Overexpression of Cutinase by Mediation of Signal Peptide

Abstract

T. fusca cutinase was extracellularly expressed by the mediation of pelB signal peptide via the Type II secretion pathway. A translational initial region degeneracy mutagenesis was carried out in the initial sequence of pelB. A fast screening method for these mutants was developed and a high cutinase production level achieved 38.0 U/mL. In addition, recombinant Escherichia coli that expressed precursor cutinase with one and two cleavage sites in signal peptide were constructed. The cutinase activity in culture medium was 95.7 U/mL and 130.3 U/mL, respectively, indicating that the increase of signal peptide cleavage site can improve the extracellular production of cutinase. We also found that extracellular expression of cutinase with pelB signal peptide depends on more than the type II secretion pathway. The phospholipid hydrolysis activity of pelB-cutinase played an important role in extracellular production. Besides, the cutinase was extracellularly expressed by the mediation of HlyAs signal peptide via the Type I secretion pathway. HlyB and HlyD are strain-specific translocation components of the alpha-hemolysin secretion system, which were coexpressed to facilitate the enzyme expression. The cutinase activity from this engineered cell was 2.5 times than that from the type II secretion pathway.