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International Journal of Molecular... Apr 2022Sucrose nonfermenting-1-related protein kinase 1 (SnRK1) is a central integrator of plant stress and energy starvation signalling pathways. We found that the...
Sucrose nonfermenting-1-related protein kinase 1 (SnRK1) is a central integrator of plant stress and energy starvation signalling pathways. We found that the -overexpression (OE) roots had a higher respiratory rate and tolerance to waterlogging than the -RNAi roots, suggesting that plays a positive role in the regulation of anaerobic respiration under waterlogging. upregulated the activity of anaerobic respiration-related enzymes including hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). also enhanced the ability to quench reactive oxygen species (ROS) by increasing antioxidant enzyme activities. We sequenced the transcriptomes of the roots of both wild-type (WT) and -RNAi plants, and the differentially expressed genes (DEGs) were clearly enriched in the defence response, response to biotic stimuli, and cellular carbohydrate metabolic process. In addition, 42 genes involved in glycolysis and 30 genes involved in pyruvate metabolism were significantly regulated in -RNAi roots. We analysed the transcript levels of two anoxia-related genes and three , and the results showed that , , and were upregulated in response to , indicating that may be involved in the ethylene signalling pathway to improve waterlogging tolerance. In conclusion, increases the expression of and further activates anoxia response genes, thereby enhancing anaerobic respiration metabolism in response to low-oxygen conditions during waterlogging.
Topics: Anaerobiosis; Fragaria; Gene Expression Regulation, Plant; Hypoxia; Plant Roots; Respiratory Rate
PubMed: 35563305
DOI: 10.3390/ijms23094914 -
Plants (Basel, Switzerland) Aug 2023Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of...
Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of kiwifruit cultivars, the underlying mechanisms remain largely unknown. Here, a comparative study was undertaken using one wild germplasm, Maorenshen ( Dunn, MRS), and one cultivar, Miliang-1 ( var. (A.Chev.) A.Chev. cv. Miliang-1, ML). Under stress, the ML plantlets were seriously damaged with wilted chlorotic leaves and blackened rotten roots, whereas the symptoms of injury in the MRS plantlets were much fewer, along with higher photosynthetic rates, chlorophyll fluorescence characteristics and root activity under stress conditions. However, neither aerenchyma in the root nor adventitious roots appeared in both germplasms upon stress exposure. The activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), as well as their transcript levels, were constitutively higher in MRS than those in ML under both normal and stress conditions. Waterlogging stress significantly enhanced the PDC and ADH enzyme activities in both germplasms, which were 60.8% and 22.4% higher in the MRS roots than those in the ML roots under waterlogging stress, respectively. Moreover, MRS displayed higher activities of antioxidant enzymes, including SOD, CAT, and APX, as well as DPPH-radical scavenging ability, and decreased HO and MDA accumulation under both normal and stress conditions. Our findings suggest that the waterlogging tolerance of the wild germplasm was associated with high PDC and ADH, as well as antioxidant ability.
PubMed: 37571025
DOI: 10.3390/plants12152872 -
Journal of Agricultural and Food... Mar 2024is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, can be persuaded into producing...
is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and α-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an α-acetolactate decarboxylase deficient strain, pyruvate could be converted to α-acetolactate and diacetyl. Summing up, by starving for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.
Topics: Pyruvic Acid; Lactococcus lactis; Thiamine; Diacetyl; L-Lactate Dehydrogenase; Lactic Acid; Butter; Lactates
PubMed: 38377583
DOI: 10.1021/acs.jafc.3c09216 -
OncoTargets and Therapy 2020Previously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine...
PURPOSE
Previously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.
METHODS
First, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial-mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.
RESULTS
knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while overexpression reversed the inhibitory effects of knockdown. Similarly, inhibition of also decreased the expression of PKM2 via reducing the c-myc-induced transcription. was co-expressed with in HCC samples, while simultaneously upregulated and led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.
CONCLUSION
/ are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.
PubMed: 33244237
DOI: 10.2147/OTT.S240535 -
Bioscience, Biotechnology, and... Jul 2017In Euglena gracilis, pyruvate:NADP oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the...
In Euglena gracilis, pyruvate:NADP oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP oxidoreductase. In contrast, pyruvate:NADP oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.
