Lin Shu1,2, Jinjie Gu1,3,2, Qinghui Wang1, Shaoqi Sun1, Youtian Cui3, Jason Fell3, Wai Shun Mak3, Justin B Siegel3, Jiping Shi1, Gary J Lye4, Frank Baganz5, Jian Hao6,7,8. 1. Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China. 2. University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China. 3. Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA. 4. Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK. 5. Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK. f.baganz@ucl.ac.uk. 6. Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China. haoj@sari.ac.cn. 7. Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK. haoj@sari.ac.cn. 8. University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China. haoj@sari.ac.cn.
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 Km 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.
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 Km 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.