Pentose Phosphate Pathway Flux Analysis for Glycopeptide Antibiotic Vancomycin Production During Glucose Limited Cultivation of Amycolatopsis orientalis

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

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Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8587-8596.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8587-8596 ◽

Cited By ~ 140

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Oskar Zelder ◽

Elmar Heinzle ◽

Christoph Wittmann

Keyword(s):

Corynebacterium Glutamicum ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Carbon Sources ◽

Pentose Phosphate ◽

Metabolic Effects ◽

Flux Analysis ◽

Lysine Production ◽

Fructose 1,6 Bisphosphatase

ABSTRACT The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfb r . The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

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Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.1604-1609.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 1604-1609 ◽

Cited By ~ 127

Author(s):

Marie Jeppsson ◽

Björn Johansson ◽

Bärbel Hahn-Hägerdal ◽

Marie F. Gorwa-Grauslund

Keyword(s):

Saccharomyces Cerevisiae ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Ethanol Yield ◽

Xylose Reductase ◽

Pentose Phosphate ◽

Xylitol Production ◽

Xylose Consumption ◽

Xylitol Yield ◽

Pathway Flux

ABSTRACT In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD+. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g−1) and the lowest xylitol yield (0.05 g g−1) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.

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Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

Cell Reports ◽

10.1016/j.celrep.2017.12.050 ◽

2018 ◽

Vol 22 (2) ◽

pp. 512-522 ◽

Cited By ~ 30

Author(s):

Susan J. Gelman ◽

Fuad Naser ◽

Nathaniel G. Mahieu ◽

Lisa D. McKenzie ◽

Gavin P. Dunn ◽

...

Keyword(s):

Oxidative Stress ◽

Pentose Phosphate Pathway ◽

Pentose Phosphate ◽

Pathway Flux

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The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

mSystems ◽

10.1128/msystems.00091-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Poonam Jyoti ◽

Manu Shree ◽

Chandrakant Joshi ◽

Tulika Prakash ◽

Suvendra Kumar Ray ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Pentose Phosphate Pathway ◽

Ralstonia Solanacearum ◽

Metabolic Flux ◽

Pentose Phosphate ◽

Flux Analysis ◽

Content Type ◽

Metabolic Network Analysis ◽

Balance Analysis

ABSTRACT In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [13C]glucose (99% [1-13C]glucose or 99% [1,2-13C]glucose or 40% [13C6]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3, 4). This work provided evidence from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood.

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