Metabolic Engineering of Klebsiella Oxytoca Based on in silico Genome-Scale Analyses to Enhance 2,3-Butanediol Production

2,3-butanediol (2,3-BD) has applicability for the manufacture of 1,3-butadiene and methyl ethyl ketone, moistening and softening agents, and cosmetics. Klebsiella oxytoca is a promising microorganism to produce 2,3-BD. In order to enhance 2,3-BD production, we engineered genetically K. oxytoca strain based on in silico genome-scale analyses. The in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by using genome-scale metabolic model of K. oxytoca, KoxGSC1457, which is composed of 1,457 reactions and 1,099 metabolites. The double knockouts of ldhA and pflB genes were targeted with the criteria of minimization of byproducts formation and maximization of 2,3-BD production. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. Optimizing the condition of fed-batch fermentation, we achieved the titer of >110 g/L 2,3-BD, with a productivity of 2.1 g/L/h and yield of 0.45 g/g glucose. [This work was supported by the Industrial Strategic Technology Development Program (No. 10050407) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).]