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Metabolites Dec 2022Pesticides as important agricultural inputs play a vital role in protecting crop plants from diseases and pests; however, the effect of pesticides on crop plant...
Pesticides as important agricultural inputs play a vital role in protecting crop plants from diseases and pests; however, the effect of pesticides on crop plant physiology and metabolism is still undefined. In this study, the effect of insecticide chlorpyrifos at three doses on rice plant physiology and metabolism was investigated. Our results revealed that chlorpyrifos cause oxidative stress in rice plants and even inhibit plant growth and the synthesis of protein and chlorophyll at high doses. The metabolomic results suggested that chlorpyrifos could affect the metabolic profiling of rice tissues and a total of 119 metabolites with significant changes were found, mainly including organic acids, amino acids, lipids, polyphenols, and flavonoids. Compared to the control, the content of glutamate family amino acids were significantly disturbed by chlorpyrifos, where defense-related proline and glutathione were significantly increased; however, glutamic acid, N-acetyl-glutamic acid and N-methyl-glutamic acid were significantly decreased. Many unsaturated fatty acids, such as linolenic acid and linoleic acid, and their derivatives lysophospholipids and phospholipids, were significantly accumulated in chlorpyrifos groups, which could act as osmolality substances to help rice cells relieve chlorpyrifos stress. Three organic acids, aminobenzoic acid, quinic acid, and phosphoenolpyruvic acid, involved in plant defenses, were significantly accumulated with the fold change ranging from 1.32 to 2.19. In addition, chlorpyrifos at middle- and high-doses caused the downregulation of most flavonoids. Our results not only revealed the effect of insecticide chlorpyrifos on rice metabolism, but also demonstrated the value of metabolomics in elucidating the mechanisms of plant responses to stresses.
PubMed: 36557326
DOI: 10.3390/metabo12121289 -
Scientific Reports Nov 2021The deazaflavin cofactor F is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy,...
The deazaflavin cofactor F is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F biosynthesis to optimize F production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space-time yield of F compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F-production system and will allow the recombinant in vivo use of F-dependent enzymes for biocatalysis and protein engineering applications.
Topics: Escherichia coli; Glyceric Acids; Phosphoenolpyruvate; Phosphotransferases (Paired Acceptors); Polyglutamic Acid; Riboflavin
PubMed: 34741069
DOI: 10.1038/s41598-021-01224-3 -
Journal of Experimental Botany Mar 2017The surface area of a mature green cucumber (Cucumis sativa L.) fruit is comparable with that of a functional leaf, but the characteristics of fruit photosynthesis and...
The surface area of a mature green cucumber (Cucumis sativa L.) fruit is comparable with that of a functional leaf, but the characteristics of fruit photosynthesis and its contribution to growth are poorly understood. Here, the photosynthetic properties of two genotypes of cucumber (dark green and light green fruits) were studied using a combination of electron microscopy, immunogold enzyme localization, chlorophyll fluorescence imaging, isotope tracer, and fruit darkening techniques. Chlorophyll content of the exocarp is similar to that of leaves, but there are no distinctive palisade and spongy tissues. The efficiency of PSII is similar to that in leaves, but with lower non-photochemical quenching (NPQ). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is found mainly in the exocarp, while phosphoenolpyruvate carboxylase (PEPC) is primarily localized to vascular bundles and placenta tissue. Rubisco and PEPC expression at both transcriptional and translational levels increases concurrently during fruit growth. The contribution of fruit photosynthesis in exocarp to its own C accumulation is 9.4%, while ~88% of respiratory CO2 in fruit was captured and re-fixed. Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, as verified by 14CO2 uptake and gas exchange, makes an important contribution to fruit growth.
Topics: Carbon Dioxide; Cucumis sativus; Fruit; Phosphoenolpyruvate Carboxylase; Photosynthesis; Ribulose-Bisphosphate Carboxylase
PubMed: 28369547
DOI: 10.1093/jxb/erx034 -
The FEBS Journal Dec 2021Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for...
Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRAS upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.
Topics: Argininosuccinate Synthase; Aspartic Acid; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Humans; Intracellular Signaling Peptides and Proteins; Metabolomics; Mutation; Phosphoenolpyruvate Carboxykinase (ATP); Phosphoenolpyruvate Carboxykinase (GTP); Proteomics; Proto-Oncogene Proteins p21(ras); Signal Transduction
PubMed: 34227245
DOI: 10.1111/febs.16111 -
Journal of Bacteriology Mar 2021The halophilic archaeon has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes...
