Regulation Mechanism Of Pichia Pastoris HOT In MAPK/HOG Signaling Pathway And Methanol Metabolism

Pichia pastoris?P.pastoris?is one of the most widely used eukaryotic expression systems.Its efficient promoter of alcohol oxidase 1(P_AOX1)is strongly induced by methanol but is depressed by some carbon sources such as glycerol.Methanol is a kind of chemical industrial material and it has defects including flammable,explosive,low rate of carbon utilization,etc.Compared with methanol,glycerol is easy for utilization and has strong reducing power,so developing a novel glycerol depressive P.pastoris system is meaningful to industrial production.However,the molecular mechanism of methanol metabolism should be understood firstly.In S.cerevisiae,as an important transcription factor in MAPK/HOG signaling pathway,HOT1 is involved in the regulation of several glycerol-related genes,so it may have a close relationship with methanol metabolism.However,the regulation of MAPK/HOG signaling pathway in P.pastoris is poorly understood.The regulation mechanism of HOT protein on MAPK/HOG signaling pathways and methanol metabolism in P.pastoris still need to be discovered.In this study,we identified two potential high-osmolarity-induced transcription proteins HOT1 and HOT2 in P.pastoris.Then,the molecular mechanism of two factors and their regulation on methanol metabolism were studied.The research was carried out with the following research results:?1?HOT proteins in P.pastoris were firstly identified.Based on the P.pastoris database,two proteins with high homology to the S.cerevisiae HOT1 sequence were found and named HOT1 and HOT2.The study revealed that deleting HOT1 or HOT2 had a nagetive impact on the growth of cells under hyperosmotic condition,and they played an important role in MAPK/HOG signaling pathway of P.pastoris.?2?Then,we detected the regulation mechanism of the upstream transcription factor HOG1 on HOT1 and HOT2:qPCR experiments indicated that HOG1 was involved in the expression of HOT1 gene at transcriptional level,and yeast two-hybrid experiment results revealed that HOG1 and HOT1 could interact with each other at protein level;while HOT2was not regulated by HOG1 at transcriptional level or protein level.?3?HOT1 and HOT2 were localized in the nucleus.It was supposed that HOT1 and HOT2 may work by changing their subcellular localization under different conditions.Therefore,EGFP gene was respectively fused into HOT1 and HOT2 to characterize their localization in cells under different conditions.The results showed that:HOT1 and HOT2were localized in the nucleus both under general osmotic stress and hyperosmotic conditions,and the deletion of HOG1 gene did not change their subcellular localization.From above results,we conjectured that the function of HOT1 and HOT2 did not depend on its localization in cells.?4?Then,the regulation of osmotic response genes by HOT1 and HOT2 was studied.Different from S.cerevisiae,the expression of GT1 and GPD1 genes in P.pastoris was not regulated by HOT1 and HOT2.P_STL1 and P_GPD1 of S.cerevisiae were introduced into P.pastoris,and it was found that HOT1 and HOT2 could not induce their expression,which indicated that the functions of HOT1 and HOT2 in P.pastoris had changed.Also,HOT1 was no longer involved in the regulation of downstream hyperosmotic response genes DOG2,DAK1,HXT1,CTT1 and HSP12 genes;HOT2 could up-regulated HXT1 gene,but was not involved in the regulation of other genes above.?5?Finally,the regulation of HOT1 and HOT2 genes on methanol metabolism was studied.The results showed that deleting HOT1 or HOT2 could promote methanol metabolism.Besides,the loss of HOT2 improved the depression ability of cells in glycerol medium.