Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway.

Autor: Frazão CJR; LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France., Topham CM; Molecular Forces Consulting, 40 rue Boyssonne, Toulouse F-31400, France., Malbert Y; TWB, 3 Rue des Satellites, Canal Biotech Building 2, Toulouse F-31400, France., François JM; LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France fran_jm@insa-toulouse.fr.; TWB, 3 Rue des Satellites, Canal Biotech Building 2, Toulouse F-31400, France., Walther T; LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, Toulouse F-31077, France.
Jazyk: angličtina
Zdroj: The Biochemical journal [Biochem J] 2018 Dec 12; Vol. 475 (23), pp. 3887-3901. Date of Electronic Publication: 2018 Dec 12.
DOI: 10.1042/BCJ20180765
Abstrakt: 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 ( k cat ) 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 ( k cat / K m ) 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 .
(© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
Databáze: MEDLINE
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