavinas/metabolismo , Zinco , Cinética , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region-based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity-related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild-type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2-bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation.
Assuntos
Malus , Malus/genética , Malus/metabolismo , Frutas/genética , Frutas/metabolismo , Malatos/metabolismo , Locos de Características Quantitativas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
MAIN CONCLUSION: γ-Aminobutyric acid alleviates acid-aluminum toxicity to roots associated with enhanced antioxidant metabolism as well as accumulation and transportation of citric and malic acids. Aluminum (Al) toxicity has become the main limiting factor for crop growth and development in acidic soils and is further being aggravated worldwide due to continuous industrial pollution. The current study was designed to examine effects of GABA priming on alleviating acid-Al toxicity in terms of root growth, antioxidant defense, citrate and malate metabolisms, and extensive metabolites remodeling in roots under acidic conditions. Thirty-seven-day-old creeping bentgrass (Agrostis stolonifera) plants were used as test materials. Roots priming with or without 0.5 mM GABA for 3 days were cultivated in standard nutrient solution for 15 days as control or subjected to nutrient solution containing 5 mM AlCl3·6H2O for 15 days as acid-Al stress treatment. Roots were sampled for determinations of root characteristics, physiological and biochemical parameters, and metabolomics. GABA priming significantly alleviated acid-Al-induced root growth inhibition and oxidative damage, despite it promoted the accumulation of Al in roots. Analysis of metabolomics showed that GABA priming significantly increased accumulations of organic acids, amino acids, carbohydrates, and other metabolites in roots under acid-Al stress. In addition, GABA priming also significantly up-regulated key genes related to accumulation and transportation of malic and citric acids in roots under acid-Al stress. GABA-regulated metabolites participated in tricarboxylic acid cycle, GABA shunt, antioxidant defense system, and lipid metabolism, which played positive roles in reactive oxygen species scavenging, energy conversion, osmotic adjustment, and Al ion chelation in roots.
Assuntos
Agrostis , Alumínio , Antioxidantes , Malatos , Raízes de Plantas , Ácido gama-Aminobutírico , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Antioxidantes/metabolismo , Ácido gama-Aminobutírico/metabolismo , Alumínio/toxicidade , Agrostis/efeitos dos fármacos , Agrostis/metabolismo , Agrostis/fisiologia , Malatos/metabolismo , Ácido Cítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted.
Assuntos
Ácido Aspártico , Malato Desidrogenase , Malatos , Mitocôndrias , Humanos , Malatos/metabolismo , Malato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Ácido Aspártico/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/diagnóstico , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Fosforilação Oxidativa , AntiportersRESUMO
Currently, the L-malic acid titer achieved through Aspergillus niger fermentation reaches 201 g/L, meeting industrial demands satisfactorily. However, the co-presence of structurally similar fumaric acid and succinic acid in fermentation products suggests a theoretical potential for further improvement in L-malic acid production. In the tricarboxylic acid cycle, fumarate reductase mediates the conversion of succinic acid to fumaric acid. Subsequently, fumarase catalyzes the conversion of fumaric acid to L-malic acid. Notably, both enzymatic reactions are reversible. Our investigation revealed that A. niger contains only one mitochondria-located fumarase FumA. Employing CRISPR-Cas9 technology, we performed a replacement of the fumA promoter with a doxycycline-induced promoter Tet. Under non-inducing condition, the conditional strain exhibited increased levels of fumaric acid and succinic acid. It strongly suggests that FumA mainly promotes the flow of fumaric acid to L-malic acid. Furthermore, a promoter PmfsA that is exclusively activated in a fermentation medium by calcium carbonate was identified through RNA-sequencing screening. Utilizing PmfsA to regulate fumA expression led to a 9.0% increase in L-malic acid titer, an 8.75% increase in yield (glucose to L-malic acid), and an 8.86% enhancement in productivity. This research serves as a significant step toward expediting the industrialization of L-malic acid synthesis via biological fermentation. Additionally, it offers valuable insights for the biosynthesis of other organic acids.IMPORTANCEThis study focuses on enhancing L-malic acid synthesis by modifying the tricarboxylic acid cycle within the mitochondria of Aspergillus niger. We emphasize the significant role of fumarase in converting fumaric acid into L-malic acid, enhancing our understanding of metabolic pathways in A. niger. The precise regulation of fumA is highlighted as a key factor in enhancing L-malic acid production. Furthermore, this research introduces a stringent conditional promoter (PmfsA), exclusively activated by CaCO3. The utilization of PmfsA for fumA expression resulted in heightened L-malic acid titers. The progress in metabolic engineering and bioprocess optimization holds promise for expediting industrial L-malic acid synthesis via biological fermentation. Moreover, it carries implications for the biosynthesis of various other organic acids.
Assuntos
Aspergillus niger , Fumarato Hidratase , Fumaratos , Aspergillus niger/genética , Aspergillus niger/metabolismo , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Malatos/metabolismo , Ácido SuccínicoRESUMO
C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.
