Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors

Abstract

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.

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

“…Although each MCS was preceded by its own T7 promoter and followed by a single terminator, the number of moles of the two expressed enzymes was not similar in the Duo strains, the ratio barely reaching 0.3 (in moles of monomer). This unbalanced enzyme production in favor of ADH has been previously observed in other ADH−BVMO cascades (Jang, Jeon, Baek, Lee, & Park, ; Milker, Fink et al, ; Müller et al, ) and may be due to the different size of the two enzymes, making the longer one more difficult to synthesize (Fernandes et al, 2017).…”

“…Regarding the enzyme ratio, another limiting factor probably interfered to explain the plateau observed Figure c, possibly the NADPH/NADP + content, O 2 supply, and/or the endogenous FAD content. NADPH limitation is well documented (Milker, Fink et al, ) and one of the most important challenges to whole‐cell processes reaching the industrial level. However, in our case, the need for NADPH, even in high‐level BVMO cells, should not have exceeded the cell capacity due to the cofactor recycling inherent to the cascade.…”

“…The most attractive solution from an economic and practical point of view is the in vivo production of all the enzymes in a single culture, within the same organism. However, many studies have shown that this situation is often difficult to control and optimize (France, Hepworth, Turner, & Flitsch, ; Ladkau, Schmid, & Bühler, ; Milker, Fink et al, ; Pennec, Hollmann, Smit, & Opperman, ). One of the main bottlenecks is the control of the enzyme ratio.…”

Whole‐Cell Cascade Biotransformations for One‐Pot Multistep Organic Synthesis

“…More importantly, racemic substituted cyclohexenol derivatives were also converted to the corresponding enantiopure chiral lactones in 64–99 % conversion. The kinetic model of the above in vivo cascade was established to identify the bottleneck in oxidation by CHMO . Bornscheuer et al.…”

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis

“…Formation of 2 mM of dihydrocarvone lactone after 3 h with an E. coli expressing the CHMO‐QC G XenB fusion protein and LK ‐ADH in whole cell was observed, while by using the three enzymes without fusion, 1.2 mM product was formed. Some research was also done on the kinetic model for the simulation and optimization of such cascades . In order to simulate the change in concentration of intermediates and products during the in vivo cascade reactions, an adapted Michaelis‐Menten parameter from in vitro experiments with isolated enzymes was estimated and used.…”