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.

Scalable, methanol‐free manufacturing of the SARS‐CoV‐2 receptor‐binding domain in engineered Komagataella phaffii

“…The improvement in specific productivity, therefore, can be primarily attributed to the elimination of methanol as a carbon source. This observation is consistent with previous transcriptomic studies about methanol metabolism in K. phaffii (Lin et al, 2021 ; Vanz et al, 2012 ). Further studies are warranted to determine the interplay between the transcript level and types and quantities of carbon source on productivity.…”

“…The improvement in specific productivity, therefore, can be primarily attributed to the elimination of methanol as a carbon source. This observation is consistent with previous transcriptomic studies about methanol metabolism in K. phaffii (Lin et al, 2021;Vanz et al, 2012).…”

Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris

“…It has been reported that the robust mutant of lipase not only upregulated MAPK signaling pathway but also reduced intracellular reactive oxygen species (ROS) levels and enhanced oxidative stress tolerance of P. pastoris ( Lin et al, 2021 ). To further investigate the mechanism of disrupting YPS1 gene to inhibit HSA-pFSHβ degradation, we examined the ROS levels within F and Δ yps1 strains after 72 h of induction.…”

“…On the one hand, Slt2/Mpk1 pathway was mutually regulated with the YPS1 gene, with Slt2 regulating YPS1 gene expression and YPS1 gene disruption capable of elevating phosphorylation of Slt2 ( Krysan et al, 2005 ; Gagnon-Arsenault et al, 2006 ; Miyazaki et al, 2011 ; Bairwa et al, 2014 ). Moreover, MAPK signaling pathway, ergosterol synthesis pathway, and the peroxisome pathway were upregulated in robust mutant of produced lipase strain, compared with wild-type strain, indicating that MAPK signaling pathway were involved in recombinant protein production ( Lin et al, 2021 ). Our results were consistent with previous studies that YPS1 gene disruption enhances Slt2 phosphorylation, thereby reducing HSA-pFSHβ degradation.…”

Overview of yeast environmental stress response pathways and the development of tolerant yeasts

“…•− ) are generated as coproducts of the mitochondrial electron transport chain, they are changed into hydrogen peroxide (H 2 O 2 ) by SODs [11]. Overexpression of SOD1 in P. pastoris reduced the ROS level caused by methanol exposure [107]. H 2 O 2 is then transformed into water and oxygen by catalase, glutathione peroxidase, and thioredoxin peroxidase, to prevent the formation of oxidant hydroxyl radicals (•OH) [54].…”