The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae

Abstract

Background: Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate.

Results: Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower K_m for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose.

Conclusions: This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Keywords: 2-Ketoisovalerate decarboxylase; Indole-3-pyruvate decarboxylase; Isobutanol; Klebsiella pneumoniae.

Fig. 1

Metabolic pathway of isobutanol synthesis and branch pathway in K. pneumoniae. Kp-IpdC is catalysing the 2-ketoisovalerate decarboxylation and further conversion to isobutanol (blue route). It was suspected that the pyruvate decarboxylase activity of Kp-IpdC reduced the available pyruvate for isobutanol synthesis (red route)

Fig. 2

The cell growth and metabolite production of K. pneumoniae ΔbudA–ΔldhA–ipdC and K. pneumoniae ΔbudA–ΔldhA–kivD in batch culture with IPTG induction. Cells were cultured in 5 L bioreactors and 1 mM of IPTG was added to the culture broth after 8 h of cultivation. Data points are the average of n = 3; error bars represent standard error about the mean

Fig. 3

The cell growth and metabolite production of K. pneumoniae ΔbudA–ΔldhA–ipdC and K. pneumoniae ΔbudA–ΔldhA–kivD in batch culture without IPTG induction. Cells were cultured in 5 L bioreactors and no IPTG was added to the culture broth in the process. Data points are the average of n = 3; error bars represent standard error about the mean

Fig. 4

A The predicted tetrameric structure of Kp-IpdC; B Structural overlay of Kp-IpdC (orange) with Ec-IpdC (gray). The root mean square deviation (RMSD) of Kp-IpdC and Ec-IpdC monomers was 1.158

Fig. 5.

3D structure of the active site of Kp-IpdC with substrate bound obtained from simulations using PyMOL. A T290L. B L546W

Fig. 6

The cell growth and metabolites production of K. pneumoniae IpdC, K. pneumoniae T290L, K. pneumoniae L546W, and K. pneumoniae KivD in batch cultures with IPTG induction. Cells were cultured in 5 L bioreactors and 1 mM of IPTG was added to the culture broth after 8 h of cultivation. Data points are the average of n = 3; error bars represent standard error about the mean

Fig. 7

The cell growth and metabolites production of K. pneumoniae IpdC, K. pneumoniae T290L, and K. pneumoniae KivD in batch cultures without IPTG induction. Cells were cultured in 5 L bioreactors and no IPTG was added to the culture broth in the process. Data points are the average of n = 3; error bars represent standard error about the mean