Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

Abstract

Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

“…Because of impurities in enzyme preparations alcohol byproducts were observed. [32,33] Fatty amines were targeted by Turner et al in a one-pot tandem cascade performed by a CAR and a transaminase, while recycling NADPH and ATP. In order to achieve high fatty amine concentrations, an in situ product removal system (ISPR) was applied and an end product yield of 0.927 g L À 1 dodecyl amine was achieved.…”

“…In similar approaches, cascade reactions were investigated to pull the aldehyde intermediate from the reaction towards follow‐up products. Because of impurities in enzyme preparations alcohol by‐products were observed . Fatty amines were targeted by Turner et al .…”

CheKiPEUQ Intro 1: Bayesian Parameter Estimation Considering Uncertainty or Error from both Experiments and Theory**

“…A common goal in kinetic studies is extraction of physico‐chemical parameter values such as pre‐exponential factors and activation energies from experimental data [1–9, 106] . Databases of values as well as improvements in theory and computation have led to increasingly accurate prior predictions of activation energies and other physico‐chemical parameters; which can be useful in the interpretation of experimental data [10–20] .…”

“…The state of the field is such that BPE is a valuable but not widely utilized method for finding parameter estimates, and the method has particular value when there are uncertainties involved, as is often the case for chemical kinetics models [2–4] . We anticipate that this publication and the accompanying software release may enable BPE to become frequently utilized by the community.…”

Development of Engineered Ferredoxin Reductase Systems for the Efficient Hydroxylation of Steroidal Substrates

“…The KshB in a KSH system is responsible for donating electrons from NADH and transferring them to KshA via a [2Fe-2S] cluster domain [5]. The cofactor NAD(P)H is essential due to its high cost for application in chemical and pharmaceutical industries [13], and the bioconversion should not occur when NADH is consumed up in vitro [14]. Therefore, the NAD(P)H regeneration systems should be established as more economical processes in preparative biotransformation.…”