Metabolic engineering of Klebsiella pneumoniae based on in silico analysis and its pilot-scale application for 1,3-propanediol and 2,3-butanediol co-production.

Klebsiella pneumoniae naturally produces relatively large amounts of 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD) along with various byproducts using glycerol as a carbon source. The ldhA and mdh genes in K. pneumoniae were deleted based on its in silico gene knockout simulation with the criteria of maximizing 1,3-PD and 2,3-BD production and minimizing byproducts formation and cell growth retardation. In addition, the agitation speed, which is known to strongly affect 1,3-PD and 2,3-BD production in Klebsiella strains, was optimized. The K. pneumoniae ΔldhA Δmdh strain produced 125 g/L of diols (1,3-PD and 2,3-BD) with a productivity of 2.0 g/L/h in the lab-scale (5-L bioreactor) fed-batch fermentation using high-quality guaranteed reagent grade glycerol. To evaluate the industrial capacity of the constructed K. pneumoniae ΔldhA Δmdh strain, a pilot-scale (5000-L bioreactor) fed-batch fermentation was carried out using crude glycerol obtained from the industrial biodiesel plant. The pilot-scale fed-batch fermentation of the K. pneumoniae ΔldhA Δmdh strain produced 114 g/L of diols (70 g/L of 1,3-PD and 44 g/L of 2,3-BD), with a yield of 0.60 g diols per gram glycerol and a productivity of 2.2 g/L/h of diols, which should be suitable for the industrial co-production of 1,3-PD and 2,3-BD.