title: Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth.
abstract: 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, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education & The College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China. ddniu@tust.edu.cn.
authors.forename: Dandan
authors.initials: D
authors.lastname: Niu
authors.forename: Kangming
authors.initials: K
authors.lastname: Tian
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Prior
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Zhengxiang
authors.initials: Z
authors.lastname: Wang
authors.forename: Fuping
authors.initials: F
authors.lastname: Lu
authors.forename: Suren
authors.initials: S
authors.lastname: Singh
mesh_terms: D061249:Batch Cell Culture Techniques; D018533:Biomass; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000096:biosynthesis*; D060847:Metabolic Engineering; D011401:Promoter Regions, Genetic; D011773:Pyruvates / Q000378:metabolism; D013237:Stereoisomerism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D011773:Pyruvates; D019344:Lactic Acid; D007770:L-Lactate Dehydrogenase