Analysis Of Metabolic Regulation Research Articles

Astaxanthin overproduction of Phaffia rhodozyma PR106 under titanium dioxide stress by transcriptomics and metabolic regulation analysis

In this study, astaxanthin yield of Phaffia rhodozyma PR106 increased significantly under titanium dioxide (TiO2) stress, and the yield of lycopene and β-carotene also increased significantly, as well as the yield of violaxanthin and lutein significantly decreased; in addition, TiO2 stress promoted cell division and changed cell morphology of PR106. Then, the mechanism of increasing astaxanthin yield was studied by transcriptomics and related metabolic regulation. The results showed that astaxanthin accumulation in PR106 was not directly related to mRNA transcription and post-translational modifications regulation under TiO2 stress; TiO2 stress accelerated glucose uptake of yeast, promoted reuse of ethanol, and increased the formation of acetyl-CoA and ATP. The more carbon flux was shifted to astaxanthin synthesis pathway and weakened carotenoids accumulation in astaxanthin branch pathway to improve the astaxanthin production of PR106. The metabolism regulation of ROS could continue in the PR106 strain.

Non-invasive evaluation of microcirculation and metabolic regulation using flow mediated skin fluorescence (FMSF): Technical aspects and methodology

This paper describes a new technique for noninvasive diagnostic analysis of metabolic regulation and the microcirculation. Flow Mediated Skin Fluorescence (FMSF) is based on monitoring the intensity of NADH fluorescence emitted from skin tissue on the forearm. The principles of the technique are discussed, in particular, concerning experimental procedures and the definition and interpretation of the measured parameters. The unique features and potential avenues for development of the FMSF technique are also outlined.

N-terminal Acetylation Levels Are Maintained During Acetyl-CoA Deficiency in Saccharomyces cerevisiae

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality control and stress tolerance. Despite its prevalence, the mechanisms regulating Nt-acetylation are still nebulous. Here, we present the first global study of Nt-acetylation in yeast cells as they progress to stationary phase in response to nutrient starvation. Surprisingly, we found that yeast cells maintain their global Nt-acetylation levels upon nutrient depletion, despite a marked decrease in acetyl-CoA levels. We further observed two distinct sets of protein N termini that display differential and opposing Nt-acetylation behavior upon nutrient starvation, indicating a dynamic process. The first protein cluster was enriched for annotated N termini showing increased Nt-acetylation in stationary phase compared with exponential growth phase. The second protein cluster was conversely enriched for alternative nonannotated N termini (i.e. N termini indicative of shorter N-terminal proteoforms) and, like histones, showed reduced acetylation levels in stationary phase when acetyl-CoA levels were low. Notably, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the steady-state levels of these proteins were regulated both by starvation and NatA activity. In summary, this study represents the first comprehensive analysis of metabolic regulation of Nt-acetylation and reveals that specific, rather than global, Nt-acetylation events are subject to metabolic regulation.

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis.

Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain polyunsaturated fatty acids, mainly from wild-caught ocean fish, is struggling to meet the increasing demand for EPA. Production of EPA by microorganisms may be an alternative, effective and economical method. The oleaginous fungus Pythium splendens RBB-5 is a potential source of EPA, and thanks to the simple culture conditions required, high yields can be achieved in a facile manner. In the study, lipid metabolomics was performed in an attempt to enhance EPA biosynthesis in Pythium splendens. Synthetic, metabolic regulation and gene expression analyses were conducted to clarify the mechanism of EPA biosynthesis, and guide optimization of EPA production. The results showed that the Δ6 desaturase pathway is the main EPA biosynthetic route in this organism, and ∆6, ∆12 and Δ17 desaturases are the rate-limiting enzymes. All the three desaturase genes were separately introduced into the parent strain to increase the flow of fatty acids into the Δ6 desaturase pathway. Enhanced expression of these key enzymes, in combination with improved regulation of metabolism, resulted in a maximum yield of 1.43g/L in the D12 transgenic strain, which represents a tenfold increase over the parent strain before optimization. This is the higher EPA production yield yet reported for a microbial system. Our findings may allow the production of EPA at an industrial scale, and the strategy employed could be used to increase the production of EPA or other lipids in oleaginous microorganisms.

Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose.

We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus.

Metabolic regulation of <i>Escherichia coli</i> cultivated under anaerobic and aerobic conditions in response to the specific pathway gene knockouts

Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic fluxes together with fermentation data. The effects of the knockout of such genes as pflA, pta, ppc, pykF, adhE, and ldhA on the metabolic changes were analyzed for the case under anaerobic condition. The effects of the knockout of such genes as pgi, zwf, gnd, ppc pck, pyk, and lpdA on the metabolic changes were also analyzed for the case under aerobic condition. The metabolic regulation analysis was made focusing on the roles of transcription factors.

