Abstract: MAPK/HOG signal pathway of yeast plays an important role in adapting the change of hyperosmotic stress outside. The current studies on this pathway, however, are practically based on Saccharomyces cerevisiae instead of Pichia pastoris, which limits the application of this excellent heterologous protein expression system. In this study, CRISPR/Cas9 system was applied to knock out the key transcription factor genes PBS2, HOG1, HOT1 and HOT2 in P. pastoris. Then, these mutant strains and GS115 strain were treated both in pre-stress and hyperosmotic condition. The results revealed that HOG1, HOT1 and HOT2 played important roles in protecting cells from hyperosmotic stress. The transcriptional levels of PBS2 and HOG1 were constitutive and they up-regulated MSN2 and HOT1, but no significant changes were found in the transcription level of HOT2. However, MSN2 and HOT1 were still induced by hyperosmotic stress indicating that more transcription factors were involved in the regulation network. Several genes in the MAPK/HOG pathway, including GT1, DOG2, DAK1, HXT1, HSP12 and CTT1, were confirmed. Hog1 was a key transcription factor for up-regulating HSP12 and CTT1. The expression of HXT1 was regulated by Hot2. However, Hot1 was not involved in the regulation network. Finally, a novel MAPK/HOG pathway model in P. pastoris was established.

Key words: Pichia pastoris, MAPK/HOG, signal pathway, hyperosmotic stress, regulation