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

Abstract

BackgroundExtracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.ResultsT. 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.ConclusionsIn 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.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential

“…On the other hand, bacterial thermophilic cutinases from T. fusca expressed in E. coli BL21, showed an optimal enzyme activity at 55-60 ο C [45,64].…”

Enhanced extracellular expression of gene-optimized Thermobifida fusca cutinase in Escherichia coli by optimization of induction strategy

“…When cutinase was expressed by the mediation of signal peptides pelB and HlyAs, the extracellular cutinase activities were 149.2 U/mL and 334 U/mL, respectively, under optimized cultivation conditions in shake flasks [19,20]. With further exploration of the latter one in a 3 L fermenter, the cutinase yield reached 725 U/mL [21].…”

“…In our previous work, the gene encoding Thermobifida fusca cutinase (Tfu 0883, NCBI accession number AAZ54921) was identified and extracellularly expressed in Escherichia coli using a variety of strategies, including utilizing different secretion systems, coexpression of key secretion transport proteins, and optimization of environmental conditions [18][19][20][21]. When cutinase was expressed by the mediation of signal peptides pelB and HlyAs, the extracellular cutinase activities were 149.2 U/mL and 334 U/mL, respectively, under optimized cultivation conditions in shake flasks [19,20].…”

Characterization of CpdC, a Large-Ring Lactone-Hydrolyzing Enzyme from Pseudomonas sp. Strain HI-70, and Its Use as a Fusion Tag Facilitating Overproduction of Proteins in Escherichia coli

“…In a separate research project, we were interested in another subfamily of ␣/␤-fold hydrolases, namely, cutinases that have numerous industrial applications, including the fiber/textile and biopolymer industry (31)(32)(33)(34). As genes for secreted proteins, the cutinase-encoding genes are generally not easily expressed in E. coli without invoking a secretion system either in E. coli (e.g., ␣-hemolysin) or in yeasts such as Pichia (35)(36)(37).…”