초록
<P><B>Abstract</B></P> <P>Thermodynamic states of the central metabolism in a metabolically engineered <I>Escherichia coli</I> strain producing mevalonate (MVA) were studied to identify metabolic reactions with regulatory function for improvement of the specific rate of MVA production. Intracellular concentrations of metabolites were determined for <I>E. coli</I> strains expressing <I>Enterococcus faecalis</I> genes <I>mvaE</I> and <I>mvaS</I> (strain MV) by gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Mixtures of <SUP>13</SUP>C-labeled metabolites served as internal standards were prepared from <I>E. coli</I> cultured in a completely <SUP>13</SUP>C-labeled medium. Based on the concentration data, the change in Gibbs energy (Δ<I>G</I>) and substrate saturation ([<I>S</I>]/<I>K</I> <SUB>M</SUB>) were calculated for each metabolic reaction and then compared between the control and MVA-producing strains. The thermodynamic and kinetic analyses showed that further activation of thermodynamically feasible reactions in the upper part of glycolysis and the pentose phosphate pathway seems difficult and that metabolic bypassing to the Entner–Doudoroff pathway was a promising strategy to improve the acetyl coenzyme A (AcCoA) and NADPH supply required for MVA biosynthesis. Strain MV-ΔGndΔGntR was constructed by deletion of the <I>gnd</I> and <I>gntR</I> genes, which respectively encode 6-phosphogluconate dehydrogenase and a negative regulator of the expression of two enzyme genes responsible for the Entner–Doudoroff pathway. Cultivation in the nongrowth phase revealed that the yield and specific production rate of MVA increased to 0.49 ± 0.01 Cmol (Cmol glucose)<SUP>−1</SUP> and 2.61 ± 0.10 mmol (g dry cell weight)<SUP>−1</SUP> h<SUP>−1</SUP>, which were 113% and 158% that of the MV strain, respectively.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Thermodynamic analysis was performed for an <I>Escherichia coli</I> strain producing mevalonate (MVA). </LI> <LI> Metabolite concentrations were determined by LC-MS/MS and GC-MS. </LI> <LI> Reactions with larger negative Δ<I>G</I> were calculated from the measured data. </LI> <LI> A promising strategy is activation of the Entner–Doudoroff pathway (EDP). </LI> <LI> Specific production rate of MVA was improved 158% by the EDP activation. </LI> </UL> </P>