Shu, Lin;
Gu, Jinjie;
Wang, Qinghui;
Sun, Shaoqi;
Cui, Youtian;
Fell, Jason;
Mak, Wai Shun;
... Hao, Jian; + view all
(2022)
The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae.
Biotechnology for Biofuels and Bioproducts
, 15
(1)
, Article 41. 10.1186/s13068-022-02144-8.
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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.
| Type: | Article |
|---|---|
| Title: | The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1186/s13068-022-02144-8 |
| Publisher version: | https://doi.org/10.1186/s13068-022-02144-8 |
| Language: | English |
| Additional information: | This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
| Keywords: | 2-Ketoisovalerate decarboxylase, Indole-3-pyruvate decarboxylase, Isobutanol, Klebsiella pneumoniae |
| UCL classification: | UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering UCL > Provost and Vice Provost Offices > UCL BEAMS UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10148388 |
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