No abstract

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

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...

Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to perform synthetically interesting reactions. Besides hydrolases, oxidoreductases represent the most applied enzyme class in industry. Oxidoreductases are attributed a high future potential, especially for applications in the chemical and pharmaceutical industries, as they enable highly interesting chemistry (e.g., the selective oxyfunctionalization of unactivated C-H bonds). Redox reactions are characterized by electron transfer steps that often depend on redox cofactors as additional substrates. Their regeneration typically is accomplished via the metabolism of whole-cell catalysts. Traditionally, studies towards productive redox biocatalysis focused on the biocatalytic enzyme, its activity, selectivity, and specificity, and several successful examples of such processes are running commercially. However, redox cofactor regeneration by host metabolism was hardly considered for the optimization of biocatalytic rate, yield, and/or titer. This article reviews molecular mechanisms of oxidoreductases with synthetic potential and the host redox metabolism that fuels biocatalytic reactions with redox equivalents. The tools discussed in this review for investigating redox metabolism provide the basis for studies aiming at a deeper understanding of the interplay between synthetically active enzymes and metabolic networks. The ultimate goal of rational whole-cell biocatalyst engineering and use for fine chemical production is discussed.