Metabolic pathway engineering for the non-growth-associated succinate production in Escherichia coli based on flux...

Non-growth-associated bio-production using microorganisms has the potential to achieve a higher target yield than growth-associated production since the latter approach does not waste the substrate for cell growth. We previously proposed a metabolic pathway engineering method (SSDesign) for non-growth-associated target production based on metabolic flux solution space using elementary mode analysis. SSDesign predicts gene knockout combinations for enforcing cells to produce a target compound under non-growing conditions. For succinate production from glucose in Escherichia coli, gene knockouts of pykA-pykF-sfcA-maeB-zwf and pykA-pykF-sfcA-pntAB-sthA were predicted as candidates. In the present study, to verify the predictions of SSDesign, succinate productivities of these multiple knockout strains were evaluated in the stationary phase under microaerobic conditions. Succinate yields of the BW25113ΔpykAΔpykFΔsfcAΔmaeBΔzwf and BW25113ΔpykAΔpykFΔsfcAΔpntABΔsthA strains were 0.48 and 0.52 mol/mol, respectively, and were higher than that of wild type strain (0.20 mol/mol). The succinate yield of BW25113ΔpykAΔpykFΔsfcAΔpntABΔsthA strain was further improved to 0.66 mol/mol by overexpression of phosphoenolpyruvate carboxylase as a potential bottleneck step in the metabolic pathway.