(643b) Improving Glucaric Acid Production By Alleviating Oxidative Stress in E. coli

Native metabolism continually produces reactive oxygen species (ROS) that promote cellular damage through redox reactions with DNA, proteins, and lipids. Endogenous scavenging enzymes and mechanisms are typically sufficient to reduce ROS to minimal levels, but genetic engineering for new applications can exhaust the cellular antioxidant capacity. While this problem has been reported in the literature, a general framework is needed to identify and address it as part of the metabolic engineering toolkit. To better understand oxidative stress in engineered metabolic pathways, we examined a heterologous pathway from glucose to glucaric acid in E. coli, with particular focus on the conversion of myo-inositol to glucuronic acid by myo-inositol oxygenase (MIOX). Overexpression of the genes encoding endogenous catalase (katE) and superoxide dismutases (sodA and sodB) led to significant increases in glucuronic acid production, suggesting that hydrogen peroxide and superoxide levels are elevated in the pathway and impinge on productivity. The increase in glucuronic acid titers also varied between E. coli strains, verifying previously observed differences in native antioxidant capacity. This work provides further evidence that engineered metabolic pathways can produce elevated ROS levels and that overexpressing ROS scavenging enzymes is an effective strategy to improve pathway performance.