Improving the expression of recombinant xylanase in Pichia pastoris with episomal expression plasmid

Abstract

Pichia pastoris is a versatile protein expression system. At present, the expression plasmids are integrated plasmids whereas the episomal plasmids are rarely used. In this study, the autonomously replicating sequence derived from yeast itself was ligated into the integrative expression vector pGAP to generate an autonomously replicative expression vector pGAPZαA-PARS. When the vector was used to express the xylanase (XynA) gene in P. pastoris, the highest enzyme activity reached 343 U/mL with glycerol as the carbon source, which was 45.9% higher than that of the integrative expression. At the same time, the specific enzyme activity of XynA was increased by 81.2%. We further studied the expression level of recombinant strains with different carbon sources such as glycerol, glucose, sucrose and mixed carbon source (sucrose︰glycerol=1︰2). The highest expression level was achieved with glycerol and the lowest with sucrose. The episomal expression vector pGAPZαA-PARS greatly promotes the application of P. pastoris in the food industry because GAP promoter does not require methanol as induction material. Meanwhile, the episomal vector can greatly improve the expression level, which lays the foundation for further research to improve the high expression of GAP promoter.