Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris
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.
KeywordsPichia pastoris RNA-seq Mig Alcohol oxidase Catabolite repression
This work was supported by Shanghai Science and Technology Innovation Plan (17JC1402400); Fundamental Research Funds for the Central Universities (22A201514040) and Grants of Young and Middle-aged Leading Science and Technology Innovation Talents from Ministry of Science and Technology of China. The authors are grateful to Prof. James M. Cregg, BioGrammatics, Inc. and Keck Graduate Institute of Applied Life Science, for helpful suggestions. The authors also thank Shanghai Huaguan Biochip Co. Ltd. for the help of RNA-seq data analysis.
Compliance with ethical standards
Conflict of interest
Lei Shi declares that she has no conflict of interest. Xiaolong Wang declares that he has no conflict of interest. Jinjia Wang declares that she has no conflict of interest. Ping Zhang declares that she has no conflict of interest. Fei Qi declares that he has no conflict of interest. Menghao Cai declares that he has no conflict of interest. Yuanxing Zhang declares that he has no conflict of interest. Xiangshan Zhou declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B (Methodological) 57:289–300Google Scholar
- Chang T, Schroder LA, Thomson JM, Klocman AS, Tomasini AJ, Strømhaug PE, Dunn WA Jr (2005) Ppatg9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris. Mol Biol Cell 16:4941–4953CrossRefPubMedPubMedCentralGoogle Scholar
- Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol R 62:334–361Google Scholar
- Hazeu W, Bruyn JC, Bos P (1972) Methanol assimilation by yeasts. Arch Microbiol 87:185–188Google Scholar
- Lin-Cereghino GP, Godfrey L, de la Cruz BJ, Johnson S, Khuongsathiene S, Tolstorukov I, Yan M, Lin-Cereghino J, Veenhuis M, Subramani S, Cregg JM (2006) Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris. Mol Cell Biol 26:883–897CrossRefPubMedPubMedCentralGoogle Scholar
- Van der Klei IJ, Yurimoto H, Sakai Y, Veenhuis M (2006) The significance of peroxisomes in methanol metabolism in methylotrophic yeast. BBA-Mol Cell Res 1763:1453–1462Google Scholar