Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici.
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.
Zhou
Shengde
S
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville 32611, USA.
Shanmugam
K T
KT
Ingram
L O
LO
D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods; D005947:Glucose / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D045022:Lactate Dehydrogenases; D019344:Lactic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D010376:Pediococcus / Q000201:enzymology* / Q000235:genetics; D011091:Polyesters; D011108:Polymers / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D014994:Xylose / Q000378:metabolism
D003470:Culture Media; D011091:Polyesters; D011108:Polymers; D019344:Lactic Acid; C033616:poly(lactide); D014994:Xylose; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; D005947:Glucose