Sung-Mok Lee

Identification and characterization of a short-chain acyl dehydrogenase from Klebsiella pneumoniae and its application for high-level production of l-2,3-butanediol

Klebsiella pneumoniae synthesize large amounts of l-2,3-butanediol (l-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an l-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward l-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of l-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.

Optimized production of 2,3-butanediol by a lactate dehydrogenase-deficient mutant of Klebsiella pneumoniae

To obtain high-yield production of 2,3-butanediol (2,3-BD) from glucose, we optimized the culture conditions for a lactate dehydrogenase-deficient mutant (ΔldhA) of Klebsiella pneumoniae using response surface methodology. 2,3-BD production was successfully improved by optimizing pH (5.6), aeration (3.50 vvm) and concentration of corn steep liquor (45.0 mL/L) as a nitrogen source, resulting in a maximum level of 2,3-BD production of 148.8 g/L and productivity of 2.48 g/L/h. 2,3-BD was also obtained with high concentration (76.24 g/L) and productivity (2.31 g/L/h) from the K. pneumoniae mutant strain using sugarcane molasses as a carbon source.

Enhancement of 1,3-propanediol production by expression of pyruvate decarboxylase and aldehyde dehydrogenase from Zymomonas mobilis in the acetolactate-synthase-deficient mutant of Klebsiella pneumoniae

Abstract The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol.

Enhanced production of 2,3-butanediol by a genetically engineered Bacillus sp. BRC1 using a hydrolysate of empty palm fruit bunches

A Bacillus species that produces 2,3-butanediol (2,3-BD), termed BRC1, was newly isolated, and a 2,3-BD dehydrogenase (Bdh) from this species was identified and characterized at the molecular and biochemical level. Sequence analysis revealed that Bdh is homologous to D-2,3-BD dehydrogenases. An analysis of the enzymatic properties of Bdh overexpressed in Escherichia coli confirmed the molecular results, showing preferred activity toward D-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Overexpression of bdh under the control of a xylose-inducible promoter resulted in increased enzyme activity and enhanced 2,3-BD production in Bacillus sp. BRC1. Additionally, a hydrolysate of cellulosic material, (empty palm fruit bunches), was successfully used for the enhanced production of 2,3-BD in the recombinant Bacillus strain.