Isoamylase, an industrially significant enzyme used primarily in the saccharification of starch, is commonly obtained through recombinant expression in Escherichia coli. To improve the yield of this important enzyme, the isoamylase from Thermobifida fusca was expressed in the reduced-genome E. coli strain MDS42. Expression conditions were initially optimized in shake flasks. The optimal induction temperature was 37 A degrees C and IPTG was superior to lactose as an inducer due to the low intracellular beta-galactosidase activity in E. coli MDS42 expression system. Then, expression conditions were optimized in a 3.6-L fermenter. In the fermenter, optimal isoamylase expression was obtained when cells were grown at 37 A degrees C and expression was induced at mid-log phase using 0.25 mM IPTG. The greatest isoamylase activity (22,983.0 U/mL of culture) and production (18.8 mg/mL) were obtained 24 h after induction of expression. These values are the highest ever reported, suggesting that E. coli MDS42 is a suitable host for the production of enzymes for industrial use.