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. Significance statementThis study examines the challenge of modeling and optimizing multi-enzyme cascades. We detail the development, testing and optimization of a deterministic model of a three enzyme cascade with four cofactor regeneration enzymes. Significantly, the model could be easily used to predict the optimal concentrations of each enzyme in order to get maximum flux through the cascade. This prediction was strongly validated experimentally. The success of our model demonstrates that robust models of systems of at least seven enzymes are † Present address: Manchester readily achievable. We highlight the importance of following good modeling practice to evaluate model quality and limitations. Examining deviations from expected behavior provided additional insight into the model and enzymes. This work provides a template for developing larger deterministic models of enzyme cascades. phosphotransferase (PAP), which allows the regeneration of ADP from AMP (24). The combination of PAP and PPK therefore allowed complete regeneration of ATP from AMP (25). In an alternative approach, adenylate kinase (which catalyzes the reversible phosphorylation of AMP by ATP) was used in place of PPK. Coupling of this enzyme with PAP pushes the equilibrium towards ATP regeneration ( Figure 1B) (26). The application of process modellingSynthetic biology has long promised the use of well-characterized parts for the rational design of new metabolic pathways (27)(28)(29). However the use of modelling to optimize these in vivo reactions is yet to be fully realized (30). In contrast, modelling of enzymes in vitro can be fairly robust and offers solutions for reaction engineering (31). This could allow the combination of enzymes, for which validated mathematical models exist, to be rapidly engineered and tested in silico (2).

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases

“…Kinetic models, in contrast, are based on kinetic rate laws for individual reactions. The relevant rate laws and parameters for each step are determined experimentally, using either a systematic approach [65,66] or a design of experiments approach [41]. Alternatively, parameters have been fitted to the entire system for up to fifteen reactions [67].…”

“…Indeed, kinetic models of enzymatic reactions have only been explored in a few studies. Key examples are cofactor recycling in vitro [65], simulation of a three-enzyme cascade J o u r n a l P r e -p r o o f system for synthesis of 6-hydroxyhexanoic acid [70], modelling of the stability of an aldolase [71], or a three-enzyme cascade for the biotransformation of sucrose to cellobiose [72].…”

“…CARs are particularly attractive for cascade reactions, as they generate the reactive aldehyde group in a single step (avoiding the full reduction and selective oxidation often necessary with chemical methods). This group is a substrate for a wide range of synthetically useful reactions (for example reductive aminases [100]), whilst many chemical and enzymatic reactions produce carboxylic acid moieties (Figure 4A; [65,98,100]). In vivo pathways using CARs to connect reactions have been successfully achieved.…”

“…Finnigan et al combined these two ideas to develop a three step enzymatic cascade from an ester to an alcohol in vitro [65]. This transformation included two parallel recycling cascades and an orthogonal cascade to remove an inhibitory by-product, requiring seven enzymes (Figure 4D).…”

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.…”