Abstract
To explore the differences in the intracellular transcriptional mechanism in carbon-derepressed and wild-type Pichia pastoris strains fed with three different carbon sources. RNA in carbon-derepressed (Δmig1Δmig2Δnrg1-Mit1; Mut) and wild-type (WT) P. pastoris fed with three different carbon sources (dextrose, glycerol, and methanol) were sequenced. Differentially expressed genes (DEGs) associated with these carbon sources were obtained and clustered into modules using weighted gene co-expression network analysis (WGCNA). Signaling pathway enrichment analysis was performed using KEGG, and protein to protein interaction (PPI) network was also constructed. A total of 2536 DEGs were obtained from three intersections, and some of them were enriched in carbon sources and involved in carbon metabolism, secondary metabolisms, and amino acid biosynthesis. Two modules, MEgreenyellow (involved in protease, oxidative phosphorylation, endoplasmic reticulum protein processing, folate carbon pool, and glycerol phospholipid metabolism pathways) and MEmidnightblue (involved in protease, endocytosis, steroid biosynthesis, and hippo signaling pathways) were significantly correlated with the strain type. Eight hub genes and two sub-networks were obtained from PPI network. Sub-network A enriched in proteasomes pathway while sub-network B enriched in ribosome pathway. The genes involved in carbon metabolism, secondary metabolic, and amino acid biosynthesis pathways changed significantly under different carbon sources. The changes in proteasome and ribosome activities play roles in carbohydrate metabolism in the methanol-free PAOX1 start-up Mut strain.
Highlights
Pichia pastoris (P. pastoris) is a widely used recombinant expression system for exogenous genes having prokaryotic advantages of having a simple operation and rapid growth, and has eukaryotic cell functions of posttranslational modifications of proteins
By combining the union Differentially expressed genes (DEGs) of the Mut strain and WT strain grown under different carbon sources, 2536 DEGs were detected as genes related to carbon sources
The results of bidirectional hierarchical clustering showed that DEG analysis only separated methanol samples from the three carbon sources, while glucose and glycerol samples were aggregated in the other branches
Summary
Pichia pastoris (P. pastoris) is a widely used recombinant expression system for exogenous genes having prokaryotic advantages of having a simple operation and rapid growth, and has eukaryotic cell functions of posttranslational modifications of proteins. Yeast expression systems include Saccharomyces cerevisiae system, The most commonly used promoter on P. pastoris expression system is the alcohol oxidase 1 promoter (AOX1), which is a carbon source-dependent promoter whose transcription expression is strongly induced by methanol, but inhibited by other carbon sources, such as glucose, glycerol, or ethanol (Liang et al 2014). P. pastoris synthesizes a large number of enzymes when using methanol as the sole carbon source. Methanol fermentation requires large amounts of oxygen, around three to four times higher than the amount needed when the carbon source is glucose (Lin et al 2000). It produces hydrogen peroxide (H2O2) during methanol metabolism, causing oxidative stress and proteolytic degradation of some proteins (Wang et al 2010)
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