Reverse Reaction of Malic Enzyme for HCO₃⁻Fixation into Pyruvic Acid to SynthesizeL-Malic Acid with Enzymatic Coenzyme Regeneration

Abstract

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).

Substrate Promiscuity of Thermoplasma acidophilum Malic Enzyme for CO2 Fixation Reaction

“…As shown in Figure S2a, the optimal yield was achieved at an initial pH of 7.5–8.5, with a decline observed at pH 7.0. Notably, this pH range differs from the reported optimal pH for the reductive carboxylation reaction by ME from Pseudomonas diminuta IFO 13182 (pH 6.0) and Thermococcus kodakarensis (pH 6.5) as well as the reported Ta GDH activity for NADPH production (pH 6.5) . The differences in optimal pH values may be attributed to the likelihood that the actual pH during the reaction in our experiment was lower than the initial pH due to the formation of H 2 CO 3 and subsequent pH reduction.…”

Metabolic engineering of Escherichia coli W3110 to produce L‐malate

“…This one-step synthesis pathway has a maximum theoretical L-malate yield of 2 mol/mol glucose, which is equivalent to the reductive TCA cycle and higher than TCA cycle Figure 3. For example, using malic enzyme from Pseudomonas diminuta coupled with coenzyme (NADH) regeneration, 38 mM L-malate was formed from 100 mM pyruvate in 24 h (Ohno et al, 2008). (1 mol/mol) and glyoxylate cycle (1.33 mol/mol) (Zelle et al, 2008).…”

“…Other studies have also attempted to produce L-malate using malic enzyme. For example, using malic enzyme from Pseudomonas diminuta coupled with coenzyme (NADH) regeneration, 38 mM L-malate was formed from 100 mM pyruvate in 24 h (Ohno et al, 2008). More recently, a synthetic metabolic engineering strategy was employed to synthesize L-malate directly from glucose, and 2.4 mM L-malate was produced using malic enzyme from Thermococcus kodakarensis (Ye et al, 2013).…”

Efficient Electrocatalytic CO2 Fixation by Nanoconfined Enzymes via a C3-to-C4 Reaction That Is Favored over H2 Production

“…The small Δ r G (−7 kJ mol −1 at 25 °C, pH 7.5) for decarboxylation of malate by NADP + allows the reaction to be run in reverse, so it presents an ideal system by which to achieve specific CO 2 reduction, provided a continuous supply of NADPH cofactor is available . Use of ME to incorporate CO 2 into pyruvate was first reported over 30 years ago and featured photo-electrochemical cofactor regeneration by FNR using methyl viologen as a low-potential electron mediator. ,− Here, we show how nanoconfinement is exploited to perform C3-to-C4 CO 2 incorporation with ease, electrochemical clarity, and high efficiency with regard to overpotential and competition with H 2 evolution.…”