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Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8a-histidyl bound FAD, which represents the most frequently encountered covalent flavin–protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophicEscherichia colistrain. | ỊFEBS Journal Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase Lars M. Blank1 2 Georgios Ionidis3 Birgitta E. Ebert1 Bruno Buhler1 and Andreas Schmid1 2 1 Laboratory of ChemicalBiotechnology TU Dortmund Germany 2 ISAS-Institute for AnalyticalSciences Dortmund Germany 3 Department of Biology Swiss Federallnstitute of Technology Zurich Switzerland Keywords flux analysis metabolic network monooxygenase quantitative physiology solvent tolerance Correspondence A. Schmid Laboratory of Chemical Biotechnology Faculty of Biochemicaland ChemicalEngineering TU Dortmund Emil Figge Str. 66 D-44227 Dortmund Germany Fax 49 231 755 7382 Tel 49 231 755 7380 E-mail schmid@bci.tu-dortmund.de These authors contributed equally to this work Received 5 June 2008 revised 5 August 2008 accepted 18 August 2008 doi 10.1111 j.1742-4658.2008.06648.x 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 energydependent biocatalysis we established a model system for biocatalysis in the presence of toxic low logPow solvents recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous octanol two-liquid phase reaction medium. Using 13C 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. .
