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Applied and Environmental Microbiology, April 2003, p. 2237-2244, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2237-2244.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Functional Replacement of the Escherichia coli D-(-)-Lactate Dehydrogenase Gene (ldhA) with the L-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici{dagger}

Shengde Zhou, K. T. Shanmugam, and L. O. Ingram*

Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611

Received 24 September 2002/ Accepted 8 January 2003

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.

* Corresponding author. Mailing address: Dept. of Microbiology and Cell Science, IFAS, P.O. Box 110700, University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail: ingram{at}ufl.edu.

{dagger} Florida Agricultural Experiment Station Journal Series no. R-09069.

Applied and Environmental Microbiology, April 2003, p. 2237-2244, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2237-2244.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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