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We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase is used 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 the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate-determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically. [ABSTRACT FROM AUTHOR]

An immense number of chemical reactions are carried out simultaneously in living cells. Nature's optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man-made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor-made microbial cell factories. [ABSTRACT FROM AUTHOR]