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

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.

Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation

“…Deficiency of either of the genes led to the de-repression of P AOX1 in response to glucose. In addition, some downstream transcriptional repressors such as Nrg1 [6] and activators (Mit1, Prm1 and Mxr1) have been identified to regulate P AOX1 [7,8]. …”

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris

“…These include the following: Mxr1p, Rop1p, Trm1p, Mit1p, and Nrg1p (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The role of these transcription factors in the regulation of amino acid metabolism has not been examined.…”

Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter

“…Lin-Cereghino et al (2006) identified methanol expression regulator 1 (Mxr1), a transcription factor (TF) with a zinc finger DNAbinding domain, homologous to the TF Adr1 from Saccharomyces cerevisiae. Recently, methanol-induced transcription factor (Mit1), a repressor of phosphoenolpyruvate carboxykinase (Rop), and Trm1 have also been described as TFs responsible for methanol regulation (Kumar & Rangarajan, 2012;Sahu, Krishna Rao, & Rangarajan, 2014;Wang et al, 2016). Further investigations by Kranthi, Kumar, Kumar, Rao, and Rangarajan, (2009) and 2010) revealed several Mxr1-binding sites in the promoter of PEX8, AOX1, and DAS.…”

“…Mutations in this gene resulted in a strain unable to grow in methanol and oleate and induce the transcription of alcohol oxidase 1 (AOX1), dihydroxyacetone synthase (DAS), peroxin 8 (PEX8), and peroxin 14 (PEX14). Wang et al (2016) suggested a transmission of the methanol induction signal among Mxr1, Mit1, and Trm1 through a cascade, being Mxr1 the responsible for the derepression of P AOX1 , whereas methanol presence would induce Trm1 and, subsequently, Mit1 expression. Subsequently, Parua, Ryan, Trang, and Young (2012) characterized a 14-3-3 family protein (regulator of numerous biological processes in many eukaryotes, Fu, Subramanian, & Masters, 2000) in P. pastoris.…”

“…Because of MXR1 is constitutively expressed at low levels (Lin-Cereghino et al, 2006), our observations suggested that the presence of multiple P AOX1 copies resulted in an insufficient number of Mxr1 molecules to fully induce transcription of methanol-regulated genes in multicopy expression strains, including the recombinant ROL. In addition, MIT1-deficient strains have marginal AOX1 messenger RNA (mRNA) when exposed to methanol, highlighting Mit1 as an essential TF for growth on methanol (Wang et al, 2016). In addition, MIT1-deficient strains have marginal AOX1 messenger RNA (mRNA) when exposed to methanol, highlighting Mit1 as an essential TF for growth on methanol (Wang et al, 2016).…”