Integrated Strategies for Enhancing the Expression of Chitosanase AqCoA in the Pichia Pastoris by A Novel High-Copy Plasmid PMC-GAP

In our previous study, the chitosanase AqCoA and its products chitooligosaccharides exhibited significant application in fungal disease protection. In this study, to enhance the expression of AqCoA, we obtained various strains with multi-copy by a novel plasmid pMC-GAP with stable transformation ability in Pichia pastoris and built an integrated model combining the gene copy number, the chaperones and protein production of AqCoA. In terms of gene dosage, the highest enzyme activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that in strain with only one copy (0.18 U/ml). In addition, we found the chaperone like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased by 61%, 31% and 42% in expression levels of the strain GAP-1AqCoA. Finally, we investigated the effect co-expression of gene copy and chaperones on protein expression. The maximum activity reached 2.319 U/ml by the strain with six chaperones intergrant plus sixteen copies, which was 13-fold higher than that by the control strain with only one copy (GAP-1AqCoA). Co-expression of gene dosage and chaperones significantly enhanced expression levels of AqCoA, which presented a powerful tool to improve foreign protein expression.