Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris

IntroductionPichia pastoris is widely used for the production of recombinant proteins, but the low production efficiency hinders its wide application in biopharmaceuticals. Moreover, many biopharmaceutical-like proteins are accompanied by degradation during secretory expression in P. pastoris. ObjectiveIn this study, we used human serum albumin and porcine follicle-stimulating hormone beta (HSA-pFSH beta) fusion protein as a model protein to investigate whether YPS1 and YPT7 gene disruption and N-acetyl-L-cysteine (NAC) supplementation have synergistic effects to inhibit the degradation of recombinant proteins. Results and discussionOur results showed that YPS1 gene disruption reduced the degradation of intact HSA-pFSH beta and increased the yield of intact protein in the culture medium and cells without affecting the integrity of the cell wall. Moreover, the beneficial effects of YPS1 gene disruption were associated with the upregulation of the MAPK signaling pathway and maintenance of redox homeostasis. YPS1 gene disruption and NAC supplementation had synergistic effects on HSA-pFSH beta production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSH beta protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSH beta strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSH beta proteins, and synergistically increase the yield of intact HSA-pFSH beta with NAC supplementation. This study provided a valuable reference for reducing recombinant protein degradation and therefore improving the yield of recombinant proteins in P. pastoris.

Automated summary

In this study, the authors investigated the synergistic effects of YPS1 and YPT7 gene disruption and NAC supplementation on the degradation of recombinant proteins in P. pastoris. The results showed that YPS1 gene disruption reduced protein degradation and increased protein yield. Additionally, YPT7 gene disruption and NAC supplementation had a synergistic effect on protein production.