Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis

Heterosis or hybrid vigor is widespread in plants and animals. Although the molecular basis for heterosis has been extensively studied, metabolic and proteomic contributions to heterosis remain elusive. Here we report an integrative analysis of time-series metabolome and proteome data in maize (Zea mays) hybrids and their inbred parents. Many maize metabolites and proteins are diurnally regulated, and many of these show nonadditive abundance in the hybrids, including key enzymes and metabolites involved in carbon assimilation. Compared with robust trait heterosis, metabolic heterosis is relatively mild. Interestingly, most amino acids display negative mid-parent heterosis (MPH), i.e., having lower values than the average of the parents, while sugars, alcohols, and nucleoside metabolites show positive MPH. From the network perspective, metabolites in the photosynthetic pathway show positive MPH, whereas metabolites in the photorespiratory pathway show negative MPH, which corresponds to nonadditive protein abundance and enzyme activities of key enzymes in the respective pathways in the hybrids. Moreover, diurnally expressed proteins that are upregulated in the hybrids are enriched in photosynthesis-related gene-ontology terms. Hybrids may more effectively remove toxic metabolites generated during photorespiration, and thus maintain higher photosynthetic efficiency. These metabolic and proteomic resources provide unique insight into heterosis and its utilization for high yielding maize and other crop plants.     

MedicCyc: a biochemical pathway database for Medicago truncatula

MOTIVATION: There is an imperative need to integrate functional genomics data to obtain a more comprehensive systems-biology view of the results. We believe that this is best achieved through the visualization of data within the biological context of metabolic pathways. Accordingly, metabolic pathway reconstruction was used to predict the metabolic composition for Medicago truncatula and these pathways were engineered to enable the correlated visualization of integrated functional genomics data. Results: Metabolic pathway reconstruction was used to generate a pathway database for M. truncatula (MedicCyc), which currently features more than 250 pathways with related genes, enzymes and metabolites. MedicCyc was assembled from more than 225,000 M. truncatula ESTs (MtGI Release 8.0) and available genomic sequences using the Pathway Tools software and the MetaCyc database. The predicted pathways in MedicCyc were verified through comparison with other plant databases such as AraCyc and RiceCyc. The comparison with other plant databases provided crucial information concerning enzymes still missing from the ongoing, but currently incomplete M. truncatula genome sequencing project. MedicCyc was further manually curated to remove non-plant pathways, and Medicago-specific pathways including isoflavonoid, lignin and triterpene saponin biosynthesis were modified or added based upon available literature and in-house expertise. Additional metabolites identified in metabolic profiling experiments were also used for pathway predictions. Once the metabolic reconstruction was completed, MedicCyc was engineered to visualize M. truncatula functional genomics datasets within the biological context of metabolic pathways. Availability: freely accessible at http://www.noble.org/MedicCyc/     

Thermodynamically based profiling of drug metabolism and drug-drug metabolic interactions: a case study of acetaminophen and ethanol toxic interaction

Drug-drug metabolic interactions can result in unwanted side effects, including reduced drug efficacy and formation of toxic metabolic intermediates. In this work, thermodynamic constraints on non-equilibrium metabolite concentrations are used to reveal the biochemical interactions between the metabolic pathways of ethanol and acetaminophen (N-acetyl-p-aminophenol), two drugs known to interact unfavorably. It is known that many reactions of these pathways are coupled to the central energy metabolic reactions through a number of metabolites and the cellular redox potential. Based on these observations, a metabolic network model has been constructed and a database of thermodynamic properties for all participating metabolites and reactions has been compiled. Constraint-based computational analysis of the feasible metabolite concentrations reveals that the non-toxic pathways for APAP metabolism and the pathway for detoxifying N-acetyl-p-benzoquinoneimine (NAPQI) are inhibited by network interactions with ethanol metabolism. These results point to the potential utility of thermodynamically based profiling of metabolic network interactions in screening of drug candidates and analysis of potential toxicity.     

Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species

The metabolic network of the central carbon metabolism represents the backbone of cellular metabolism and provides the precursors and cofactors required for synthesis of secondary metabolites. It is therefore pivotal to map the operating metabolic network in the central carbon metabolism in order to design metabolic engineering strategies towards construction of more efficient producers of specific metabolites. In this context, methods that allow rapid and reliable mapping of the central carbon metabolism are valuable. In the present study, a (13)C labelling-based method was used to identify the primary metabolic pathways of the poorly characterized antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727. Surprisingly, it was found that Nonomuraea sp. ATCC 39272 predominantly metabolizes glucose via the Entner-Doudoroff (ED) pathway. This represents the first time that the ED pathway has been recognized as the main catabolic pathway in an actinomycete. The Nonomuraea genes encoding the key enzymes of the ED pathway were subsequently identified, sequenced and functionally described.     

