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. 2022 May 12;23(1):366.
doi: 10.1186/s12864-022-08592-8.

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

Yi-Fan Yu  1 , Jiashuo Yang  1 , Fengguang Zhao  1 , Ying Lin  1 , Shuangyan Han  2
Affiliations

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

Yi-Fan Yu et al. BMC Genomics. .

Abstract

Background: Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).

Results: The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.

Conclusions: This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA-protein crosslinking.

Keywords: Dissimilation pathway; Formaldehyde; Glutathione; Methanol metabolism; Oxidative phosphorylation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Phenotypic differences between knockout strains and wild strains. A The biomass difference between knockout strain and wild strain under the conditions of YPD or 1% YPM for 12 h. **p < 0.01 B the phenotypic difference between knockout strain and wild strain under 4% YPM plate culture condition for 7 days
Fig. 2
Fig. 2
Comparative transcriptome. A. Number of DEGs between dissimilation pathway knockout and wild-type strains under methanol culture conditions (three parallel experiments). B. Venn diagrams of DEGs between dissimilation pathway knockout and wild-type strains under methanol culture conditions
Fig. 3
Fig. 3
Analysis of differential metabolites between Δfld and GS115 in positive (A) and negative (B) ion mode
Fig. 4
Fig. 4
Oxidative phosphorylation pathway under methanol culture conditions. A Dialogue with oxidative phosphorylation in Pichia pastoris [28]. B DEGs of oxidative phosphorylation between dissimilation pathway knockout and wild-type strains
Fig. 5
Fig. 5
Fold change between the RNA-seq and qRT-PCR of DEGs
Fig. 6
Fig. 6
Methanol metabolism pathway and comparison of transcription levels in Pichia pastoris. The arrow indicates the comparative transcription level of the dissimilation pathway knockout and wild-type strains (q ≤ 0.05)
Fig. 7
Fig. 7
Comparative transcriptome analysis of glutathione and amino acid metabolism under methanol culture conditions [28]. The arrow indicates the comparative transcription level of the dissimilation pathway knockout and wild-type strains (q ≤ 0.05)
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