A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s−1), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (kcat/Km) between natural and synthetic OHB substrates (84 and 31 s−1 mM−1, respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis.
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December 2018
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Research Article|
December 12 2018
Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway
Cláudio J.R. Frazão;
Cláudio J.R. Frazão
1LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France
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Christopher M. Topham;
Christopher M. Topham
2Molecular Forces Consulting, 40 rue Boyssonne, Toulouse F-31400, France
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Yoann Malbert;
Yoann Malbert
3TWB, 3 Rue des Satellites, Canal Biotech Building 2, Toulouse F-31400, France
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Jean Marie François;
1LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France
3TWB, 3 Rue des Satellites, Canal Biotech Building 2, Toulouse F-31400, France
Correspondence: Jean Marie François (fran_jm@insa-toulouse.fr)
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Thomas Walther
Thomas Walther
*
1LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France
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Publisher: Portland Press Ltd
Received:
September 19 2018
Revision Received:
November 07 2018
Accepted:
November 07 2018
Accepted Manuscript online:
November 08 2018
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2018
Biochem J (2018) 475 (23): 3887–3901.
Article history
Received:
September 19 2018
Revision Received:
November 07 2018
Accepted:
November 07 2018
Accepted Manuscript online:
November 08 2018
Citation
Cláudio J.R. Frazão, Christopher M. Topham, Yoann Malbert, Jean Marie François, Thomas Walther; Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway. Biochem J 12 December 2018; 475 (23): 3887–3901. doi: https://doi.org/10.1042/BCJ20180765
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