Topics: Aerobiosis; Amino Acid Sequence; Anaerobiosis; Carboxy-Lyases; Cloning, Molecular; Culture Media; Decarboxylation; Energy Metabolism; Escherichia coli; Euglena gracilis; Fermentation; Gene Expression; Gene Expression Regulation; Glucose; Ketone Oxidoreductases; Kinetics; Mitochondria; Oxidation-Reduction; Protozoan Proteins; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 28463550
DOI: 10.1080/09168451.2017.1318696 -
The Plant Journal : For Cell and... Apr 2019Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO and are well known to play a key role in energy supply via...
Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.
Topics: Acetaldehyde; Aldehydes; Carboxy-Lyases; Cucumis melo; Fruit; Gene Expression Profiling; Gene Expression Regulation, Plant; Plant Proteins; Pyruvic Acid
PubMed: 30556202
DOI: 10.1111/tpj.14204 -
Microbial Pathogenesis Jun 2021Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for...
Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.
Topics: Animals; Escherichia coli; Guinea Pigs; Mycoplasma Infections; Mycoplasma synoviae; Pyruvate Dehydrogenase (Lipoamide); Pyruvate Dehydrogenase Complex; Rabbits; Recombinant Proteins
PubMed: 33794298
DOI: 10.1016/j.micpath.2021.104851 -
The Biochemical Journal Apr 2018Ca can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca release under conditions of ATP concentration decline... (Review)
Review
Ca can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb-nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca- and low pH-activated external NADH and NADPH dehydrogenases participate. Ca can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.
Topics: Adaptation, Physiological; Calcium; Cytosol; Energy Metabolism; Oxidative Stress; Oxygen; Plants
PubMed: 29686143
DOI: 10.1042/BCJ20180169 -
Metabolic Engineering Sep 2020Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and...
Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.
Topics: Aerobiosis; Aspergillus oryzae; Citric Acid Cycle; Ethanol; Metabolic Engineering; Mitochondria; Mutation; Oxygen Consumption; Pyruvic Acid
PubMed: 32623009
DOI: 10.1016/j.ymben.2020.06.010 -
MSystems Aug 2022Traditional fermentation processes are driven by complex fungal microbiomes. However, the exact means by which fungal diversity affects fermentation remains unclear. In...
Traditional fermentation processes are driven by complex fungal microbiomes. However, the exact means by which fungal diversity affects fermentation remains unclear. In this study, we systematically investigated the diversity of a fungal community and its functions during the multibatch fermentation process. Metabolomics analysis showed that the metabolic profiles of the were enhanced with an increase in the fermentation time, as determined from the characteristic volatile flavors. High-throughput sequencing technology revealed that the major fungal species involved in sauce-flavor fermentation are sp. (41.75%, average relative abundance), sp. (13.07%), thermophilic species (9.16%), sp. (6.80%), Aspergillus sp. (4.69%), sp. (3.76%), sp. (3.74%), and Zygosaccharomyces sp. (1.41%). In addition, the fungal diversity increased as the number of fermentation batches increased. Moreover, the increased fungal diversity contributed to the modularity of the fungal communities, wherein sp., sp., and sp. maintained the stability of the fungal community. In addition, metatranscriptomics sequencing technologies were used to reconstruct the key metabolic pathways during fermentation, and it was found that the increased microbial diversity significantly promoted glucose-mediated carbon metabolism. Finally, functional gene analysis showed that functional microorganisms, such as Zygosaccharomyces and , can enhance fermentation as a result of the high expression of pyruvate decarboxylase and propanol-preferring alcohol dehydrogenase during the metabolism of pyruvate. These results indicate that fungal biodiversity can be exploited to enhance fermentation-based processes via network interactions and metabolism during multiple-batch fermentation. Biodiversity and network interactions act simultaneously on the microbial community structure in the fermentation process, thereby rendering the microbiome dynamics challenging to manage and predict. Understanding the complex fermentation community and its relationship to community functions is therefore important in the context of developing improved fermentation biotechnology systems. Our work demonstrates that multiple-batch fermentation steps increase microbial diversity and promote community stability. Crucially, the enhanced modularity in the microbial network increases the metabolism of flavor compounds and ethanol. This study highlights the power of biodiversity and network interactions in regulating the function of the microbiome in food fermentation ecosystems.
Topics: Fermentation; Microbiota; Carbohydrate Metabolism; Mycobiome; Pichia; Saccharomyces
PubMed: 35862822
DOI: 10.1128/msystems.00401-22