[Zurück]

S. Milker, M. Fink, N. Oberleitner, A. Ressmann, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff:
"Kinetic modeling of an enzymatic redox cascade in vivo reveals cofactor-caused bottlenecks";
ChemCatChem, 9 (2017), S. 3420 - 3427.

This paper describes the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli, using a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis-Menten parameters from in vitro experiments with isolated enzymes, and used these values to simulate the change in concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated the fastest enzyme to be rate-determining due to the unexpectedly low concentration of the active form, opening up reversible reaction channels towards side products. We also provide substantial experimental evidence, that a low intracellular concentration of flavin and nicotinamide cofactors drastically throttled the performance of the in vivo cascade.

kinetic modeling . biocatalytic cascade . enzyme estimations in vivo . cascade optimization . cofactor limitation

http://publik.tuwien.ac.at/files/publik_266586.pdf