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A systems approach to understand and engineer whole-cell redox biocatalysts

Birgitta E. Ebert

Published by Shaker Verlag Feb 2012, 2012
ISBN 10: 3844007946 / ISBN 13: 9783844007947
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Bibliographic Details

Title: A systems approach to understand and ...

Publisher: Shaker Verlag Feb 2012

Publication Date: 2012

Binding: Buch

Book Condition: Neu


Neuware - Redox-cofactor dependent biotransformations are preferably done in whole microbial cells to exploit the active metabolism for redox-cofactor regeneration. The general assumption that these redox-reactions operate independently of the host metabolism was questioned in this thesis. The potential mutual impact of the redox-cofactor consuming biotransformation and the redoxcofactor regenerating metabolism was investigated by systems biology approaches coupling computational modeling with experimental techniques. Metabolic modeling depicted a dependency of NADH-availability on metabolic network structure. Probing the in vivo redoxbiocatalytic performance of appropriate E. coli single gene deletion strains verified the simulation results and further indicated a potential NADH-limitation of the recombinant styrene monooxygenase activity. Additional simulations identified metabolic engineering targets that increase the NADH-yield on glucose. NADH-yield and regeneration rate were successfully increased after appropriate in vivo redesign of the E. coli metabolic network. The inability of the recombinant styrene monooxygenase to harness this NADH-surplus points to further hidden limitations. The potential of Pseudomonas putida for whole-cell redox biocatalysis was analyzed by challenging the strain with increased redox and energy demands. These stresses caused an uncoupling of carbon catabolism and biomass formation and improved NADH regeneration rates. While energy demanding conditions amplified the glycolytic activity, perturbed NADH-demands exerted no control over the glycolytic flux. This insensitivity resulted in NADH-limitation of the recombinant NADH-oxidase questioning the potential of this strain for whole-cell redox biocatalysis. A parameter sensitivity analysis of perturbed steady states indicated targets to tackle this undesired robustness and additionally revealed that substantially increased NADH regeneration rates had no negative impact on the stability of the P. putida metabolism. 13 C-based metabolic flux analysis is a powerful technique to infer metabolic system behavior and design principles. Enhancement of existing software allows now 13 C-based metabolic flux analysis in a high-throughput manner by non-experts and flexible extension with other services to build custom analysis workflows. This automated software version will broaden the application of this analysis. Summarizing, the increased insights of the interplay of redox and energy stress and metabolic operation will foster metabolic engineering of superior whole-cell redox biocatalysts. 195 pp. Englisch. Bookseller Inventory # 9783844007947

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