Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3)

T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase.In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.Generally, extracellular expression of proteins has an absolute advantage in a large-scale industrial production. Escherichia coli, the most widely used host, has five protein secretion pathways, in which, the vast majority of recombinant proteins are secreted using the SecB-dependent type II pathway. In this process, the pre-protein is first transferred across the inner membrane, folded in the periplasm, and then secreted into the culture medium, usually by nonspecific periplasmic leakage [1]. Due to this two-step process, the recombinant protein is sometimes partially located in the extracellular medium and partially in the periplasm, affecting the total extracellular yields [2,3].By contrast, type I secretion systems (TISS) export their native passenger proteins or recombinant proteins/peptides