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 . 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 such as repeats-in-toxin (RTX) proteins, proteases and lipases directly into the culture medium without accumulation in the periplasmic space [4–9]. The best-characterized and most widely used TISS is the α-hemolysin (HlyA) secretion pathway from uropathogenic E. coli . The secretory machinery of this pathway consists of three components: HlyB, an ATP-binding cassette (ABC transporter); HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The three components can form a trans-membrane channel connecting both inner and outer membranes of cell, through which HlyA can be transferred directly to the extracellular medium. In this pathway, the signal peptide, which is located in the C-terminus of HlyA, is not cleaved after translocation and remains in the target protein as the final product .
In addition to HlyA, proteins ranging in size from 50 to over 4000 amino acids, with C-terminally fused HlyA signal sequence (HlyAs), can also be recognized and secreted via the HlyB-HlyD-TolC translocator [1, 11]. The α-hemolysin secretion pathway has mainly been reported in uropathogenic E. coli. HlyB and HlyD have been generally considered strain specific proteins, TolC, in contrast, is a component of multiple trans-membrane systems in many microorganisms. Thus, co-expression of HlyB/D is often performed to facilitate the extracellular translocation of proteins utilizing the α-hemolysin secretion pathway during expression in host strains other than uropathogenic E. coli . So far, this approach has most often been applied to the expression of proteins involved in immunological and vaccine research, especially in the recombinant antigen presentation through live-attenuated bacterial vaccine strains [11, 13–15], there were only several cases of successful extracellular expression of industrial enzymes, such as lipase , protease , cyclodextrin glucanotransferase , alkaline phosphatase . Recent reports about α-hemolysin secretion pathway have focused on the effects of translated protein folding rate and mutagenesis of HlyAs, HlyB or HlyD on the efficiency of translocation [15, 17, 19].
Cutinase is a multi-functional esterase, which shows hydrolytic activity (cutin and a variety of soluble synthetic esters, insoluble triglycerides and polyesters), synthetic activity and transester activity [20–28]. Therefore, it is an important industrial enzyme. Especially, due to the capacity of hydrolyzing the cutin structure in the cuticle of cotton fiber, it has potential application in environmentally friendly bioscouring . Previously, cutinase from Thermobifida fusca was cloned and extracellularly expressed in E. coli BL21(DE3) through type II secretory system in our laboratory . Large amounts of cutinase were found in the periplasmic space . In the present study, we showed that the T. fusca cutinase could be efficiently secreted to the external medium through α-hemolysin secretion pathway without a periplasmic intermediate. In addition, detailed characterization showed that the recombinant enzyme, even with c-terminal signal peptide attached, has similar properties to that of wild-type cutinase.