Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B12 producing strain Pseudomonas denitrificans using 13C-labeled glucose

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).

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Metabolic Flux Analysis of Simultaneous Production of Vitamin B12 and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-021-03584-y ◽

2021 ◽

Author(s):

Yuhan Zhang ◽

Xiaolian Li ◽

Ziqiang Wang ◽

Yunshan Wang ◽

Yuanyuan Ma ◽

...

Keyword(s):

Vitamin B12 ◽

Propionic Acid ◽

Metabolic Flux Analysis ◽

Fermentation Process ◽

Metabolic Flux ◽

Flux Analysis ◽

Propionibacterium Freudenreichii ◽

Simultaneous Production

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

International Journal of Molecular Sciences ◽

10.3390/ijms21197313 ◽

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.

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

Biotechnology and Bioengineering ◽

10.1002/bit.24344 ◽

2011 ◽

Vol 109 (3) ◽

pp. 763-771 ◽

Cited By ~ 72

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

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

Scientific Reports ◽

10.1038/s41598-019-49717-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603577113 ◽

2016 ◽

Vol 113 (47) ◽

pp. E7428-E7437 ◽

Cited By ~ 22

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.

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