Pichia pastoris expression system is characterized with simple culture conditions,low nutrient requirements,clear genetic background and high density fermentation,so it has been widely used in the expression of exogenous genes.The PAOX1(promoter of alcohol oxidase 1),a methanol inducible promoter,plays an important role for the efficient expression of exogenous genes.But some studies showed that PAOX1 was tightly regulated by methanol and repressed by glycerol,which led to the typically 2-step fermentation method in P.pastoris expression system: glycerol medium for cells growth and methanol medium for exogenous protein expression.However,this fermentation processes not only greatly extend fermentation period,but also lead to substantial loss of raw materials.So it could be an important way to improve P.pastoris expression system through studying the mechanism referring to glycerol inhibition PAOX1.In our previous studies,the first glycerol transporter in P.pastoris was found and named glycerol transporter 1(GT1).Its functions in glycerol repression of PAOX1 were studied.The results showed that the expression of AOX1 in Δgt1 mutant was higher than that in wide type in glycerol and methanol medium,which suggested that GT1 was involved in glycerol repression of PAOX1 pathway.Further studies showed that GT1 overexpression could increase intracellular glycerol level and repress the expression of methanol regulatory factors MXR1(methanol expression regulator 1).Some studies proved that MXR1 could bind on PAOX1 with the 5-CYCCNR-3 to promote the expression of AOX1.While in the mutant deleting mxr1,the expression of AOX1 would be repressed tightly.The above results showed that GT1 could inhibit the expression of PAOX1 by inhibiting mxr1.And then,that MXR1 transcription factor binding regions were found on gt1 promoter.The results of EMSA experiments showed that MXR1 could combine with PGT1 to inhibit the expression of gt1 and relieve the inhibition of GT1 to PAOX1 in methanol and methanol mixed glycerol medium.All the above results showed that,in glycerol repression of PAOX1 signaling pathway,gt1 and mxr1 inhibited each other to regulate the expression of PAOX1.