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Redox-cofactor dependent biotransformations are typically conducted in whole microbial cells to leverage active metabolism for redox-cofactor regeneration. This thesis challenges the assumption that redox-reactions function independently of host metabolism. It investigates the mutual impact between redox-cofactor consuming biotransformations and regenerating metabolism through systems biology approaches that combine computational modeling with experimental techniques. Metabolic modeling revealed that NADH availability is dependent on the metabolic network structure. In vivo performance tests of E. coli single gene deletion strains confirmed simulation results, indicating a potential NADH limitation for recombinant styrene monooxygenase activity. Further simulations identified metabolic engineering targets to enhance NADH yield from glucose, which was successfully increased through in vivo redesign of the E. coli metabolic network. However, the recombinant styrene monooxygenase's inability to utilize the surplus NADH suggests hidden limitations. The potential of Pseudomonas putida for whole-cell redox biocatalysis was evaluated under increased redox and energy demands, which uncoupled carbon catabolism from biomass formation, improving NADH regeneration rates. Despite amplified glycolytic activity, perturbed NADH demands did not control glycolytic flux, leading to NADH limitation of recombinant NADH-oxidase. A parameter sensiti
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A systems approach to understand and engineer whole-cell redox biocatalysts, Birgitta E. Ebert
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- 2012
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