Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

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 similar to 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.