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Applied and Environmental Microbiology, March 2008, p. 1436-1446, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.02234-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain{triangledown}

Bruno Bühler,1 Jin-Byung Park,2 Lars M. Blank,1,3 and Andreas Schmid1,3*

Laboratory of Chemical Biotechnology, Dortmund University of Technology, D-44227 Dortmund, Germany,1 Department of Food Science and Technology, Ewha Womans University, Seoul 120-750, Korea,2 Institute for Analytical Sciences, D-44139 Dortmund, Germany3

Received 1 October 2007/ Accepted 22 December 2007

Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 µmol per min per g cells (dry weight), the absence of limitations by O2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.

* Corresponding author. Mailing address: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund, Emil-Figge-Str. 66, D-44227 Dortmund, Germany. Phone: 49 231 755 7380. Fax: 49 231 755 7382. E-mail: andreas.schmid{at}bci.tu-dortmund.de

{triangledown} Published ahead of print on 11 January 2008.

Applied and Environmental Microbiology, March 2008, p. 1436-1446, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.02234-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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