Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of alpha-ketobutyrate. In this study, a genetically engineered strain THRD Delta rhtA Delta ilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing alpha-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRD Delta rhtA Delta ilvIH/pWSK29-ilvA allowed the production of 16.2 g/L alpha-ketobutyrate. Accumulation of alpha-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRD Delta rhtA Delta ilvIH for alpha-ketobutyrate production. The initial temperature was maintained at 35 degrees C to guarantee normal cell growth, and then elevated to 40 degrees C to induce the expression of TD. Under optimized conditions, the alpha-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential.