Threonine Supplementation Reduces Methylglyoxal Overflow by Increasing Glycolysis Flux at the Payoff Phase: A Metabolic Modeling Analysis

Abstract

Methylglyoxal (MGO) is a metabolic byproduct of sugar metabolism involved in the formation of advanced glycation end products (AGEs) and its accumulation disrupts protein function, redox balance and cellular viability. Yet the metabolic rewiring required to divert MGO overflow and prevent these cytotoxic effects remains largely unknown. To address these gaps, we used flux sampling and flux balance analysis in genome scale metabolic model iJO1366 of E. coli. High glucose increased MGO flux from 0.16 mmol/gDCW/h to 1.87 mmol/gDCW/h in simulation. Large scale in silico screening of metabolic reactions identified that increasing threonine uptake, thereby increasing ethanol flux, reduced glucose induced increase in MGO flux from 1.87 mmol/gDCW/h to 0.405 mmol/gDCW/h. In silico inhibition of ethanol production (NAD+ regeneration) inhibited the MGO lowering potential of threonine. Threonine supplementation actively drives the acetaldehyde to ethanol flux to provide localized NAD+ relief, which subsequently enhances the flux of payoff phase in glycolysis (glyceraldehyde 3-phosphate), thereby efficiently draining the stagnated DHAP pool and shutting down the overflow towards MGO production. Unexpectedly, under reduced oxygen conditions, high glucose caused only a minimal increase in MGO flux, which was not reduced by threonine supplementation. Our study also decoded the mechanistic details behind this paradox. Though this model driven hypothesis requires further validation in vivo, these stoichiometric predictions have applications in metabolic engineering, for commercial MGO production. Furthermore, in vivo studies focused on bacterial stress, gut microbiome imbalances and AGE related diseases might benefit from these simulations