Production of Optically Pure d -Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110

Abstract

ABSTRACT 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.