Fig. 1

Summary of investigations performed in this study and the key results. a The orthogonality (OS) for various substrate-product pairs were evaluated. The most orthogonal pair [ethylene glycol (EG) to glycolate], was selected to demonstrate the use of orthogonality as a metric to establish successful production systems. b A stoichiometric metabolic model was built to characterize the production system and its metabolic behaviour using flux balance analysis (FBA) and flux variability analysis (FVA). Both aerobic and oxygen-limiting conditions were used to investigate the effect of oxygen level on growth and production. c Two strains employing the selected pathway, each with different enzyme mutants, were then tested in shake flasks to confirm that the pathway could indeed sustain cell growth and glycolate production. d The best performing strain (LMSE11) was then tested in bioreactors, using a two-stage growth/production system. As per the identified metabolic valve (glycolate oxidase) and FBA results, a decrease in oxygen level was predicted to switch the system from growth to production. Thus, two secondary air flow rates were tested to evaluate the effect of oxygen level on the production stage. e Using data collected from the shake flask and bioreactor experiments, metabolic modeling was used to further characterize the production system and its response to oxygen level. f Finally, insights gained from modeling and earlier experiments were used to inform the design and testing of two strategies to improve glycolate production in the bioreactors. Colours indicate the type of analysis performed: blue for computational and green for experimental. Key results are reported below the dotted lines