초록
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<SUP>o</SUP>'=+7.3kJmol<SUP>-1</SUP>) 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+2HCO<SUB>3</SUB><SUP>-</SUP>+2H→2 malate+2H<SUB>2</SUB>O; ΔG<SUP>o</SUP>'=-121.4kJmol<SUP>-1</SUP>). TkME exhibited both pyruvate carboxylation (malate-forming) and pyruvate reduction (lactate-forming) activities. By increasing HCO<SUB>3</SUB><SUP>-</SUP> 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%.