NADH Availability Limits Asymmetric Biocatalytic Epoxidation in Growing Recombinant Escherichia coli

Chair of Chemical Biotechnology, University of Dortmund, D-44227 Dortmund, Germany; Department of Food Science & Technology, Ewha Womans University, Seoul, 120-750, Korea, ISAS-Institute for Analytical Sciences, D-44139 Dortmund, Germany

^* To whom correspondence should be addressed. Email: andreas.schmid{at}bci.uni-dortmund.de.

	Abstract

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 TCA cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis related NAD(P)H consumption was 3.2 – 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 above 21 µmol per min per g cell 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.