Efficient production of isobutanol from glycerol in Klebsiella pneumoniae: Regulation of acetohydroxyacid synthase, a rate-limiting enzyme in isobutanol biosynthesis at gene expression level

Abstract

Abstract Background: Glycerol is inexpensive and abundant, and can be used for the industrial production of value-added products. Isobutanol is an important industrial chemical that has many applications, and its biosynthesis from different carbon sources has been studied using various microorganisms. So far, isobutanol production by Klebsiella pneumoniae has been mainly studied using glucose. In this study, we produced isobutanol from glycerol based on the K. pneumoniae ΔldhAΔadcmutant harboring pBR-iBO used in previous study using K. pneumoniae, which has an excellent ability to assimilate glycerol even under limited conditions. Results: We investigated the effect of different acetohydroxyacid synthase (AHAS) isoenzymes (rate-limiting enzymes in isobutanol biosynthesis), plasmid copy number, and different promoters as a method to increase isobutanol production by K. pneumoniae. The K. pneumoniae Cu ΔldhAΔbudA, pUC-tac-BN-ISO strain produced 2.56-fold more isobutanol than previously reported for glycerol-derived isobutanol production. Also, the in vitro enzyme activity of AHAS I (ilvBN) was greater than that of the other two isoenzymes (ilvIHand ilvGM). Evaluation of process factors indicated that an agitation speed of 200 rpm with the culture maintained at pH 6 were favorable conditions for isobutanol production (1.02 g/L). Conclusion: We obtained enhanced isobutanol production from glycerol by metabolic engineering of K. pneumoniae. Our results demonstrated enhanced production of isobutanol from glycerol, and suggest future avenues for research in this area.