Assuntos
Carbono , NADH Desidrogenase , Carbono/metabolismo , NADH Desidrogenase/metabolismo , Zea mays/genética , Zea mays/metabolismo , Malatos/metabolismo , NADP/metabolismo , Dióxido de Carbono/metabolismo , Proteômica , Fotossíntese , Oxirredução , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Folhas de Planta/metabolismoRESUMO
Although saline-alkali stress can improve tomato quality, the detailed molecular processes that balance stress tolerance and quality are not well-understood. Our research links nitric oxide (NO) and γ-aminobutyric acid (GABA) with the control of root malate exudation and fruit malate storage, mediated by aluminium-activated malate transporter 9/14 (SlALMT9/14). By modifying a specific S-nitrosylated site on pyruvate-dependent GABA transaminase 1 (SlGABA-TP1), we have found a way to enhance both plant's saline-alkali tolerance and fruit quality. Under saline-alkali stress, NO levels vary in tomato roots and fruits. High NO in roots leads to S-nitrosylation of SlGABA-TP1/2/3 at Cys316/258/316, reducing their activity and increasing GABA. This GABA then reduces malate exudation from roots and affects saline-alkali tolerance by interacting with SlALMT14. In fruits, a moderate NO level boosts SlGABA-TP1 expression and GABA breakdown, easing GABA's block on SlALMT9 and increasing malate storage. Mutants of SlGABA-TP1C316S that do not undergo S-nitrosylation maintain high activity, supporting malate movement in both roots and fruits under stress. This study suggests targeting SlGABA-TP1Cys316 in tomato breeding could significantly improve plant's saline-alkali tolerance and fruit quality, offering a promising strategy for agricultural development.
Assuntos
Álcalis , Frutas , Malatos , Óxido Nítrico , Raízes de Plantas , Solanum lycopersicum , Ácido gama-Aminobutírico , Solanum lycopersicum/genética , Solanum lycopersicum/efeitos dos fármacos , Malatos/metabolismo , Óxido Nítrico/metabolismo , Álcalis/farmacologia , Ácido gama-Aminobutírico/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Frutas/genética , Frutas/efeitos dos fármacos , 4-Aminobutirato Transaminase/metabolismo , 4-Aminobutirato Transaminase/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
In photosynthetic tissues in the light, the function of energy production is associated primarily with chloroplasts, while mitochondrial metabolism adjusts to balance ATP supply, regulate the reduction level of pyridine nucleotides, and optimize major metabolic fluxes. The tricarboxylic acid cycle in the light transforms into a noncyclic open structure (hemicycle) maintained primarily by the influx of malate and the export of citrate to the cytosol. The exchange of malate and citrate forms the basis of feeding redox energy from the chloroplast into the cytosolic pathways. This supports the level of NADPH in different compartments, contributes to the biosynthesis of amino acids, and drives secondary metabolism via a supply of substrates for 2-oxoglutarate-dependent dioxygenase and for cytochrome P450-catalyzed monooxygenase reactions. This results in the maintenance of redox and energy balance in photosynthetic plant cells and in the formation of numerous bioactive compounds specific to any particular plant species. The noncoupled mitochondrial respiration operates in coordination with the malate and citrate valves and supports intensive fluxes of respiration and photorespiration. The metabolic system of plants has features associated with the remarkable metabolic plasticity of mitochondria that permit the use of energy accumulated during photosynthesis in a way that all anabolic and catabolic pathways become optimized and coordinated.
Assuntos
Malatos , Fotossíntese , Malatos/metabolismo , Fotossíntese/fisiologia , Oxirredução , Mitocôndrias/metabolismo , Cloroplastos/metabolismo , Plantas/metabolismo , Citratos/metabolismoRESUMO
Jujube (Ziziphus jujuba Mill.), the most economically important fruit tree in Rhamnaceae, was domesticated from sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). During domestication, fruit sweetness increased and acidity decreased. Reduction in organic acid content is crucial for the increase in sweetness of jujube fruit. In this study, the determination of malate content among 46 sour jujube and 35 cultivated jujube accessions revealed that malate content varied widely in sour jujube (0.90-13.31 mg g-1) but to a lesser extent in cultivated jujube (0.33-2.81 mg g-1). Transcriptome sequencing analysis showed that the expression level of Aluminum-Dependent Malate Transporter 4 (ZjALMT4) was substantially higher in sour jujube than in jujube. Correlation analysis of mRNA abundance and fruit malate content and transient gene overexpression showed that ZjALMT4 participates in malate accumulation. Further sequencing analyses revealed that three genotypes of the W-box in the promoter of ZjALMT4 in sour jujube associated with malate content were detected, and the genotype associated with low malate content was fixed in jujube. Yeast one-hybrid screening showed that ZjWRKY7 binds to the W-box region of the high-acidity genotype in sour jujube, whereas the binding ability was weakened in jujube. Transient dual-luciferase and overexpression analyses showed that ZjWRKY7 directly binds to the promoter of ZjALMT4, activating its transcription, and thereby promoting malate accumulation. These findings provide insights into the mechanism by which ZjALMT4 modulates malate accumulation in sour jujube and jujube. The results are of theoretical and practical importance for the exploitation and domestication of germplasm resources.