Shu, Lin; Gu, Jinjie; Wang, Qinghui; Sun, Shaoqi; Cui, Youtian; Fell, Jason; Mak, Wai Shun; Siegel, Justin B.; Shi, Jiping; Lye, Gary J.; Baganz, Frank; Hao, Jian

BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS

2022, 15,

Shu, Lin; Gu, Jinjie; Wang, Qinghui; Sun, Shaoqi; Cui, Youtian; Fell, Jason; Mak, Wai Shun; Siegel, Justin B.; Shi, Jiping; Lye, Gary J.; Baganz, Frank; Hao, Jian

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.