Functional Replacement of the Escherichia coli d- (−)-Lactate Dehydrogenase Gene ( ldhA ) with the l- (+)-Lactate Dehydrogenase Gene ( ldhL) from Pediococcus acidilactici

Abstract

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.