Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris

The alcohol oxidase 1 (AOX1) promoter (P(AOX1)) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)(2)Cys(6)-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating P(AOX1) activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of P(AOX1) in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated P(AOX1) in response to methanol, were bound to P(AOX1) at different sites and did not interact with each other. However, these factors cooperatively activated P(AOX1) through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (P(MIT1)), thus increasingly expressing Mit1 and subsequently activating P(AOX1).