Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint-based modeling and experimental verification.
Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.
Laboratory of Chemical Biotechnology, TU Dortmund, D-44221 Dortmund, Germany. lars.blank@bci.tu-dortmund.de
Lars M
LM
Blank
Birgitta E
BE
Ebert
Bruno
B
Bühler
Andreas
A
Schmid
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003198:Computer Simulation; D004926:Escherichia coli / Q000378:metabolism*; D025461:Feedback, Physiological / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000032:analysis / Q000378:metabolism; D006019:Glycolysis; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000032:analysis / Q000378:metabolism; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway; D015202:Protein Engineering
D001426:Bacterial Proteins; D009243:NAD; D005947:Glucose