초록
<P>L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD<SUP>+</SUP>-dependent L-lactate dehydrogenase. Naturally, <I>Escherichia coli</I> does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the <I>E. coli</I> strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, Δ<I>ldhA</I>::<I>diflldD</I>::P<SUB><I>ldh</I></SUB>-<I>ldh</I><SUB>Lca</SUB>), 090B2 (B0013-070, Δ<I>ldhA</I>::<I>diflldD</I>::P<SUB><I>ldh</I></SUB>-<I>ldh</I><SUB>Strb</SUB>) and 090B3 (B0013-070, Δ<I>ldhA</I>::<I>diflldD</I>::P<SUB><I>ldh</I></SUB>-<I>ldh</I><SUB>Bcoa</SUB>) were developed from a previously developed D-lactate over-producing strain, <I>E. coli</I> strain B0013-070 (<I>ack-ptappspflBdldpoxBadhEfrdA</I>) by: (1) deleting <I>ldhA</I> to block D-lactate formation, (2) deleting <I>lldD</I> to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the <I>ldhA</I> promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), <I>E. coli</I> 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by <I>E. coli</I> is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production.</P><P><B>Highlights</B></P><P>The enzymatic thermodynamics was used as a tool for metabolic regulation. ► minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. ► maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.</P>