[Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1]

Abstract

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.