Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology

Hanke, Tanja; Richhardt, Janine; Polen, Tino; Sahm, Hermann; Bringer, Stephanie; Bott, Michael

doi:10.1016/j.jbiotec.2011.12.020

Cited by 42 publications

(33 citation statements)

References 38 publications

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“…DNA microarray analysis. RNA preparation, cDNA labeling, and DNA microarray analysis were performed as described recently by Hanke et al (14). The samples for DNA microarray analysis were taken after 8 h and 14.25 h, and three biological replicates were performed.…”

Section: Methodsmentioning

confidence: 99%

“…The cultivations were performed in triplicate starting from independent precultures. RNA was prepared from the six samples and used for comparative DNA microarray analysis as described recently (14). In total, 454 genes showed differential expression: 227 genes had an mRNA ratio (sample point II to sample point I) of Ն2.0, and 227 genes had an mRNA ratio of Յ0.5.…”

Section: Intracellular Carbon Fluxes Of Phosphorylated Compoundsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Intracellular Carbon Fluxes Of Phosphorylated Compoundsmentioning

confidence: 99%

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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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“…(47,48). In this context it is interesting that G. oxydans possesses two different F 1 F o -type ATP synthases (49), one of which might use Na ϩ as coupling ion (8,38). The comparative DNA microarray analysis of the cyo deletion mutant versus the reference strain showed an upregulation of genes (GOX2167 to GOX2175) coding for the putative Na ϩ -dependent F 1 F o -ATPase (see Table S1 in the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

“…For DNA microarray analysis of strain G. oxydans ⌬upp ⌬cyoBACD versus the reference strain G. oxydans ⌬upp, cells were cultivated in baffled shake flasks with mannitol as the carbon source and harvested at an OD 600 of 1.5. RNA extraction, cDNA labeling and DNA microarray analysis were performed as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

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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Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling

Carniel

Santos

Júnior

et al. 2023

Biotechnology Journal

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Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be a biocatalyst to biodepolymerize PET. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. We found that this yeast tolerates high EG concentrations (up to 2 M) based on maximum non-inhibitory concentration (MNIC) tests.Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 vs. 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group.Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product.

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Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology

Cited by 42 publications

References 38 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

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

Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling

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