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.     

鸟苷产生菌的代谢途径分析

代谢工程要解决的主要问题就是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径以获得产物的最大转化率。利用途径分析方法对枯草芽孢杆菌生产鸟苷的途径进行了分析,建立了3种基础模型,鸟苷理论摩尔产率分别是0．625、0．75和0．667,确定了枯草芽孢杆菌生产鸟苷的最佳途径的通量分布。     

Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme's role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway.     

Parameter estimation and dynamic control analysis of central carbon metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The central carbon metabolic pathway is the most important among metabolic pathways in all microorganisms since it produces energy and precursors for biosynthesis. In this study, a dynamic model for central carbon metabolism in Escherichia coli (E. coli) consisting of the phosphotransferase (PTS) system, glycolysis, pentose-phosphate pathway (PPP), and storage materials was obtained by ameliorating the model proposed by Chassagnole et al. (2002). In order to improve the performance of the model, principal parameters were estimated through the experimental measurements of intracellular concentrations of metabolites under transient conditions. Through dynamic metabolic control analysis (MCA), the tendencies of the metabolic fluxes at branch points were investigated, and the key parameters and enzyme kinetics that most dominantly affected the productivity of the desired metabolites were determined.     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.     

Improving the phenotype predictions of a yeast genome‐scale metabolic model by incorporating enzymatic constraints

Genome‐scale metabolic models (GEMs) are widely used to calculate metabolic phenotypes. They rely on defining a set of constraints, the most common of which is that the production of metabolites and/or growth are limited by the carbon source uptake rate. However, enzyme abundances and kinetics, which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between and within metabolic pathways. The developed method and model are expected to increase the use of model‐based design in metabolic engineering.     

An algorithm for linear metabolic pathway alignment

Metabolic pathway alignment represents one of the most powerful tools for comparative analysis of metabolism. It involves recognition of metabolites common to a set of functionally-related metabolic pathways, interpretation of biological evolution processes and determination of alternative metabolic pathways. Moreover, it is of assistance in function prediction and metabolism modeling. Although research on genomic sequence alignment is extensive, the problem of aligning metabolic pathways has received less attention. We are motivated to develop an algorithm of metabolic pathway alignment to reveal the similarities between metabolic pathways. A new definition of the metabolic pathway is introduced. The algorithm has been implemented into the PathAligner system; its web-based interface is available at http://bibiserv.techfak.uni-bielefeld.de/pathaligner/.     

MetaCyc and AraCyc. Metabolic pathway databases for plant research

MetaCyc (http://metacyc.org) contains experimentally determined biochemical pathways to be used as a reference database for metabolism. In conjunction with the Pathway Tools software, MetaCyc can be used to computationally predict the metabolic pathway complement of an annotated genome. To increase the breadth of pathways and enzymes, more than 60 plant-specific pathways have been added or updated in MetaCyc recently. In contrast to MetaCyc, which contains metabolic data for a wide range of organisms, AraCyc is a species-specific database containing only enzymes and pathways found in the model plant Arabidopsis (Arabidopsis thaliana). AraCyc (http://arabidopsis.org/tools/aracyc/) was the first computationally predicted plant metabolism database derived from MetaCyc. Since its initial computational build, AraCyc has been under continued curation to enhance data quality and to increase breadth of pathway coverage. Twenty-eight pathways have been manually curated from the literature recently. Pathway predictions in AraCyc have also been recently updated with the latest functional annotations of Arabidopsis genes that use controlled vocabulary and literature evidence. AraCyc currently features 1,418 unique genes mapped onto 204 pathways with 1,156 literature citations. The Omics Viewer, a user data visualization and analysis tool, allows a list of genes, enzymes, or metabolites with experimental values to be painted on a diagram of the full pathway map of AraCyc. Other recent enhancements to both MetaCyc and AraCyc include implementation of an evidence ontology, which has been used to provide information on data quality, expansion of the secondary metabolism node of the pathway ontology to accommodate curation of secondary metabolic pathways, and enhancement of the cellular component ontology for storing and displaying enzyme and pathway locations within subcellular compartments.     

丁香属次生代谢产物及其与系统演化和地理环境的关联

环境塑造的植物次生代谢产物富于变化,也可能带有系统演化的信息。由于完整或具有系统学代表性的专属植物收集存在较大困难,使得次生代谢产物与系统学的关联研究尚不多见。通过文献汇总获得了存在于丁香属(Syringa)植物根、茎、叶和花中的10类377个次生代谢产物,主要涉及甲戊二羟酸途径、脱氧木酮糖磷酸酯途径以及莽草酸途径。在...     

