Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici

Appl Environ Microbiol. 2003 Apr;69(4):2237-44. doi: 10.1128/aem.69.4.2237-2244.2003.

Abstract

The microbial production of L-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-(+) and D-(-) isomers. For most uses of PLA, the L-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a D-(-)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an L-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in L-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native D-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased L-lactate dehydrogenase activity. SZ85 produced L-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for L-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Culture Media
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Gene Deletion
  • Genetic Engineering / methods
  • Glucose / metabolism
  • L-Lactate Dehydrogenase / genetics*
  • L-Lactate Dehydrogenase / metabolism*
  • Lactate Dehydrogenases*
  • Lactic Acid / metabolism*
  • Molecular Sequence Data
  • Pediococcus / enzymology*
  • Pediococcus / genetics
  • Polyesters
  • Polymers / metabolism
  • Recombination, Genetic*
  • Sequence Analysis, DNA
  • Stereoisomerism
  • Xylose / metabolism

Substances

  • Culture Media
  • Polyesters
  • Polymers
  • Lactic Acid
  • poly(lactide)
  • Xylose
  • Lactate Dehydrogenases
  • L-Lactate Dehydrogenase
  • D-lactate dehydrogenase
  • Glucose

Associated data

  • GENBANK/AY205156
  • GENBANK/AY205157