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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 + -dependent L-lactate dehydrogenase. Naturally,  Escherichia coli  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  E. coli  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 ldhA :: diflldD ::P  ldh  - ldh  Lca ), 090B2 (B0013-070, Δ ldhA :: diflldD ::P  ldh  - ldh  Strb ) and 090B3 (B0013-070, Δ ldhA :: diflldD ::P  ldh  - ldh  Bcoa ) were developed from a previously developed D-lactate over-producing strain,  E. coli  strain B0013-070 ( ack-ptappspflBdldpoxBadhEfrdA ) by: (1) deleting  ldhA  to block D-lactate formation, (2) deleting  lldD  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  ldhA  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),  E. coli  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  E. coli  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.   Highlights  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.

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth