Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50 degrees C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2gh(-1) g cells(-1)) at 37 degrees C (B. subtilis strain DA12). The D-LA titer at 48 degrees C was about 0.6 M at a yield of 0.99 (g D-LAg-1 glucose consumed). Strain DA12 also fermented glucose at 48 degrees C in mineral salts medium to lactate at a yield of 0.89 gg(-1) glucose and the D-lactate titer was 180 +/- 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48 degrees C that could minimize process cost.