Metabolic flux analysis ofShewanellaspp. reveals evolutionary robustness in central carbon metabolism

2009 ◽  
Vol 102 (4) ◽  
pp. 1161-1169 ◽  
Author(s):  
Yinjie J. Tang ◽  
Hector Garcia Martin ◽  
Paramvir S. Dehal ◽  
Adam Deutschbauer ◽  
Xavier Llora ◽  
...  
Keyword(s):  
Carbon Metabolism ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Central Carbon ◽  
Evolutionary Robustness

scholarly journals Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator

2011 ◽  
Vol 286 (41) ◽  
pp. 35782-35794 ◽  
Author(s):  
Semen A. Leyn ◽  
Xiaoqing Li ◽  
Qingxiang Zheng ◽  
Pavel S. Novichkov ◽  
Samantha Reed ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Carbon Metabolism ◽  
Metabolic Flux ◽  
Gene Expression Pattern ◽  
Bioinformatic Analysis ◽  
Central Carbon Metabolism ◽  
Comparative Genomic ◽  
Binding Motif ◽  
Flux Analysis ◽  
Central Carbon

Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis ΔhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [13C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

scholarly journals Reproductive Potential of Yeast Cells Depends on Overall Action of Interconnected Changes in Central Carbon Metabolism, Cellular Biosynthetic Capacity, and Proteostasis

2020 ◽  
Vol 21 (19) ◽  
pp. 7313
Author(s):  
Roman Maslanka ◽  
Renata Zadrag-Tecza
Keyword(s):  
Calorie Restriction ◽  
Carbon Metabolism ◽  
Metabolic Flux ◽  
Reproductive Potential ◽  
Central Carbon Metabolism ◽  
Yeast Cells ◽  
Reproductive Capacity ◽  
Hexokinase 2 ◽  
Central Carbon

Carbon metabolism is a crucial aspect of cell life. Glucose, as the primary source of energy and carbon skeleton, determines the type of cell metabolism and biosynthetic capabilities, which, through the regulation of cell size, may affect the reproductive capacity of the yeast cell. Calorie restriction is considered as the most effective way to improve cellular physiological capacity, and its molecular mechanisms are complex and include several nutrient signaling pathways. It is widely assumed that the metabolic shift from fermentation to respiration is treated as a substantial driving force for the mechanism of calorie restriction and its influence on reproductive capabilities of cells. In this paper, we propose another approach to this issue based on analysis the connection between energy-producing and biomass formation pathways which are closed in the metabolic triangle, i.e., the respiration-glycolysis-pentose phosphate pathway. The analyses were based on the use of cells lacking hexokinase 2 (∆hxk2) and conditions of different glucose concentration corresponding to the calorie restriction and the calorie excess. Hexokinase 2 is the key enzyme involved in central carbon metabolism and is also treated as a calorie restriction mimetic. The experimental model used allows us to explain both the role of increased respiration as an effect of calorie restriction but also other aspects of carbon metabolism and the related metabolic flux in regulation of reproductive potential of the cells. The obtained results reveal that increased respiration is not a prerequisite for reproductive potential extension but rather an accompanying effect of the positive role of calorie restriction. More important seems to be the changes connected with fluxes in central carbon metabolic pathways resulting in low biosynthetic capabilities and improved proteostasis.

Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of 13C metabolic flux analysis

2011 ◽  
Vol 109 (3) ◽  
pp. 763-771 ◽  
Author(s):  
Martin Rühl ◽  
Beat Rupp ◽  
Katharina Nöh ◽  
Wolfgang Wiechert ◽  
Uwe Sauer ◽  
...  
Keyword(s):  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
13C Metabolic Flux Analysis ◽  
Central Carbon

scholarly journals Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chao Wu ◽  
Ryan A. Herold ◽  
Eric P. Knoshaug ◽  
Bo Wang ◽  
Wei Xiong ◽  
...  
Keyword(s):  
Growth Rate ◽  
Carbon Metabolism ◽  
Metabolic Flux ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Central Carbon Metabolism ◽  
Nitrogen Fertilizers ◽  
Ammonium Excretion ◽  
Diazotrophic Bacteria ◽  
Central Carbon

