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

Xiaolong Wang,Qi Wang,Jinjia Wang,Peng Bai,Lei Shi,Wei Shen,Mian Zhou,Xiangshan Zhou,Yuanxing Zhang,Menghao Cai

doi:10.1074/jbc.m115.692053

Abstract

The alcohol oxidase 1 (AOX1) promoter (PAOX1) 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)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 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 PAOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated PAOX1 in response to methanol, were bound to PAOX1 at different sites and did not interact with each other. However, these factors cooperatively activated PAOX1 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 (PMIT1), thus increasingly expressing Mit1 and subsequently activating PAOX1.

Highlights

Metabolic pathways and enzymes involved in the MUT pathway are similar in different methylotrophic yeasts [6, 11, 12]
Identification of an Essential Transcription Factor methanol-induced transcription factor 1 (Mit1) for the Growth of P. pastoris in Methanol—In our previous study, we identified a methanol-induced zinc finger (ZF) protein by performing RNA-Seq and designated it as P. pastoris Mit1
We have shown that the ZF domain of Mit1 is necessary for DNA binding (Fig. 4) and that the UR2 domain is necessary for its subcellular localization (Fig. 3F). ⌬RR1, ⌬UR1, and ⌬UR4 cells showed growth defects in the presence of methanol, and PAOX1 activity in these cells decreased to 88, 64, and 30%, respectively, compared with that in wild type (WT) cells (Fig. 3, C and E)

Summary

Introduction

Metabolic pathways and enzymes involved in the MUT pathway are similar in different methylotrophic yeasts [6, 11, 12]. The promoters of these genes are repressed in the presence of glucose and ethanol, AOD1 promoter (PAOD1) and MOX promoter (PMOX) show ϳ3–30% and 60 – 80% derepression, respectively, whereas PAOX1 shows almost complete repression in glycerol [6, 13] These differences cannot be attributed to variances in the sequences of these promoters because PAOX1 has the same regulatory profile as PMOX when introduced into H. polymorpha [14]. Prm functions as a positive regulator of genes involved in the MUT pathway in P. pastoris [18, 19] Each of these transcription regulators plays a critical role in activating methanol-inducible promoters in methylotrophic yeasts. The results of this study will help clarify in detail the mechanisms underlying the regulation of PAOX1 in P. pastoris and will provide a reference for elucidating the mechanisms underlying the response of methylotrophic yeasts to methanol

Methods

Results

Conclusion