Metabolic response of <i>Pseudomonas putida</i> during redox biocatalysis in the presence of a second octanol phase

Blank, Lars M.; Ionidis, Georgios; Ebert, Birgitta E.; Bühler, Bruno; Schmid, Andreas

doi:10.1111/j.1742-4658.2008.06648.x

Cited by 139 publications

(155 citation statements)

References 82 publications

(115 reference statements)

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“…In contrast, manifold overexpression of nearly all enzymes of the glycolytic enzymes in yeast did not increase the glycolytic activity (44). Similar to the observation made with E. coli, Pseudomonas strains challenged with redox biocatalysis react to this metabolic burden by increasing the glycolytic rate but simultaneously decreasing the specific growth rate (10). As redox biocatalysis is intrinsically coupled to both increased ATP and redox cofactor demand, it is not clear which of these two loads triggers the increased metabolic activity.…”

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confidence: 52%

“…To separate cells and toxic compounds that are frequently also characterized by a low water solubility, the latter are solved and extracted, in a second organic phase (32,36). However, solvents with good extraction characteristics for the biocatalytic compounds can be similarly toxic to the cells (10). Microorganisms react to this toxification by different mechanisms, including changes in cell membrane composition, active removal of the toxic substrate by energy-driven efflux pumps, and alteration of the energy metabolism, which significantly increase the cellular energy expenditure.…”

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confidence: 99%

“…P. putida KT2440 was chosen as a model organism. We consider this species a favorable host for whole-cell redox biocatalysis for the following reasons: P. putida strains have proven to have a high NADH regeneration capacity (10) and they are characterized by a low cellular energy demand (28). Low energy expenditure for cell maintenance processes is an advantage for NADH-dependent biocatalysis, as it implies a low consumption of NADH for ATP generation and consequently a high net NADH regeneration rate available for biocatalysis.…”

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confidence: 99%

“…The latter calculations were based on the stoichiometric model of P. putida's central carbon metabolism described in reference 10, which was extended with the glyoxylate shunt (see Table S1 in the supplemental material) and constrained with the extracellular flux parameter specific growth rate and specific net carbon uptake rate (calculated from the conversion rate of glucose, gluconate, and 2-ketogluconate) and the determined metabolic flux ratios. Specifically, the following metabolic flux ratios were used: fraction of pyruvate derived through the Entner-Doudoroff pathway (only amenable for [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose-labeled experiments), fraction of oxaloacetate originating from pyruvate, fraction of phosphoenolpyruvate (PEP) originating from oxaloacetate, upper and lower bounds of the fraction of pyruvate originating from malate, and the upper bound of the phosphoenolpyruvate fraction derived through the pentose phosphate pathway. As P. putida KT2440 possesses two nicotinamide nucleotide transhydrogenases that interconvert NADH and NADPH, we did not calculate separate NADH and NADPH regeneration rates but give a pooled rate for both cofactors.…”

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Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Ebert

Kurth

Grund

et al. 2011

Appl Environ Microbiol

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Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.Knowledge of the physiological response of bacteria to environmental stresses and metabolic burdens (e.g., the presence of toxic compounds or recombinant protein overexpression) is of importance in fundamental research to elucidate regulatory mechanisms or basic principles of the metabolic organization and functioning of microbial metabolism. However, a sound understanding of the microbial behavior is equally essential for the development of bioprocesses, as the physiology of the host organisms determines production efficiency (6, 43). Detailed knowledge about the metabolic response and adaptation of microorganisms to specific and often challenging process parameters is key to strain selection. Furthermore, this knowledge allows for the rational engineering of superior production strains and effective process design and control. The interplay of microbial physiology and process performance has long been neglected in bioprocess optimization efforts, which have traditionally focused on biochemical engineering aspects, such as reactor setup and control. However, this issue is becoming increasingly important to establish bio-based processes with high productivity and product yield. These parameters are essential for bio-based processes to be competitive with chemical alternatives not only in the synthesis of high-value pharmaceuticals, but especially for the production of low-value bulk and commodity chemicals, such as biofuels, organic solvents, or plastic mono...

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confidence: 52%

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confidence: 99%

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Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Ebert

Kurth

Grund

et al. 2011

Appl Environ Microbiol

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117

124

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“…Blank et al (2008) reported net glucose uptake rates of 10-15 mmol glucose g À1 CDW h À1 for exponentially growing Pseudomonas putida strains in batch cultures in the presence of solvents, while for Pseudomonas sp. VLB120DC grown in a chemostat a rate of about 3 mmol glucose g À1 CDW h À1 has been published (Park et al, 2007).…”

Section: Influence Of Oxygen Supply On Metabolic Activity Of the Biofmentioning

confidence: 99%

Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis

Gross

Lang

Bühler

et al. 2009

Biotech & Bioengineering

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Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120DC biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)-styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60-80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the main parameters influencing the biotransformation rate. Productivity was linearly dependent on the specific membrane area and on the tube wall thickness. On average volumetric productivities of 24 g L À1 aq day À1 with a maximum of 70 g L À1 aq day À1 and biomass concentrations of 45 g BDW L À1 aq have been achieved over long continuous process periods (!50 days) without reactor downtimes.

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