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Applied and Environmental Microbiology, January 2003, p. 399-407, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.399-407.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Production of Optically Pure D-Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110{dagger}

Shengde Zhou, T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 19 June 2002/ Accepted 24 September 2002

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of L-lactic acid to D-lactic acid. Although the largest demand is for the L enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of D-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. D-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this D-lactic acid was ~98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of D-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, Bldg. 981, Museum Road, 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 Journal Series no. R-08894.

Applied and Environmental Microbiology, January 2003, p. 399-407, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.399-407.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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