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

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 CO2 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.

OXPHOS deficiencies affect peroxisome proliferation by downregulating genes controlled by the SNF1 signaling pathway

How environmental cues influence peroxisome proliferation, particularly through other organelles, remains largely unknown. Yeast peroxisomes metabolize all fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, the products of these pathways enter mitochondria for ATP production, and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH shuttling and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits the expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by high AMP and low ATP. Consistent with this mechanism, in OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2 and NRG1) controlled by SNF1 signaling. We uncovered here the mechanism by which peroxisomal and mitochondrial metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol and the nucleus. We discuss the physiological relevance of this work in view of human OXPHOS deficiencies.

Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis

The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

GENOMIC DIVERSITY, CHROMOSOMAL REARRANGEMENTS, AND INTERSPECIES HYBRIDIZATION IN THE OGATAEA POLYMORPHA SPECIES COMPLEX

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the Ogataea polymorpha species complex, including representatives of the species O. polymorpha, O. parapolymorpha, O. haglerorum, and O. angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Methanol sensor Wsc1 and MAP kinase suppress degradation of methanol-induced peroxisomes in methylotrophic yeast

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol shows circadian dynamics on plant leaves to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations, and transmits the information to induce genes for methanol metabolism together with huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK negatively regulate pexophagy in the presence of >0.15% methanol. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, KpRlm1 and a phosphatase were found suppress pexophagy by controlling phosphorylation level of KpAtg30, the key factor of pexophagy. We reveal at the molecular level how the single methanol sensor KpWsc1 commits the cell to peroxisome synthesis and degradation according to the methanol concentration, and discuss the physiological significance of regulating pexophagy for survival in the phyllosphere.

Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2

ABSTRACT Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

Physiological, genomic, and sulfur isotopic characterization of methanol metabolism by Desulfovibrio carbinolicus

Methanol is often considered as a non-competitive substrate for methanogenic archaea, but an increasing number of sulfate-reducing microorganisms (SRMs) have been reported to be capable of respiring with methanol as an electron donor. A better understanding of the fate of methanol in natural or artificial anaerobic systems thus requires knowledge of the methanol dissimilation by SRMs. In this study, we describe the growth kinetics and sulfur isotope effects of Desulfovibrio carbinolicus, a methanol-oxidizing sulfate-reducing deltaproteobacterium, together with its genome sequence and annotation. D. carbinolicus can grow with a series of alcohols from methanol to butanol. Compared to longer-chain alcohols, however, specific growth and respiration rates decrease by several fold with methanol as an electron donor. Larger sulfur isotope fractionation accompanies slowed growth kinetics, indicating low chemical potential at terminal reductive steps of respiration. In a medium containing both ethanol and methanol, D. carbinolicus does not consume methanol even after the cessation of growth on ethanol. Among the two known methanol dissimilatory systems, the genome of D. carbinolicus contains the genes coding for alcohol dehydrogenase but lacks enzymes analogous to methanol methyltransferase. We analyzed the genomes of 52 additional species of sulfate-reducing bacteria that have been tested for methanol oxidation. There is no apparent relationship between phylogeny and methanol metabolizing capacity, but most gram-negative methanol oxidizers grow poorly, and none carry homologs for methyltransferase (mtaB). Although the amount of available data is limited, it is notable that more than half of the known gram-positive methanol oxidizers have both enzymatic systems, showing enhanced growth relative to the SRMs containing only alcohol dehydrogenase genes. Thus, physiological, genomic, and sulfur isotopic results suggest that D. carbinolicus and close relatives have the ability to metabolize methanol but likely play a limited role in methanol degradation in most natural environments.