Abstract
BackgroundPichia 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.ResultsIn 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.ConclusionsThis 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.
Highlights
IntroductionThe robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions
We found that the mechanism of high heterologous protein expression ability in the robust mutant was mainly owed to its enhanced oxidative stress tolerance
The robust mutant expressed an up to 2.5-fold higher level of heterologous protein than the wild type (WT) [19]. To determine whether this phenomenon could occur in the expression of other proteins, a green fluorescence protein (GFP) was expressed in the robust mutant and its WT
Summary
The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions. With its proven ability to express over 400 proteins, from human endostatin to spider dragline silk protein, Pichia pastoris has become a consistent choice for heterologous protein production [1,2,3]. Many methods have been used to improve the expression of heterologous proteins in P. pastoris, mainly by codon optimization, increasing gene dosage, promoting protein folding and secretion, etc. Increasing the copy number of target genes can improve the production of target heterologous proteins [9, 10]. Based on the above rationales, to prevent the degradation of overexpressed heterologous protein by the proteasome, the disruption of Pep protease increases phytase secretion in P. pastoris [12]. We successfully improved the expression level of lipase in P. pastoris by co-expressing PDI [16]
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