Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris

Abstract

Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism. Approximately 23% (1197 genes) were significantly downregulated; these were mainly correlated with the physiological characteristics of the cell. The methanol catabolism and peroxisome biogenesis pathways were remarkably enhanced, and the genes AOX1 and AOX2 were upregulated higher than 30-fold, which was consistent with the experimental results of AOX expression. The Mig proteins had a slight effect on autophagy when cells were grown in glycerol. The expression analysis of transcription factors showed that deletion of MIG1 and MIG2 significantly upregulated the binding of an essential transcription activator, Mit1p, with the AOX1 promoter, which suggested that Mig proteins might regulate the AOX1 promoter through the regulation of Mit1p. This work provides a reference for the further exploration of the methanol induction and catabolite repression mechanisms of AOX expression in methylotrophic yeasts.

Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

“…K. phaffii possesses two homologs of Sc Mig1: Mig1-1 and Mig1-2. These two TFs were previously shown to be involved in the regulation of the methanol metabolism in K. phaffii : derepression of AOX1 was observed in mig1-1Δ and mig1-1Δmig1-2Δ knock-out strains on glycerol (Wang et al 2017 ), and the genes of the methanol metabolism, as well as the peroxisomes biogenesis pathway, were upregulated on glycerol in mig1-1Δmig1-2Δ (Shi et al 2018 ). Finally, Mig1-1 and Mig1-2 localized in the nucleus of cells grown on glucose or glycerol, but when K. phaffii cells were transferred to methanol, Mig1-1 and Mig1-2 predominantly localized to the cytoplasm (Wang et al 2017 ).…”

Convergence between Regulation of Carbon Utilization and Catabolic Repression in Xanthophyllomyces dendrorhous

“…Contrasting results have been found in previous studies in which the inactivation or deletion of Mig1 partially eliminated glucose repression, and it is probable that a second repressor persists as an independent glucose control mechanism (22). Other evidence of a general role for Mig1 has been described in Kluyveromyces marxianus wherein histidine biosynthesis was positively regulated (23), in Pichia pastoris for autophagy and peroxisome biogenesis (24), and in S. cerevisiae for acetate metabolism (25).…”

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology

“…The mechanisms of activity and repression of P AOX1 are not fully dissolved. Several transcription factors regulating P AOX1 have been discovered (Shi et al, ; X. Wang et al, ). Among these, methanol expression regulator 1 ( MXR1 , GenBank: ABD57365.1) is the most important transcriptional activator of P AOX1 , and a deficiency in MXR1 prevents the utilization of methanol by P. pastoris (Lin‐Cereghino, Godfrey, Bernard, et al, ).…”

“…The mechanisms of activity and repression of P AOX1 are not fully dissolved. Several transcription factors regulating P AOX1 have been discovered (Shi et al, 2017;X. Wang et al, 2016).…”