title: Long-term adaptive evolution ofLeuconostoc mesenteroidesfor enhancement of lactic acid tolerance and production.
abstract: BACKGROUND
  Lactic acid has been approved by the United States Food and Drug Administration as Generally Regarded As Safe (GRAS) and is commonly used in the cosmetics, pharmaceutical, and food industries. Applications of lactic acid have also emerged in the plastics industry. Lactic acid bacteria (LAB), such asLeuconostocandLactobacillus, are widely used as lactic acid producers for food-related and biotechnological applications. Nonetheless, industrial mass production of lactic acid in LAB is a challenge mainly because of growth inhibition caused by the end product, lactic acid. Thus, it is important to improve acid tolerance of LAB to achieve balanced cell growth and a high titer of lactic acid. Recently, adaptive evolution has been employed as one of the strategies to improve the fitness and to induce adaptive changes in bacteria under specific growth conditions, such as acid stress.
  
  
  RESULTS
  Wild-typeLeuconostoc mesenteroideswas challenged long term with exogenously supplied lactic acid, whose concentration was increased stepwise (for enhancement of lactic acid tolerance) during 1 year. In the course of the adaptive evolution at 70 g/L lactic acid, three mutants (LMS50, LMS60, and LMS70) showing high specific growth rates and lactic acid production were isolated and characterized. Mutant LMS70, isolated at 70 g/L lactic acid, increased d-lactic acid production up to 76.8 g/L, which was twice that in the wild type (37.8 g/L). Proteomic, genomic, and physiological analyses revealed that several possible factors affected acid tolerance, among which a mutation of ATPase ε subunit (involved in the regulation of intracellular pH) and upregulation of intracellular ammonia, as a buffering system, were confirmed to contribute to the observed enhancement of tolerance and production of d-lactic acid.
  
  
  CONCLUSIONS
  During adaptive evolution under lethal stress conditions, the fitness ofL. mesenteroidesgradually increased to accumulate beneficial mutations according to the stress level. The enhancement of acid tolerance in the mutants contributed to increased production of d-lactic acid. The observed genetic and physiological changes may systemically help remove protons and retain viability at high lactic acid concentrations.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.|Department of Applied Chemistry and Biological Engineering, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.
authors.forename: Si Yeon
authors.initials: SY
authors.lastname: Ju
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.|Department of Applied Chemistry and Biological Engineering, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.
authors.forename: Jin Ho
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.|Department of Applied Chemistry and Biological Engineering, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749 South Korea.
authors.forename: Pyung Cheon
authors.initials: PC
authors.lastname: Lee
keywords: Acid tolerance; Leuconostoc mesenteroides; Long-term adaptive evolution; d-lactic acid