title: Direct conversion of glucose to malate by synthetic metabolic engineering.
abstract: 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%.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Ye
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.forename: Yumi
authors.initials: Y
authors.lastname: Morimoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001639:Bicarbonates; D002245:Carbon Dioxide; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008293:Malates / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D009249:NADP; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D001639:Bicarbonates; D008293:Malates; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase; D005947:Glucose