Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

Abstract

Background: Pichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions.

Results: In this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris.

Conclusions: This study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.

Keywords: Heterologous protein expression; Lipase; Oxidative stress tolerance; Pichia pastoris; Yeast.

Fig. 1

The characteristics of the WT and the robust mutant. A The cell growth and fluorescence intensity of green fluorescence protein (GFP) in the WT and the robust mutant under glucose condition; B the cell growth and fluorescence intensity of GFP in the WT and the robust mutant under methanol condition; C ROS levels of the WT and the robust mutant during fermentation process under the induction of methanol

Fig. 2

The correlation of inter-group samples by PCA analysis. Three biological replicates were used in each group

Fig. 3

Transcriptomic analysis. A The Venn diagram of differentially expressed genes in three comparison groups. B–D Bubble diagrams of GO enrichment result, each ten GO terms from bottom to top represents biological process, cellular component, and molecular function, respectively. B GO enriched analysis of comparison group Mut_Met vs. WT_Met; C GO enriched analysis of comparison group Mut_Met vs. Mut_Glu; D GO enriched analysis of comparison group WT_Met vs. WT_Glu. The GO terms marked in red include oxidation–reduction process (GO:0055114), oxidoreductase activity (GO:0016491), oxidoreductase activity, acting on CH–OH group of donors (GO:0016614), and oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen (GO:0016705)

Fig. 4

Heat map profiles of key genes. A Genes related to MAPK pathway; B genes related to peroxisome; C genes related to antioxidants and transcription factors responded to oxidative stress; D the ergosterol synthesis pathway

Fig. 5

The predicted regulatory model of P_AOX1 activation. Green arrows indicate activation; red blunt-end arrows indicate repression

Fig. 6

Expression levels of key genes. Comparative of the robust mutant with the wild type under identical methanol conditions. AOX1: alcohol oxidase 1; Form, formaldehyde; DAS1, dihydroxyacetone synthase; DHA: dihydroxyacetone; DHAP: dihydroxyacetone phosphate; E4P: erythrose-4-phosphate; FBA1-2: fructose 1,6-bisphosphate aldolase; FDH1: formate dehydrogenase 1; Form, formaldehyde; F1,6BP: fructose 1,6-bisphosphate; F6P: fructose-6-phosphate; GAP: glyceraldehyde-3-phosphate; R5P: ribose-5-phosphate; Rul5P: ribulose-5-phosphate; S1,7BP: sedoheptulose-1,7-bisphosphate; S7P: sedoheptulose-7-phosphate; X5P: xylulose-5-phosphate; GS-CH₂OH: S-(hydroxymethyl)glutathione; GS-CHO: S-formylglutathione; HCOOH: formic acid; G6P: glucose-6-phosphate; FDP: fructose-1, 6-diphosphate; PEP: phosphoenolpyruvate; OAA: oxaloacetic acid; TCA: tricarboxylic acid cycle. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Overexpression of antioxidant genes to improve the heterologous protein expression ability of P. pastoris. A The antioxidant defence system of yeasts. O₂^·−: superoxide anion; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GLR1: glutathione reductase; Prx: peroxiredoxin/thioredoxin peroxidase; Trx: thioredoxin; TRR1: thioredoxin reductase; G6PD: glucose-6-phosphate dehydrogenase; 6PD: 6-phosphogluconate dehydrogenase; GSH: glutathione; γ-GC: gamma glutamylcysteine; l-glu: l-glutamic acid; l-cys: l-cysteine. B Relative transcription levels of antioxidant system-related genes. The expression ratio of a gene was analysed using the 2^−ΔΔCt method; C the intracellular ROS level of overexpressed strains and their blank control during methanol-induced fermentation; D foldchange of lipase activity levels in overexpressed strains. *p < 0.05, **p < 0.01