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

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 ϳ98% 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.

Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

“…1). Previous reports indicate that E. coli with a ppc knockout does not grow on glucose as the sole carbon source (1,17,22,24,33). In our present study, ALS961 (YYC202 ppc) similarly did not grow aerobically in shake flasks with GAM medium.…”

“…PEP carboxylase is the primary anaplerotic pathway in E. coli, and as expected, a ppc mutation prevents growth on glucose minimal medium (1,18,22,24,33). Although PEP carboxykinase (encoded by the pckA gene) and malic enzymes (encoded by maeB and sfcA) also catalyze reversible C-3 carboxylation/ C-4 decarboxylation reactions, these two pathways appear to be responsible for decarboxylation in E. coli rather than for carboxylation (22).…”

“…The second strategy examined for prevention of succinate accumulation was a knockout of frdABCD (3,33). In E. coli, two distinct enzymes catalyze succinate oxidation and fumarate reduction (4): succinate-ubiquinone oxidoreductase functions aerobically as part of the TCA cycle, and menaquinol-fumarate oxidoreductase (QFR) is mainly used for anaerobic respiration and succinate production.…”

“…Similarly, the ldhA mutant of JP203 containing pLS65 encoding Lactobacillus casei L-LDH was grown aerobically to 7.2 g/liter DCW and generated 45 g/liter L-lactate, with a 0.7 g/g yield and a productivity of 0.67 g/liter · h after an anaerobic switch (6). A pflB frdBC adhE ackA mutant (SZ63) grown in minimal media generated 48 g/liter D-lactate of 99% optical purity, close to the theoretical maximum yield of 1.0 g/g (33). After part of the chromosomal ldhA coding region was replaced with Pediococcus acidilactici ldhL, encoding L-LDH, the new strain (SZ85) generated 50 g/liter L-lactate at a 95% yield, with a productivity of 0.42 g/liter · h (34).…”

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

“…E. coli also metabolizes pentose sugars through the pentose phosphate pathway, which feeds intermediates into the EMP pathway towards homolactate production, in contrast to the use of phosphoketolase by lactic acid bacteria, which leads to production of a mixture of lactic acid and acetic acid during pentose fermentation. Because E. coli ferments all the sugars present in plant biomass in a simple salt-based medium, this organism has been engineered for production of optically pure lactic acid by deleting competing pathways (Chang et al, 1999;Dien et al, 2001;Zhou et al, 2003aZhou et al, ,b, 2005. E. coli produces only the D-())-LDH enzyme, and the resulting product is 100% D-()) lactic acid (Zhou et al, 2003a).…”