Comparative Transcriptome and Metabolome Analyses Reveal the Methanol Dissimilation Pathway of Pichia Pastoris

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), which is further metabolized either in the assimilation or dissimilation pathway. In the dissimilation pathway, formaldehyde is oxidized to CO 2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH). In addition, formaldehyde induces DNA-protein crosslinks (DPCs). Formaldehyde dehydrogenase is critical to minimize formaldehyde-mediated DNA lesions. Although phenotypes have been studied in engineered strains by modified dissimilation, there is a clear lack of systematic studies at the whole-omics level, especially transcriptomics and metabolomics. Results: Focusing on the dissimilation pathway being cut off, we compared the transcriptomes and metabolomes from a formaldehyde dehydrogenase-deficient strain ( Δfld ), an S-hydroxymethyl glutathione dehydrogenase-deficient strain ( Δfgh ), a formate dehydrogenase deficient-strain ( Δfdh ) and the wild type (GS115). First, the differences between strains were most apparent after FLD knockout. When methanol was used as the sole carbon source, the differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Second, analysis of differentially expressed genes (DEGs) between knockout and wild type strains under methanolic incubation showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while proteasomes, autophagy and peroxisomes were upregulated. Transcription of alcohol metabolism was upregulated. It is worth noting that the degree of variation 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 was carried out in Pichia pastoris on the basis of transcriptomics and metabolomics. Truncation of the dissimilation pathway affected methanol metabolism, and knockdown of FLD impaired formaldehyde assimilation. The significant downregulation of oxidative phosphorylation may reveal that FLD and FGH are key enzymes in the energy utilization of cellular methanol metabolism. In addition, formaldehyde can not only bind glutathione but also react with amino acids, especially cysteine. The upregulation of the proteasome and autophagy may solve the problem of DNA-protein crosslinking caused by formaldehyde.