Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a Δ
            <i>edd</i>
            Δ
            <i>eda</i>
            Mutant on Mannitol

Richhardt, Janine; Bringer, Stephanie; Bott, Michael

doi:10.1128/aem.01166-12

Cited by 43 publications

(37 citation statements)

References 26 publications

(19 reference statements)

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“…Despite the long-time use in biotechnology, the regulation of carbon and energy metabolism in G. oxydans is largely unknown. Based on the genome sequence of strain 621H (DSM2343) (3), we recently applied DNA microarrays to analyze the influence of pH and oxygen limitation on global gene expression and to study mutants defective in the PPP or the EDP (14,15).…”

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confidence: 99%

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

Hanke¹,

Nöh²,

Noack³

et al. 2013

Appl Environ Microbiol

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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.

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confidence: 99%

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

Hanke¹,

Nöh²,

Noack³

et al. 2013

Appl Environ Microbiol

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“…Subsequently, the synthesized pyruvate is likely to be converted to acetate (Fig. 2D) by pyruvate decarboxylase (GOX1081) and aldehyde dehydrogenase (GOX2018) acting sequentially in the cytoplasm (29,38). Indeed, no alternative pathway for the production of acetate from pyruvate appears to exist in G. oxydans 621H, which lacks phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), and pyruvate:quinone oxidoreductase (EC 1.2.2.2) (29).…”

Section: Discussionmentioning

confidence: 99%

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

Sheng

Zhang

et al. 2015

Appl Environ Microbiol

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b D-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used D-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on D-lactate was therefore investigated. Although two putative NAD-independent D-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing D-lactate, GOX1253 was the only enzyme able to support the D-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward D-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward D-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in D-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on D-lactate, but also provides new insights for applications of the important industrial microbe and the novel D-lactate oxidase.

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“…After conjugative transfer of pAJ-cyoBACD from E. coli S17-1 to G. oxydans ⌬upp, colonies were screened by PCR for chromosomal integration of pAJ-cyoBACD via a single homologous crossover event using the oligonucleotides cyoBACD_ mut_forw and cyoBACD_mut_rev. Positive clones were inoculated overnight in 20 ml of mannitol medium containing cefoxitin and kanamycin and used as a preculture for fluorouracil-enforced vector excision, as described previously (5). Fluorouracil-resistant colonies were screened by PCR for the chromosomal deletion of cyoBACD using the oligonucleotide pair cyoBACD_mut_forw/cyoBACD_mut_rev and cyoBACD_forw/ cyoBACD_rev.…”

Section: Methodsmentioning

confidence: 99%

“…After complete consumption of mannitol, the OTRs of all three strains decreased to 2 to 5 mmol liter Ϫ1 h Ϫ1 , showing that only a small fraction of fructose was either oxidized in the periplasm to ketofructose or in the cytoplasm to CO 2 and acetate. During the cultivations, the pH dropped from 6 to 4.5, presumably due to acetate formation (5).…”

Section: Mmol Litermentioning

confidence: 99%

“…Important applications are in the production of vitamin C and of 6-amino-L-sorbose, an intermediate in the synthesis of the antidiabetes drug miglitol (3). Only small fractions of the carbon and energy sources are metabolized in the cytoplasm, either via the pentose phosphate pathway or via the Entner-Doudoroff pathway, and it was demonstrated that the former one is of major importance for growth on mannitol and glucose (4,5). Moreover, it was recently shown that the pentose phosphate pathway has partially cyclic operation during growth on glucose (6).…”

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Evidence for a Key Role of Cytochrome bo3 Oxidase in Respiratory Energy Metabolism of Gluconobacter oxydans

Richhardt

Luchterhand

Bringer

et al. 2013

Journal of Bacteriology

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The obligatory aerobic acetic acid bacterium Gluconobacter oxydans oxidizes a variety of substrates in the periplasm by membrane-bound dehydrogenases, which transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo 3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen. In this study, mutants lacking either of these terminal oxidases were characterized. Deletion of the cydAB genes for cytochrome bd had no obvious influence on growth, whereas the lack of the cyoBACD genes for cytochrome bo 3 severely reduced the growth rate and the cell yield. Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints of a low-oxygen affinity of cytochrome bd oxidase were obtained, which were supported by measurements of oxygen consumption in a respirometer. The H ؉ /O ratio of the ⌬cyoBACD mutant with mannitol as the substrate was 0.56 ؎ 0.11 and more than 50% lower than that of the reference strain (1.26 ؎ 0.06) and the ⌬cydAB mutant (1.31 ؎ 0.16), indicating that cytochrome bo 3 oxidase is the main component for proton extrusion via the respiratory chain. Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields, both in the wild type and the ⌬cyoBACD mutant, suggesting that cytochrome bo 3 might be a rate-limiting factor of the respiratory chain.

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Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a Δ edd Δ eda Mutant on Mannitol

Cited by 43 publications

References 26 publications

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

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

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

Evidence for a Key Role of Cytochrome bo3 Oxidase in Respiratory Energy Metabolism of Gluconobacter oxydans

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