Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Abstract

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris

“…We recently demonstrated that Mxr1p, a master regulator of the Mut pathway in P. pastoris, regulates gene expression by binding to promoter sequences containing 59-CYCCNY39motif (Kranthi et al, 2009(Kranthi et al, , 2010. The DNA-binding domain of Mxr1p shares 55 % amino acid sequence identity with that of the S. cerevisiae zinc finger transcription factor Adr1p (Lin-Cereghino et al, 2006, Fig.…”

“…1). We reported earlier that Adr1p and Mxr1p bind to promoter sequences containing 59-CYCCNY-39 and 59-CYCCNR-39 respectively (Kranthi et al, 2009(Kranthi et al, , 2010. In view of the high degree of homology among the DNA-binding domains of ROP, Mxr1p and Adr1p (Fig.…”

“…The DNA-binding domain of Mxr1p shares 55 % amino acid sequence identity with that of the S. cerevisiae zinc finger transcription factor Adr1p (Lin-Cereghino et al, 2006, Fig. 1), whose recognition sequence contains 59-CYCCNR -39 rather than 59-CYCCNY -39 motifs (Kranthi et al, 2009). A BLAST search of the P. pastoris genome sequence database with the amino acid sequence of Mxr1p led to the identification of a gene encoding a putative zinc finger protein (GenBank accession number CAY70604/XM_002492738.1).…”

“…Mxr1p regulates the expression of target genes by binding to MXR response elements (MXREs) comprising of a core 59-CYCCNY-39 motif (Kranthi et al, 2009(Kranthi et al, , 2010. The P. pastoris genome encodes a large number of zinc finger proteins, many of which are still not well characterized (De Schutter et al, 2009).…”

Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris

“…In addition the positive acting transcription factor Mxr1p interacting with the AOX1 promoter and responsible for the activation of many genes in response to methanol was identified by Lin-Cereghino et al (2006). Kranthi et al (2009) identified 6 Mxr1p binding sites within the AOX1 promoter. In addition, a comprehensive study of Hartner et al (2008) described the influence of mutations, deletions and multiplications of putative transcription factor binding sites on the regulatory properties of the AOX1 promoter.…”