imprint:
Springer Science and Business Media LLC, 2022
Published in:Biotechnology for Biofuels and Bioproducts
Language:
English
DOI:
10.1186/s13068-022-02144-8
ISSN:
2731-3654
Origination:
Footnote:
Description:
<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Background</jats:title>
<jats:p><jats:italic>Klebsiella pneumoniae</jats:italic> contains an endogenous isobutanol synthesis pathway. The <jats:italic>ipdC</jats:italic> gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate.</jats:p>
</jats:sec><jats:sec>
<jats:title>Results</jats:title>
<jats:p>Compared with 2-ketoisovalerate decarboxylase from <jats:italic>Lactococcus lactis</jats:italic> (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower <jats:italic>K</jats:italic><jats:sub>m</jats:sub> for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of <jats:italic>ipdC</jats:italic> 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 <jats:italic>K. pneumoniae</jats:italic> for in vivo testing. Isobutanol production by <jats:italic>K. pneumoniae</jats:italic> 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.</jats:p>
</jats:sec><jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>This research provides a new way to improve the efficiency of the biological route of isobutanol production.</jats:p>
</jats:sec>