Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3

Abstract

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy

“…Likewise, Kar2 (BiP), which can activate Ire1 (Schroder et al, 2003), was not upregulated in this study. In accordance with the results of a study performed by Li et al (2019), protein disulfide isomerase (PDI, PAS_chr4_0844), which catalyzes appropriate disulfide bond formation during protein folding, is only significantly upregulated (Mares and Ramos, 2018) prior to and at the end of methanol induction (p-Value < 0.05 and log2 fold changes > 0.75) ( Figure 6 and Supplementary Table FIGURE 6 | Changes in proteins involved in the UPR and ERAD pathways. S3), while another PDI (PAS_chr1−3_0125) is significantly down-regulated during GM and the methanol induction stage (p-Value < 0.05 and log2 fold changes < −0.60) ( Figure 6 and Supplementary Table S3).…”

“…From the GM phase, almost all genes controlling the development and function of peroxisomes are gradually upregulated compared with the glycerol growth stage (detection sample is G_3) (Figure 4 and Supplementary Table S1). However, it should be stated that the latter results differ from those published by Li et al (2019). Other peroxidated molecules are also generated during the oxidation of methanolreactive oxygen species (e.g., hydrogen peroxide); these latter molecules require removal to prevent or minimize cellular damage (Vanz et al, 2012).…”

“…Starting from the adaptation period following the addition of the GM phase, genes involved in methanol metabolism, carbon metabolism, and glycolysis/gluconeogenesis were both significantly upregulated and significantly downregulated (Jordà et al, 2013). These results indicate that pathways related to carbon metabolism are especially significant during heterologous expression activities (Rußmayer et al, 2015;Li et al, 2019).…”

“…As shown in the heat map reporting the gene number changes for each KEGG metabolic pathway, no genes were significantly upregulated during the glycerol growth stage compared with yeast cell culture stage, the genes that were significantly upregulated from the GM phase were mainly involved in peroxisome, starch and sucrose metabolism, and fatty acid degradation (p-Value < 0.05 and log2 fold changes > 0.50). However, in contrast to a fermentation study performed without a GM stage by Li et al (2019), in this current study, genes that were enriched in the MAPK signaling pathway were significantly upregulated in the methanol induction stage (p-Value < 0.05 and log2 fold changes > 0.50), and genes involved in autophagy in yeast, mitophagy in yeast, autophagy-other, and fructose and mannose metabolism were significantly upregulated almost throughout the entirety of the methanol induction phase (p-Value < 0.05 and log2 fold changes > 0.75). Among these processes, the upregulation of genes in the MAPK signaling pathway was the most significant (p-Value < 10 −5 and log2 fold changes > 0.75) ( Figure 2).…”

“…The H 2 O 2 produced by the latter metabolism affected O 2 formation following the activity of the ROS pathway (Van Der Klei et al, 2006). Methanol is oxidized to formaldehyde that goes onto form CO 2 through dissimilation (Li et al, 2019); however, some of the latter formaldehyde enters the pentose phosphate pathway, and one molecule of methanol finally produces one-third of a molecule of glyceraldehyde 3phosphate (GAP) for biomass synthesis (Figure 3).…”