Unstructured kinetic models to simulate an arabinose switch that decouples cell growth from metabolite production

Harley Edwards,Peng Xu

doi:10.1016/j.synbio.2020.07.003

Harley Edwards, Peng Xu

Open Access

https://doi.org/10.1016/j.synbio.2020.07.003

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Abstract

Modeling synthetic gene circuits to implement dynamic flux balancing is crucial in teaching and exploring metabolic engineering strategies to repartition metabolic precursors and construct efficient microbial cell factories. Microbial fitness and production rates are often complex phenotypes that are governed by highly non-linear, multivariable functions which are intrinsically linked through carbon metabolism. The solution of such dynamic system can be difficult for synthetic biologists to visualize or conceptualize. Recently, researchers (Santala et al., Metab. Eng. Comm., 2018) have implemented an arabinose based genetic switch to dynamically partition the central carbon flux between cell growth and product formation. The autonomous switch allowed dynamic shift from arabinose-associated cell growth to acetate-associated product (wax ester) formation. This system clearly demonstrates the effectiveness of using a genetic switch to decouple cell growth from product formation in a one-pot bioreactor to minimize operational cost. Coupled with Michaelis-Menten kinetics, and Luedeking-Piret equations, we were able to reconstruct and analyze this metabolic switch in silica and achieved graphical solutions that qualitatively match with the experimental data. By assessing physiologically-accessible parameter space, we observed a wide range of dynamic behavior and examined the different limiting cases. Graphical solutions for this dynamic system can be viewed simultaneously and resolved in real time via buttons on the graphical user interface (GUI). Metabolic bottlenecks in the system can be accurately predicted by varying the respective rate constants. The GUI serves as a diagnosis toolkit to troubleshoot genetic circuits design constraints and as an interactive workflow of using this arabinose based genetic switch to dynamically control carbon flux, which may provide a valuable computational toolbox for metabolic engineers and synthetic biologists to simulate and understand complex genetic-metabolic system.

Highlights

Microbes use a wide range of substrates with different energetic and redox states
One of the important feature of this metabolic switch is to use arabinose as the signaling molecule to tune the cell growth rate, so that the substrate consumption is diverted to product formation that leads to significant improvement in the pathway yield
By altering the initial concentration of arabinose (Fig. 1), the authors were able to achieve this tunability and effectively control the point at which carbon utilization would shift from cell growth to product formation

Summary

Introduction

Microbes use a wide range of substrates with different energetic and redox states. These substrates may be assimilated to form biomass or final products. Cosubstrate utilization may serve to bypass some catabolic, energeticallycostly steps, and facilitates more readily available anabolic precursors (i.e. acetyl-CoA) to synthesize final product [9], since the co-utilized substrate may form a metabolic bypass with less enzymatic steps, which otherwise could not be attained by the breakdown of the initial carbon source [10] These feedstocks hold great promise in creating a sustainable, energy efficient commodity chemical production platform needed to supply a growing global population [11,12]. In an effort to further increase product yield, optimizing metabolic flux has long been accepted as a viable strategy by systems biologists when it comes to increasing carbon conversion along a metabolic pathway [13,14,15]. Much of the effort concerns with the static regulation of metabolic flux without considering the hierarchically-organized regulatory architecture that is built into the cell metabolism [17,18]

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