In vitro metabolic fate of a novel structural class: evidence for the formation of a reactive intermediate on a benzothiophene moiety

The characterization of the metabolic pathways of new chemical entities with a special emphasis on detecting potentially reactive metabolites is increasingly being performed early in the drug discovery process. In the present study, the preliminary in vitro metabolic routes of a series of novel 2-substituted benzothiophene-containing discovery molecules were determined in fresh and cryopreserved hepatocyte suspensions. The objectives of this investigation were: (1) to use systematic LC/MS and LC/MS/MS analyses to provide a preliminary characterization of the in vitro metabolism of these compounds, with a particular focus on metabolites potentially arising from reactive intermediates, and (2) to identify potential lead molecules not associated with such metabolic pathways. This benzothiophene-containing series of compounds was characterized by the formation of five metabolites, at least two of which (dihydrodiol formation and glutathione adduct of the dihydrohydroxyl) were indicative of the formation of a reactive arene oxide intermediate. Tandem mass spectral analysis of the metabolites formed from a variety of structurally similar compounds demonstrated this reactive arene oxide intermediate to form on the 2-substituted benzothiophene moiety. Substitution of the benzothiophene with other functional groups eliminated these potentially toxic metabolites. The data presented here demonstrate the utility of performing metabolic route screens early in the drug discovery process prior to lengthy and costly radiolabeled studies, and furthermore, implicate a 2-substituted benzothiophene moiety as a substrate for formation of a reactive arene oxide intermediate.     

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.     

Lessons learned from metabolic engineering of cyanogenic glucosides

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits. The interplay of a multitude of biosynthetic pathways and the possibility of metabolic cross-talk combined with an incomplete understanding of the regulation of these pathways, explain why metabolic engineering of plant secondary metabolism is still in its infancy and subject to much trial and error. Cyanogenic glucosides are ancient defense compounds that release toxic HCN upon tissue disruption caused e.g. by chewing insects. The committed steps of the cyanogenic glucoside biosynthetic pathway are encoded by three genes. This unique genetic simplicity and the availability of the corresponding cDNAs have given cyanogenic glucosides pioneering status in metabolic engineering of plant secondary metabolism. In this review, lessons learned from metabolic engineering of cyanogenic glucosides in Arabidopsis thaliana (thale cress), Nicotiana tabacum cv Xanthi (tobacco), Manihot esculenta Crantz (cassava) and Lotus japonicus (bird’s foot trefoil) are presented. The importance of metabolic channelling of toxic intermediates as mediated by metabolon formation in avoiding unintended metabolic cross-talk and unwanted pleiotropic effects is emphasized. Likewise, the potential of metabolic engineering of plant secondary metabolism as a tool to elucidate, for example, the impact of secondary metabolites on plant–insect interactions is demonstrated.     

慢性疲劳综合征中学生运动前后尿液差异代谢物的比较

目的：探讨慢性疲劳综合征（CFS）中学生运动前后尿液的差异代谢物及其代谢通路特征,阐述慢性疲劳综合征的代谢机制。方法：依据美国疾病预防控制中心关于CFS的筛选标准,选取8名17~19岁男性CFS中学生作为受试对象,同时选择来自同一学校的8名同龄、同性别健康中学生作为对照组。受试者进行一次改良的哈佛台阶运动（上下台阶30次/min,持续3 min）,采集运动前后的尿液,以液相-质谱联用（LC-MS）法检测其差异代谢物;采用多维统计法对所检测到的代谢物进行主成分分析（PAC）和正交偏最小二乘判别分析（OPLS-DA）;通过MetPA数据库分析与差异代谢物相关的代谢通路。结果：与对照组相比较,运动前CFS组筛选出肌酸、吲哚乙醛、植物鞘氨醇和焦谷氨酸4个差异代谢物,其含量均显著下降（P<0.05或P<0.01）;运动后CFS组检测出11个差异代谢物,依次是壬二酸、甲基腺苷、乙酰肉碱、癸酸、皮质酮、肌酸、左炔诺孕酮、泛酸、焦谷氨酸、黄嘌呤核苷和黄尿酸,其中除甲基腺苷和肌酸比对照组显著升高（P<0.05）外,其他9个差异代谢物均较对照组显著降低（P<0.05或P<0.01）。将上述15个差异代谢物分别输入MetPA数据库进行代谢通路权重得分分析,结果显示,运动前CFS组只检测出精氨酸-脯氨酸代谢通路紊乱,标记代谢物为肌酸;而运动后检测出精氨酸-脯氨酸代谢、泛酸与辅酶A生物合成、类固醇激素生物合成3条代谢通路存在障碍,标记代谢物依次是：肌酸、泛酸和皮质酮。结论：运动干预前,CFS中学生的精氨酸-脯氨酸代谢通路紊乱被检出;施加运动后,又检测出其类固醇激素生物合成代谢通路与泛酸和辅酶A代谢通路也存在代谢紊乱情况。