Direct conversion of glucose to malate by synthetic metabolic engineering

• Published in: Journal of biotechnology Vol. 164; no. 1; pp. 34 - 40
• Main Authors: Ye, Xiaoting, Honda, Kohsuke, Morimoto, Yumi, Okano, Kenji, Ohtake, Hisao
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.03.2013
• Subjects: Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bicarbonates; biotechnology; Carbon Dioxide; Carbon dioxide fixation; carboxylation; Escherichia coli - genetics; glucose; Glucose - metabolism; glycolysis; heat treatment; Hydrogen-Ion Concentration; Malate Dehydrogenase - genetics; Malate Dehydrogenase - metabolism; malates; Malates - metabolism; Malic enzyme; manufacturing; metabolic engineering; Metabolic Engineering - methods; Metabolic Networks and Pathways; NADP; pyruvic acid; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Synthetic metabolic engineering; Temperature; Thermococcus - enzymology; Thermococcus - genetics; Thermococcus kodakarensis; Thermodynamic equilibrium; Thermodynamics; Thermophilic enzyme; Thermophilic enzyme; Carbon dioxide fixation; Synthetic metabolic engineering; Malic enzyme; Thermodynamic equilibrium
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2012.11.011

► An in vitro metabolic pathway for conversion of glucose to malate was constructed. ► Reversible carboxylation was coupled with a non-ATP-forming glycolysis pathway. ► Thermococcus malic enzyme showed both lactate-forming and malate-forming activities. ► Reaction specificity was redirected to malate by increasing the HCO3− concentration. Synthetic metabolic engineering enables us to construct an in vitro artificial synthetic pathways specialized for chemical manufacturing through the simple heat-treatment of the recombinant mesophiles having thermophilic enzymes, followed by rational combination of those biocatalytic modules. In this work, we constructed a synthetic pathway capable of direct conversion of glucose to malate. The reversible carboxylation of pyruvate catalyzed by a malic enzyme derived from Thermococcus kodakarensis (TkME) (ΔG°′=+7.3kJmol−1) was coupled with a thermodynamically favorable non-ATP-forming Embden–Meyerhof pathway to balance the consumption and regeneration of redox cofactors and to shift the overall equilibrium toward malate production (glucose+2HCO3−+2H→2 malate+2H2O; ΔG°′=−121.4kJmol−1). TkME exhibited both pyruvate carboxylation (malate-forming) and pyruvate reduction (lactate-forming) activities. By increasing HCO3− concentration, the reaction specificity could be redirected to malate production. As a result, the direct conversion of glucose to malate was achieved with a molar yield of 60%.