Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Abstract

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...

Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways

“…Approximately 14 -17% of the total 6PG was generated from Glc-6-P through Zwf (Table 2). Net fluxes in the lower glycolysis and through the ED pathway confirmed previous results proposed for pseudomonads, where the ED pathway is the main pathway for glucose processing (3,7,8,10,52,54,55).…”

Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid

“…Given this, the maximum NAD(P)H generation rate can be estimated from q FDCA , yielding approximately 1 mmol (g CDW) À1 h À1 for P. putida S12_hmfH and 3 mmol (g CDW) À1 h À1 for P. putida S12_B38 during fed-batch FDCA production. The maintenance requirement of P. putida is approximately 0.8 mmol NADH (g CDW) À1 h À1 in mineral glucose medium (unstressed) and 2.7 mmol NADH (g CDW) À1 h À1 under extreme solvent stress in a second phase of toluene (Isken et al 1999;Ebert et al 2011). Given the harsh conditions associated with FDCA production (aldehyde toxicity, H 2 O 2 formation, osmotic stress) and the fact that the maximum growth rate is greatly reduced (Koopman et al 2010a), it may be hypothesized that the maintenance demand during FDCA production is near the solvent-stressed value of 2.7 mmol NADH (g CDW) À1 h À1 .…”

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

“…It has been stated that oxygenase-based biocatalytic reactions are often faced with problems of high energy demand and redox cofactor consumption (Ebert et al, 2011). This adverse effect has been accentuated in OST bacteria because they require additionally high energy and a high NADH level to sustain the solvent tolerance property (Kuhn et al, 2010).…”