Effect Of MXR1 Phosphorylation On AOX1 Expression

Pichia pastoris?P.pastoris?is one of the most widely used eukaryotic microbial expression systems.Compared with other expression systems,the most significant advantage of the pastoris expression system is its inducible promoter-alcohol oxidase 1(P_AOX1),which is strictly induced by methanol.It was found that this promoter can only be activated when methanol is the only carbon source.AOX1 transcription is severely repressed in the presence of carbon sources such as glycerol or glucose.Through studying its induction mechanism,it was found that methanol expression regulator 1?MXR1?is required for P_AOX1 to function as a promoter.At present,the mechanism of how MXR1 regulates P_AOX1 is not well understood.This study reveals the mechanism of methanol-induced glycerol repression of AOX1transcription by studying the mechanism by which MXR1 activates AOX1 transcription.First,a protein that interacts with P.pastoris MXR1,Protein phosphotyrosine phosphatase?PTP?,was found by PULL-DOWN.The protein interacts with MXR1 in methanol medium and has been dephosphorylated by in vitro experiments to hydrolyze the phosphate group of phosphorylated MXR1 215th serine.After the expression of PTP was high and low,the glycerol,wild-type,high-expressing and knockout strains were extracted from the methanol medium.The MXR1S215 specific site phosphorylation antibody was used to detect the phosphorylation of MXR1 in different media and in methanol.The amount of methanol consumed in the medium.The phosphorylation level of wild strain MXR1 in methanol medium was found to be much higher than its phosphorylation level in glycerol medium.When PTP was highly expressed,MXR1 was not phosphorylated in glycerol or methanol medium,and the phosphorylation level of MXR1 protein in PTP knockout strain was significantly higher than that in wild type strain,indicating that phosphatase PTP regulates MXR1 phosphorylation.The effect of PTP high and low expression mutants on methanol metabolism was detected.It was found that the PTP high expression strain inhibited the metabolism of methanol,the AOX1 transcription level was almost zero,and the AOX1 enzyme activity was not detected.It also indicated that the high expression of PTP inhibited the transcription of AOX1.Thereby inhibiting the metabolism of methanol when culturing with methanol as the sole carbon source.Furthermore,the effect of 215th serine phosphorylation modification on methanol metabolism in MXR1 was studied.The CRISPR/Cas9 system was used to successfully mutate the 215th serine of MXR1 to alanine on the P.pastoris genome.In methanol medium,the expression of genes related to methanol metabolism in wild type,site-directed mutagenesis and knockout strains was detected by real-time PCR.Several MXR1 targeting genes AOX1,AOX2and DAS1 were detected by real-time PCR.,the expression level of DAS2 after induction with methanol-containing medium.Deletion of the MXR1 gene resulted in a significant decrease in the transcriptional levels of AOX1,DAS1,and DAS2.In the MXR1S215A mutant,the transcription of AOX1,AOX2,DAS1,and DAS2 was also reduced by nearly 50%.The above results indicate that the transcription of AOX1,DAS1,and DAS2 is regulated by MXR1,and the serine phosphorylation modification of 215th of MXR1 is not important for this function.The above indicates that PTP regulates serine phosphorylation at position 215 of MXR1and regulates the methanol metabolism pathway.However,when PTP is highly expressed,the inhibition of methanol metabolism is not only regulated by serine phosphorylation at position215 of MXR1,but may also be related to other regulatory.