Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

Abstract

In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13 C-based metabolic flux analysis ( 13 C-MFA) in combination with transcriptomics and enzyme assays. For 13 C-MFA, cells were cultivated with specifically 13 C-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. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13 C-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.

Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells

“…We therefore propose a model for the immediate response to acute oxidative stress in which oxidative PPP is immediately activated, leading to increased NADPH production and reversal of non-oxidative PPP compared to unstressed steady state ( Figure 7C). This early redirection of flux is independent of the inhibition of glycolytic flux by GAPDH or PKM2, which, however, might favor carbon cycling in the PPP and thus maximize NADPH generation (Hanke et al, 2013;Kruger and von Schaewen, 2003;Siedler et al, 2012).…”

Evaluating the Feasibility of Microbial Electrosynthesis Based on Gluconobacter oxydans

“…From this result three possible reasons for the current decrease by increasing pH have to be differentiated: 1) microbial physiology, that is, metabolic activity change; 2) altered cell‐mediator interaction, for example, due to membrane integrity changes and 3) thermodynamic shifts, for example, impeded electron transfer. The metabolism of G. oxydans shifts from pH 6.5 to pH 3.7 from a partly cytoplasmic metabolism with biomass build up through the pentose phosphate pathway to a mostly periplasmic metabolism based on membrane‐bound enzymes . Thus, at pH 6.5 more electrons could be kept in the cytoplasm, possibly not being accessible for the mediator, while at pH 3.7 the electrons are highly present in the periplasm and therefore easier to take up by the mediator, resulting in higher current densities at acidic pH.…”

13C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling

“…Thus, 13 C-MFA depends on more distant labeling measurements, for example, of intermediates in the lower glycolytic pathway such as 3PG and PEP (Ahn and Antoniewicz, 2013). LC-MS and LC-MS/MS based approaches have provided direct labeling information on pentose phosphate pathway intermediates and fragments (Hanke et al, 2013; Rühl et al, 2012); however, these methods are often costly and laborious, as analysis of these intracellular metabolites requires rapid quenching and efficient extraction techniques, and in many cases large sample sizes due to low concentrations of these metabolites inside cells.…”