Production of α-ketobutyrate using engineered Escherichia coli via temperature shift

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 α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-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 THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-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. Biotechnol. Bioeng. 2016;113: 2054-2059. © 2016 Wiley Periodicals, Inc.