The halophilic archaeon has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis, and in acetyl coenzyme A (acetyl-CoA) formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase, and pyruvate-ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Furthermore, we show that -during aerobic growth on glucose-excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway and of anaplerosis and report the first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea. In this work, we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism The study includes transcriptional analyses, growth experiments with deletion mutants. and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl coenzyme A (acetyl-CoA) and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Furthermore, we analyzed the key players involved in the acetate switch, i.e., in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects of glucose degradation, anaplerosis, and acetate switch in and thus expand our understanding of the unusual sugar metabolism in archaea.
Topics: Acetate-CoA Ligase; Acetates; Acetyl Coenzyme A; Archaeal Proteins; Glucose; Haloferax volcanii; Phosphoenolpyruvate Carboxylase; Phosphoglycerate Mutase; Phosphopyruvate Hydratase; Pyruvic Acid
PubMed: 33558390
DOI: 10.1128/JB.00690-20 -
Journal of Industrial Microbiology &... Nov 2015Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was...
Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.
Topics: Actinobacillus; Aspergillus; Bioreactors; Carbon Cycle; Citric Acid; Escherichia coli; Glucose; Hydrogen-Ion Concentration; Phosphoenolpyruvate Carboxykinase (ATP); Phosphoenolpyruvate Carboxylase; Transformation, Genetic; Xylose
PubMed: 26403577
DOI: 10.1007/s10295-015-1688-4 -
Plant Physiology Jun 2022C4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations despite sharing key biochemical modules. Portulaca is a...
C4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations despite sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4 but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4 + CAM. It has been hypothesized that C4 + CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of Portulaca amilis and sampled mRNA from P. amilis and Portulaca oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and posttranscriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive genes for primary carbon fixation, and it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, but we found evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.
Topics: Crassulacean Acid Metabolism; Droughts; Phosphoenolpyruvate Carboxylase; Photosynthesis; Portulaca
PubMed: 35285495
DOI: 10.1093/plphys/kiac116 -
The Journal of Physiology Jan 2023Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass...
Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8 weeks. Subsequently, glucose and fatty acid metabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification in intact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.
Topics: Rats; Animals; Adipose Tissue, Brown; Uncoupling Protein 1; Glycerol; Glycerol Kinase; Phosphoenolpyruvate; Tyrosine 3-Monooxygenase; Mitochondrial Proteins; Diet; Obesity; Triglycerides; Adipocytes, Brown; Glucose; Fatty Acids; Thermogenesis
PubMed: 36419345
DOI: 10.1113/JP283721 -
NPJ Biofilms and Microbiomes Aug 2022In addition to catalyzing coupled transport and phosphorylation of carbohydrates, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulates various...
In addition to catalyzing coupled transport and phosphorylation of carbohydrates, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulates various physiological processes in most bacteria. Therefore, the transcription of genes encoding the PTS is precisely regulated by transcriptional regulators depending on substrate availability. As the distribution of the mannose-specific PTS (PTS) is limited to animal-associated bacteria, it has been suggested to play an important role in host-bacteria interactions. In Vibrio cholerae, mannose is known to inhibit biofilm formation. During host infection, the transcription level of the V. cholerae gene encoding the putative PTS (hereafter referred to as manP) significantly increases, and mutations in this gene increase host survival rate. Herein, we show that an AraC-type transcriptional regulator (hereafter referred to as ManR) acts as a transcriptional activator of the mannose operon and is responsible for V. cholerae growth and biofilm inhibition on a mannose or fructose-supplemented medium. ManR activates mannose operon transcription by facilitating RNA polymerase binding to the promoter in response to mannose 6-phosphate and, to a lesser extent, to fructose 1-phosphate. When manP or manR is impaired, the mannose-induced inhibition of biofilm formation was reversed and intestinal colonization was significantly reduced in a Drosophila melanogaster infection model. Our results show that ManR recognizes mannose and fructose in the environment and facilitates V. cholerae survival in the host.
Topics: Animals; Cytarabine; Drosophila melanogaster; Fructose; Gene Expression Regulation, Bacterial; Humans; Mannose; Phosphates; Phosphoenolpyruvate Sugar Phosphotransferase System; Vibrio cholerae
PubMed: 35987769
DOI: 10.1038/s41522-022-00331-x -
Metabolic Engineering Communications Jun 2020is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer....
is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in are not completely known. In , both pyruvate kinase (PYK) and pyruvate phosphate dikinase (PPDK) are highly expressed based on transcriptomic and proteomic data. Both enzymes catalyze the formation of pyruvate from phosphoenolpyruvate (PEP). PYK is typically the last step of EMP glycolysis pathway while PPDK is reversible and is found mostly in C4 plants and some microorganisms. It is not clear what role PYK and PPDK play in metabolism and fermentation pathways and whether both are necessary. In this study we deleted the gene in wild type and homoethanologen strains of and showed that it is not essential for growth or ethanol production.
PubMed: 32025490
DOI: 10.1016/j.mec.2020.e00122