A New Insight into the Main Metabolic Regulation of Escherichia coli Based on Systems Biology Approach

The systems biology approach was considered for the analysis of metabolic regulation in Escherichia coli. In particular, it was shown that the feed-forward and feed-back regulations are formed in the metabolic regulation by enzymatic and transcriptional regulations, where these contribute to the robustness of the cell system. Moreover, the effects of the specific pathway gene knockout such as Δpgi, Δzwf, and Δpyk on the metabolic regulations were clarified based on systems biology approach. Mathematical modeling with computer simulation was also made for the main metabolism.

Response to dietary L-glutamine supplementation in weaned piglets: a serum metabolomic comparison and hepatic metabolic regulation analysis

Response to dietary L-glutamine supplementation in weaned piglets: a serum metabolomic comparison and hepatic metabolic regulation analysis

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.

Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

BackgroundA metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C.ResultsThis study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions.ConclusionResults suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.

メタボローム, トランスクリプトーム解析を活用した下面発酵酵母の代謝制御機能及び遺伝子機能の解明

[in Japanese]

Network-level analysis of metabolic regulation in the human red blood cell using random sampling and singular value decomposition.

BackgroundExtreme pathways (ExPas) have been shown to be valuable for studying the functions and capabilities of metabolic networks through characterization of the null space of the stoichiometric matrix (S). Singular value decomposition (SVD) of the ExPa matrix P has previously been used to characterize the metabolic regulatory problem in the human red blood cell (hRBC) from a network perspective. The calculation of ExPas is NP-hard, and for genome-scale networks the computation of ExPas has proven to be infeasible. Therefore an alternative approach is needed to reveal regulatory properties of steady state solution spaces of genome-scale stoichiometric matrices.ResultsWe show that the SVD of a matrix (W) formed of random samples from the steady-state solution space of the hRBC metabolic network gives similar insights into the regulatory properties of the network as was obtained with SVD of P. This new approach has two main advantages. First, it works with a direct representation of the shape of the metabolic solution space without the confounding factor of a non-uniform distribution of the extreme pathways and second, the SVD procedure can be applied to a very large number of samples, such as will be produced from genome-scale networks.ConclusionThese results show that we are now in a position to study the network aspects of the regulatory problem in genome-scale metabolic networks through the use of random sampling.Contact: palsson@ucsd.edu

Expression of Peroxidases During Seedling Growth in Ebenus Cretica L. as Affected by Light and Temperature Treatments

Peroxidase (POD) activity and isoform patterns were investigated during seedling growth (up to 20 days) of Ebenus cretica L. Seeds germinated to approx. 100% after a 24-h imbibition. Seedling growth proceeded smoothly, in both light and dark conditions. No seedling growth was noticed at 4°C. A positive effect of light and increasing temperature (4, 10, 16, 22 and 28°C) on seedlings growth, lignin content and POD activity was observed. Lignin content was 2.5 times higher in seedlings grown under light than in seedlings grown under darkness. Seedlingsȁ9 POD activity was higher in acid pH (5.5) in comparison to neutral pH (7.0). These activities were higher in seedlings grown under darkness than in those grown under light; since additional POD isoforms were expressed in dark conditions. The increase in POD activity was accompanied with the appearance of new POD isoforms correlated with the growth of the seedlings. Four soluble anionic POD isoforms (named A1, A2, A3 and A4) and three soluble cationic POD isoforms (named C1, C2 and C3) were displayed depending on the treatment and the course of growth. POD isoforms were detected in gel after PAGE. The fast migrating (A4) isoform, which appeared in the dark-grown seedlings as well as on day 20 at 28°C in the temperature treatment, was separated by DEAE–Sepharose column chromatography. A slow migrating C1 isoform slightly appeared in both 4 and 10°C temperature treatments and could be related to the low temperature treatments, while A1, A2, A3 and C2 to the growth stage of seedlings. The expression of seven POD isoforms during seedling growth seems to be related to different developmental events of growth and could be used as useful biochemical markers in the analysis of metabolic regulation in seedling growth of Ebenus cretica.

Quantitative Proteomic Analysis of Metabolic Regulation by Copper Ions in Methylococcus capsulatus (Bath)

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them.

Metabolic flux analysis based on 13C-labeling experiments and integration of the information with gene and protein expression patterns.

