In silico co-factor balance estimation using constraint-based modelling informs metabolic engineering in Escherichia coli
Fig 2
Toy illustration of ATP and NAD(P)H reactions and reaction categories accounted for by the CBA protocol.
(A) All reactions in the E.coli Core Model that directly contribute to the intracellular levels of ATP and NAD(P)H pools (blue or yellow circle, accordingly). Arrows pointing inwards on the left display reactions leading to ATP or NAD(P)H build-up (i.e. co-factor production), while arrows pointing outwards on the right show reactions that drain the co-factor pools (i.e. co-factor consumption). The thickness of the arrows represent the varying fluxes of these reactions. The CBA protocol identifies all co-factor related reactions producing and/or consuming ATP or NAD(P)H, it records their fluxes and distributes them across five core categories: (1) co-factor production, (2) biomass production, (3) waste release, (4) cellular maintenance and (4) target production (this category is target product specific). (B) Theoretical example of how the classification of ATP reactions is handled by the CBA protocol. Co-factor fluxes (here illustrated by the varying arrow thickness) are dependent on the co-factor stoichiometric coefficient and flux calculated by FBA. ATP production accounts for all reactions that generate a positive ATP flux. The ATP waste category accounts for both ATP produced during acetate production, but also ATP consumed in ATP-hydrolysing reactions (also known as ‘ATP burning’ reactions), such as ATPM and ADK1. ATP biomass includes the ATP flux consumed during biomass formation. The ATP target category is pathway-specific, accounts for only those synthetic reactions introduced into the stoichiometric model, and will lead to a positive or negative flux according to whether the synthetic pathway leads to the formation or drain of intracellular ATP, respectively. If the synthetic pathway is ATP-neutral, the net value for this category will be zero. ATP maintenance includes any ATP consumed in additional metabolic activities and not considered in the aforementioned categories. (C) Theoretical example of how the classification of redox reactions is handled by the CBA protocol, similarly to (B). The NAD(P)H waste category also accounts for reactions GND, PDH, AKGDH, ICDHyr, which produce NAD(P)H but simultaneously release CO2, and reactions such as LDH_D and ADHEr that consume NAD(P)H and release fermentation products. For categories including both positive and negative co-factor fluxes, the net is calculated for that category. Figure design inspired by [38].