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 β (HSA-pFSHβ) 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-pFSHβ 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-pFSHβ production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSHβ protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSHβ strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSHβ proteins, and synergistically increase the yield of intact HSA-pFSHβ 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.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.