Our previous study demonstrated that when Thermobifida fusca cutinase was expressed in Escherichia coil without mediation of a signal peptide, it could release to the culture medium via the enhanced membrane permeability, which was based on its limited phospholipid hydrolysis activity. The goal of the present work was to achieve highly efficient extracellular production of the recombinant signal peptide-free cutinase in E. coli. The codons of the T. fusca cutinase gene were optimized for expression in E. coli, and a recombinant expression system was constructed using this optimized gene. After that, the induction strategy was optimized using high-cell-density cultivation in a 3-L fermentor. Results showed that the optimal induction condition was at a dry cell weight (DCW) of 13 gL(-1), and an IPTG/Iactose combination induction strategy is proposed, in which IPTG is added once in a final concentration of 25.0 mu M, and lactose is fed at a rate of 0.5 gL(-1) h(-1). In this condition, an extracellular cutinase activity of 2258.5 U mL(-1) (5.1 gL(-1)) was achieved, which represented the highest cutinase production ever reported, and demonstrated the potential of this system for the industrial production of cutinase. (C) 2015 Elsevier Ltd. All rights reserved.