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.

Lanthanide-Dependent Methanol and Formaldehyde Oxidation in Methylobacterium aquaticum Strain 22A

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica

Background Crude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate. Results To prevent methanol conversion to CO2 via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from Yarrowia lipolytica. The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δfld1 strain. Conclusions In silico, the expression of heterologous formaldehyde assimilation pathways enable Y. lipolytica to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable Y. lipolytica to efficiently use methanol as co-substrate with glycerol.

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum

ABSTRACT GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Sclerotinia sclerotiorum Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in S. sclerotiorum. Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of Ssfdh1; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors. IMPORTANCE S. sclerotiorum is a pathogenic fungus with sclerotium and infection cushion development, making S. sclerotiorum one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of Ssfdh1. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.

Formaldehyde Detection by a Combination of Formaldehyde Dehydrogenase and Chitosan on a Sensor Based on an Organic Field-Effect Transistor

Formaldehyde is utilized for the preservation of materials due to its strong bactericidal effects. As formaldehyde is also a harmful substance that causes health hazards, the quantitative monitoring of formaldehyde in natural and living environments is desirable. For the rapid and easy detection of formaldehyde, in this study we applied an organic field-effect transistor (OFET)-based sensor that can function as a potentiometric device for electrochemical measurements. A polyion-complex gel of formaldehyde dehydrogenase (FDH) and chitosan (CT) was constructed on a gold electrode. When the FDH/CT gel-coated electrode was connected to an OFET device it could detect formaldehyde in an aqueous solution, in which the amino groups of chitosan would protonate during the enzymatic reaction. The limit of detection was calculated to be 3.1 µM (93 ppb), demonstrating the applicability of the film-type OFET sensor to environmental monitoring.