title: Subtoxic product levels limit the epoxidation capacity of recombinant E. coli by increasing microbial energy demands.
abstract: The utilization of the cellular metabolism for cofactor regeneration is a common motivation for the application of whole cells in redox biocatalysis. Introduction of an active oxidoreductase into a microorganism has profound consequences on metabolism, potentially affecting metabolic and biotransformation efficiency. An ambitious goal of systems biotechnology is to design process-relevant and knowledge-based engineering strategies to improve biocatalyst performance. Metabolic flux analysis (MFA) has shown that the competition for NAD(P)H between redox biocatalysis and the energy metabolism becomes critical during asymmetric styrene epoxidation catalyzed by growing Escherichia coli containing recombinant styrene monooxygenase. Engineering TCA-cycle regulation allowed increased TCA-cycle activities, a delay of acetate formation, and enhanced NAD(P)H yields during batch cultivation. However, at low biomass and product concentrations, the cellular metabolism of both the mutants as well as the native host strains could cope with increased NADH demands during continuous two-liquid phase biotransformations, whereas elevated but still subtoxic product concentrations were found to cause a significantly increased NAD(P)H demand and a compromised efficiency of metabolic operation. In conclusion, operational conditions determine cellular energy and NAD(P)H demands and thus the biocatalytic efficiency of whole-cell redox biocatalysts.
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, D-44227 Dortmund, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kuhn
authors.forename: Frederik S O
authors.initials: FS
authors.lastname: Fritzsch
authors.forename: Xiumei
authors.initials: X
authors.lastname: Zhang
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
authors.forename: Bruno
authors.initials: B
authors.lastname: Bühler
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D001709:Biotechnology / Q000379:methods; D001711:Biotransformation; D004734:Energy Metabolism; D004852:Epoxy Compounds / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D049490:Systems Biology
chemical_list: D004852:Epoxy Compounds; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; C013690:styrene oxide; D010105:Oxygenases; C026721:styrene monooxygenase; D005947:Glucose