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

Autor: Shu L; Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China.; University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China., Gu J; Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China.; Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA.; University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China., Wang Q; Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China., Sun S; Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China., Cui Y; Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA., Fell J; Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA., Mak WS; Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA., Siegel JB; Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA., Shi J; Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People's Republic of China., Lye GJ; Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK., Baganz F; Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK. f.baganz@ucl.ac.uk., Hao J; 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.; Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK. haoj@sari.ac.cn.; University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China. haoj@sari.ac.cn.
Jazyk: angličtina
Zdroj: Biotechnology for biofuels and bioproducts [Biotechnol Biofuels Bioprod] 2022 May 02; Vol. 15 (1), pp. 41. Date of Electronic Publication: 2022 May 02.
DOI: 10.1186/s13068-022-02144-8
Abstrakt: 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
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