NADPH‐dependent reductive biotransformation with <i>Escherichia coli</i> and its <i>pfkA</i> deletion mutant: Influence on global gene expression and role of oxygen supply

Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael

doi:10.1002/bit.25271

Cited by 4 publications

(4 citation statements)

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“…Under conditions of normal aerobic growth, a reductive pathway involving a NADPH-dependent specific enzyme appears to maintain SoxR in a reduced inactive form (Figure ). Several studies suggested that the SoxRS regulon is responsive to intracellular NADPH/NADP + . − Recently, the analysis of fluorescence-activated cell sorting revealed that the transcription in SoxR is linked to the level of NADPH . NADPH-dependent SoxR reduction is enzyme-mediated, allowing for a rapid adjustment to changes in cellular conditions. , The identity of SoxR reductase has not been characterized, and there still remain reducing systems other than these systems.…”

Section: Discussionmentioning

confidence: 99%

Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

2014

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The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, functions as a sensor of oxidative stress in Escherichia coli. The transcriptional activity of SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. Electrochemistry measurements on DNA-modified electrodes have shown a dramatic shift in the reduction potential of SoxR from -290 to +200 mV with the promoter DNA-bound [ Gorodetsky , A. A. , Dietrich , L. E. P. , Lee , P. E. , Demple , B. , , Newman , D. K. , and Barton , J. K. ( 2008 ) DNA binding shifts the reduction potential of the transcription factor SoxR , Proc. Natl. Acad. Sci. U.S.A. 105 , 3684 - 3689 ]. To determine the change of the SoxR reduction potential using the new condition, the one-electron oxidation-reduction properties of [2Fe-2S] cluster in SoxR were investigated in the absence and presence of the DNA. The [2Fe-2S] cluster of SoxR was completely reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CRP) in the presence of a NADPH generating system (glucose 6-dehydrogenase and glucose-6 phosphate), indicating that CRP can serve as an NADPH-dependent electron carrier for SoxR. The reduction potential of SoxR was measured from equilibrium data coupled with NADPH and CRP in the presence of electron mediators. The reduction potentials of DNA-bound and DNA-free states of SoxR were -320 and -293 mV versus NHE (normal hydrogen electrode), respectively. These results indicate that DNA binding causes a moderate shift in the reduction potential of SoxR.

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Section: Discussionmentioning

confidence: 99%

Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

2014

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“…Disrupting the EMPP by knocking out the phosphofructokinase I (PfkA) gene also resulted in re-routing the glycolytic flux through the PPP (~ 60% of the glycolytic flux) and the native EDP (~ 14% of glycolytic flux) [16]. The ΔpfkA recombinant E. coli strains showed enhanced production of 1,3-diaminopropane [17], hydrogen and ethanol [18], lycopene [19], and methyl 3-hydroxybutyrate [20]. However, these strains showed a more significant decrease in growth rate compared with the Δpgi mutants owing to the partial cyclization of PPP, which may produce excess NADPH [21].…”

Section: Biotechnology For Biofuels and Bioproductsmentioning

confidence: 99%

“…The Δ pfkA recombinant E . coli strains showed enhanced production of 1,3-diaminopropane [ 17 ], hydrogen and ethanol [ 18 ], lycopene [ 19 ], and methyl 3-hydroxybutyrate [ 20 ]. However, these strains showed a more significant decrease in growth rate compared with the Δ pgi mutants owing to the partial cyclization of PPP, which may produce excess NADPH [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an Entner–Doudoroff pathway-activated Escherichia coli

Kim

Cho

Bang

et al. 2022

Biotechnol Biofuels

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Background Escherichia coli have both the Embden–Meyerhof–Parnas pathway (EMPP) and Entner–Doudoroff pathway (EDP) for glucose breakdown, while the EDP primarily remains inactive for glucose metabolism. However, EDP is a more favorable route than EMPP for the production of certain products. Results EDP was activated by deleting the pfkAB genes in conjunction with subsequent adaptive laboratory evolution (ALE). The evolved strains acquired mutations in transcriptional regulatory genes for glycolytic process (crp, galR, and gntR) and in glycolysis-related genes (gnd, ptsG, and talB). The genotypic, transcriptomic and phenotypic analyses of those mutations deepen our understanding of their beneficial effects on cellulosic biomass bio-conversion. On top of these scientific understandings, we further engineered the strain to produce higher level of lycopene and 3-hydroxypropionic acid. Conclusions These results indicate that the E. coli strain has innate capability to use EDP in lieu of EMPP for glucose metabolism, and this versatility can be harnessed to further engineer E. coli for specific biotechnological applications.

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“…Our results demonstrate that Δ pgi mutants can grow under anaerobic conditions and co-produce H 2 and ethanol at near-theoretical yields. Additionally, the data obtained show that the developed strains can be used as an interesting platform when generation of considerable reducing power is needed in anaerobic glucose metabolism [17, 18]. …”

Section: Introductionmentioning

confidence: 99%

Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

Sekar

Seol

Park

2017

Biotechnol Biofuels

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BackgroundBiologically, hydrogen (H2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H2 production yield is unsatisfactorily low as <4 mol H2/mol glucose. To address this challenge, simultaneous production of H2 and ethanol has been suggested. Co-production of ethanol and H2 requires enhanced formation of NAD(P)H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden–Meyerhof–Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition.ResultsHere, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli Δpgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgi mutant could produce 1.69 mol H2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol−1 glucose, indicating that some carbon is metabolized through the Entner–Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol−1 glucose along with 1.74 mol H2 mol−1 glucose, which are close to the theoretically maximal yields, 1.67 mol mol−1 each for ethanol and H2. However, the attempt to delete the ED pathway in the Δpgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful.ConclusionsBy deletion of pgi and overexpression of heterologous zwf and gnd in E. coli ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB, two important biofuels, ethanol and H2, could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0768-2) contains supplementary material, which is available to authorized users.

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NADPH‐dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: Influence on global gene expression and role of oxygen supply

Cited by 4 publications

References 45 publications

Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

Characterization of an Entner–Doudoroff pathway-activated Escherichia coli

Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

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