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Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway.
Biochemical Journal 2018 November 9
A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine, composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction of 2-keto-4-hydroxybutyrate (OHB) into (L)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In this 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 step-wise manner into these regions of the protein. This rational engineering led to the production of a Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat ) on OHB that was increased by more than 2,000 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 an homoserine-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert homoserine to OHB resulted in 89 % increased DHB production as compared to 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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