The recent progress on metabolic systems engineering was reviewed, in particular focusing on the metabolic flux analysis (MFA) based on the isotopomer distribution obtained using NMR and/or GC-MS. After the brief explanation of how to estimate the metabolic flux distribution (MFD) based on 13C-labeling experiments, the metabolic regulation analysis was made based on the protein expression patterns obtained by two-dimensional electrophoresis (2DE) together with enzyme activity data, and the gene expression patterns obtained by RT-PCR analysis. The particular application was considered for Escherichia coli. The effect of culture conditions such as different carbon sources (glucose, gluconate, glycerol, acetate, etc.) and different dissolved oxygen (DO) concentration, etc. on the metabolism was investigated. The effect of some single-gene knockout such as pgi-, pyk-, and gnd- was also investigated. It was found to be quite useful to integrate the information obtained from metabolic flux analysis, gene and protein expressions as well as intracellular metabolite concentrations to understand the overall picture of metabolic regulation.

Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements.

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP(+) ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP(+)-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.

Network-based analysis of metabolic regulation in the human red blood cell

Reconstruction of cell-scale metabolic networks is now possible. A description of allowable metabolic network functions can be obtained using extreme pathways, which are the convex basis vectors of the solution space containing all steady state flux distributions. However, only a portion of these allowable network functions are physiologically possible due to kinetic and regulatory constraints. Methods are now needed that enable us to take a defined metabolic network and deduce candidate regulatory structures that control the selection of these physiologically relevant states. One such approach is the singular value decomposition (SVD) of extreme pathway matrices ( P), which allows for the characterization of steady state solution spaces. Eigenpathways, which are the left singular vectors from the SVD of P, can be described and categorized by their biochemical function. SVD of P for the human red blood cell showed that the first five eigenpathways, out of a total of 23, effectively characterize all the relevant physiological states of red blood cell metabolism calculated with a detailed kinetic model. Thus, with five degrees of freedom the magnitude and nature of the regulatory needs are defined. Additionally, the dominant features of these first five eigenpathways described key metabolic splits that are indeed regulated in the human red blood cell. The extreme pathway matrix is derived directly from network topology and only knowledge of V max values is needed to reach these conclusions. Thus, we have implemented a network-based analysis of regulation that complements the study of individual regulatory events. This topological approach may provide candidate regulatory structures for metabolic networks with known stoichiometry but poorly characterized regulation.

Model Complexity has a Significant Effect on the Numerical Value and Interpretation of Metabolic Sensitivity Coefficients

Logarithmic sensitivity coefficients were promulgated for the analysis of metabolic regulation about 20 years ago. Interest in their use has risen significantly since their introduction. However, no comprehensive evaluation of the utility of these metabolic sensitivity coefficients is available for realistic metabolic models. In this study, logarithmic sensitivity coefficients calculated from three progressively simpler metabolic models of red blood cell metabolism were compared. Two simpler models were obtained from a comprehensive red cell model by first removing volume regulation, and second by removing two pathways. The comparisons of sensitivity coefficients obtained for these three models showed that model complexity has significant effects on the numerical values and interpretation of the metabolic sensitivity coefficients. Additionally, it was found that the physiochemical volume regulatory mechanism, namely electroneutrality and osmotic balances, play an important role in the red cell metabolic flux control. In general, there is no proportionate relationship between sensitivity coefficients calculated from the three different red cell metabolic models or other simple red cell models reported in the early literature. Some sensitivity coefficients determined by different models even have opposite signs. Thus, analysis of incomplete metabolic models can be seriously misleading and produce inappropriate indicators of the characteristics of a full model for the same metabolic network.

Taking enzyme kinetics out of control; putting control into regulation.

The matrix formulation of metabolic control analysis, which states that multiplying the elasticity matrix for any system by the corresponding control matrix yields an identity matrix, can be transformed into a statement that multiplying a matrix expressing internal regulatory properties by a matrix expressing external regulatory properties also yields an identity matrix. This transformation supplies the formal basis for metabolic regulation analysis, and provides the key to determining the control structure of a system without the need to know the exact changes in enzyme activities that are made to measure control coefficients.

Determination of Flux Control Coefficients from transient metabolite concentrations.

Flux Control Coefficients have been used in the analysis of metabolic regulation for quantifying the effect of an enzyme on the overall steady-state flux. However, the experimental determination of these coefficients is very time-consuming, involving either determining the individual enzyme kinetics or perturbing the enzyme activity by genetic or other means. We developed a methodology that enables the determination of the Flux Control Coefficients from transient metabolite concentrations without knowing kinetic parameters. The transient states can be generated by changing the incubation conditions or adding the initial substrate. This approach is suitable for investigating metabolic regulation in vivo or multiple enzyme systems in vitro. It is particularly helpful if used in conjunction with n.m.r. measurements. The approach is based on a relationship between transient metabolite concentrations and the Flux Control Coefficients. The methodology has been improved from our previous results, and it is illustrated by three examples with simple pathway topologies.