Abstract Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner–Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

scholarly journals Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

2016 ◽  
Vol 113 (47) ◽  
pp. E7428-E7437 ◽  
Author(s):  
Drew G. Michael ◽  
Ezekiel J. Maier ◽  
Holly Brown ◽  
Stacey R. Gish ◽  
Christopher Fiore ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Carbon Metabolism ◽  
Regulatory Networks ◽  
Metabolic Flux ◽  
Xylose Fermentation ◽  
Fold Increase ◽  
Central Carbon Metabolism ◽  
Central Carbon ◽  
Ethanol Production Rate ◽  
Transcriptional State

The ability to rationally manipulate the transcriptional states of cells would be of great use in medicine and bioengineering. We have developed an algorithm, NetSurgeon, which uses genome-wide gene-regulatory networks to identify interventions that force a cell toward a desired expression state. We first validated NetSurgeon extensively on existing datasets. Next, we used NetSurgeon to select transcription factor deletions aimed at improving ethanol production in Saccharomyces cerevisiae cultures that are catabolizing xylose. We reasoned that interventions that move the transcriptional state of cells using xylose toward that of cells producing large amounts of ethanol from glucose might improve xylose fermentation. Some of the interventions selected by NetSurgeon successfully promoted a fermentative transcriptional state in the absence of glucose, resulting in strains with a 2.7-fold increase in xylose import rates, a 4-fold improvement in xylose integration into central carbon metabolism, or a 1.3-fold increase in ethanol production rate. We conclude by presenting an integrated model of transcriptional regulation and metabolic flux that will enable future efforts aimed at improving xylose fermentation to prioritize functional regulators of central carbon metabolism.

scholarly journals Metabolic mechanism of Saccharomyces cerevisiae under different physiological conditions based on non-stationary 13C metabolic flux analysis

2020 ◽  
Author(s):  
Huan Li ◽  
Min Chen ◽  
Peng Liu ◽  
Shuai Wang ◽  
JY Xia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Energy Charge ◽  
Flux Analysis ◽  
13C Metabolic Flux Analysis ◽  
Metabolic Flux Distribution ◽  
Central Carbon

Abstract Crabtree effect is well known for Saccharomyces cerevisiae, and is defined as glucose-induced repression of respiratory flux. Even though a number of hypotheses have been formulated, its triggering mechanisms are still unknown. At present, the information about intracellular metabolic flux can be obtained by the 13C isotope labeling experiments. 13C metabolic flux analysis(13C-MFA) is a traditional method for calculating metabolic flux based on isotopic steady state. Another new method (INST-13C-MFA: Isotopically nonstationary metabolic flux analysis) based on isotope non-steady state is being used by researchers. In this review, we have chemostatized S. cerevisiae at three different dilution rates (D=0.12, 0.22, 0.32 h-1) and obtained the metabolic flux distribution of the intracellular central carbon metabolic of S. cerevisiae using INST-13C-MFA. Combined with the metabolome and metabolic fluxome data, we found obvious metabolic flux shift under the three different physiological states. In this process, pyruvate decarboxylase, ethanol dehydrogenase and acetyl-CoA synthase(AcCoA) catalyzed reactions were key points. Negative correlation between relative flux of embden meyerh of pathway(EMP) and tricarboxylic acid cycle(TCA) and biomass yield, while positive correlation for pentose phosphate pathway(PPP) were observed. Yield of acetate and glycerol did not change significantly, while that of ethanol increased sharply. In the central carbon metabolism (CCM), most of the carbon flux (70%) was directed to the EMP. At the same time, the energy charge increased with dilution rate, and the cell's energy supply mode gradually shifted from oxidative respiration to substrate level phosphorylation mode.

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