In silico metabolic engineering of E. coli for enhanced anaerobic ATP availability: Investigating the impact of cofactor modulation on PntAB flux via kinetic modelling

Abstract

The transition towards a sustainable bio-based economy relies on efficient Microbial Cell Factories (MCFs) to replace traditional petrochemical processes. While anaerobic fermentations are economically favorable and offer higher theoretical carbon yields than aerobic respiration, they are severely constrained by low ATP production, restricting the synthesis of complex, ATPdemanding compounds. The ICEMAN project proposes rewiring native NADH-generating enzymes to produce NADPH. This redox imbalance is hypothesized to reverse the membrane-bound PntAB transhydrogenase into a redox-driven proton pump, enabling greater ATP conservation.

To evaluate this strategy in silico, a dynamic kinetic model of Escherichia coli central carbon metabolism was rigorously curated. Refining the PntAB formulation improved thermodynamic consistency, parameter collinearity, and environmental dependency. A two-phase optimization pipeline parameterized robust, condition-specific wild-type baselines against in vivo 13C-Metabolic Flux Analysis datasets.

Kinetic simulations validated the ICEMAN strategy under fermentative conditions. The permissive anaerobic proton motive force (pmf) allowed the engineered NADPH surplus to drive PntAB in reverse, increasing global ATP availability across primary backgrounds. The GAPDHNADPH ΔsthA mutant emerged as the optimal candidate, achieving a 24.1% increase in normalized ATP production. Deleting the soluble transhydrogenase SthA proved universally critical across all strains to prevent futile NADPH dissipation. Simulations of PDH-NADPH strains revealed severe carbon flux dilution into competing fermentative pathways, yielding modest energetic improvements (3.1%) and underscoring the necessity of a strictly engineered “homoethanol” chassis. Conversely, the massive aerobic pmf acted as a rigid thermodynamic barrier, preventing PntAB reversal and inducing severe redox stress. This emphasizes the need for robust rate laws and accurate representation of complex in vivo regulation at the PntAB node.

Ultimately, this work provides systems-level guidelines for developing energy-optimized anaerobic E. coli strains, highlighting the critical need to integrate strict thermodynamic constraints into future kinetic modeling frameworks.