MAPK/HOG signaling pathway induced stress‐responsive damage repair is a mechanism for Pichia pastoris to survive from hyperosmotic stress

BACKGROUND MAPK/HOG signaling pathway plays a key role in the response of yeast to external hyperosmotic stress. Over the past few decades, the regulation mechanism for this pathway in the robust yeast, Saccharomyces cerevisiae, has been elucidated. However, the weak ability of the biotechnical workhorse, Pichia pastoris, in surviving hyperosmotic stress suggests a unique regulatory mechanism needing further investigation. RESULT Here, we identified crucial genes in the MAPK/HOG pathway of P. pastoris and investigated their effects on cell growing in osmotically stressed environments by knocking out these genes using a novel CRISPR/Cas9 system. Using real‐time polymerase chain reaction (RT‐PCR) and yeast two‐hybrid assay, transcription factors Hot1, Msn4 and Sko1 were demonstrated to be regulated by Pbs2 and Hog1 either at mRNA or protein level. We also examined the subcellular localization of these transcription factors, reflecting their translocation between cytoplasm and nucleus. The transcriptions of putative osmo‐responsive genes were then studied by RT‐PCR. We found the induction of glycerol‐related genes, such as GT1 and GPD1, was marginal when cells experienced high osmolarity. The ability of P. pastoris to increase intracellular glycerol level was determined and found to be much weaker than that in S. cerevisiae. By contrast, stress‐induced damage repair genes, including CTT1 and HSP12, were dramatically increased. CONCLUSION We conclude that P. pastoris could barely balance hyperosmotic stress by increasing intracellular glycerol concentrations, and stress‐induced damage repair is still an important mechanism for P. pastoris survival under hyperosmotic stress. This study demonstrates a description of the MAPK/HOG pathway in P. pastoris and provides a trigger for improving its robustness. © 2020 Society of Chemical Industry (SCI)