Phenotypic and quantitative genetic analysis
We knocked out enzyme genes of the dissimilation pathway (FLD (PAS_chr3_1028), FGH (PAS_chr3_0867) and FDH (PAS_chr3_0932)) separately in GS115 by CRISPR/Cas9 and found no homologous sequences in the genome through BLAST (Fig. 1) . Glucose was used as the main carbon source, and there was no obvious growth difference among strains. Methanol was used as the main carbon source, and the growth of dissimilation pathway knockout strains was worse than that of wild-type strain (GS115) (Fig. 2. A). We conducted pairwise transcriptome comparisons between wild-type and dissimilation pathway knockout strains after culture for 12 hours with glucose and 1% methanol as the main carbon sources. Sample clusters with high similarity between the strains under glucose culture conditions were identified. The samples of strains under methanol culture conditions were significantly different. The transcriptional profiles of ∆fld, ∆fgh, ∆fdh and GS115 were developed and designated KO_FLD, KO_FGH, KO_FDH and GS115 respectively. We found that outlier samples (KO_FLD) showed the lowest correlation coefficient, which means a less similar gene expression level. GS115 and KO_FDH have the smallest sample variation.
DEGs between wild-type and dissimilation pathway knockout groups
Transcriptome comparisons were designed to reflect the effects of metabolic pathways induced by gene knockout or methanol perturbations between dissimilation pathway knockout and wild-type strains in three groups: GS115 versus KO_FLD (GL), GS115 versus KO_FGH (GG) and GS115 versus KO_FDH (GD). The results of the significantly DEGs detected, based on the gene expression levels of individual samples, are as follows (Fig. 2. B). The order of the knockout affected the level of transcription. Screening for highly expressed genes (log2FC≥2, q≤0.05) in GL revealed the upregulation of some dehydrogenase transcripts after FLD knockout, such as pyridoxine 4-dehydrogenase (PAS_chr4_0550), alcohol dehydrogenase (NADP+) (PAS_chr3_0006), NADPH2 dehydrogenase (PAS_chr3_1184), D-arabinose 1-dehydrogenase (PAS_chr2-1_0775), which may imply that other dehydrogenases compensate for the function of formaldehyde dehydrogenase, in GG, ubiquitin C (PAS_chr4_0762), AN1-type domain-containing protein (PAS_chr4_0567), 20S proteasome subunit alpha 2 (PAS_chr1-1_0433), which is in ubiquitin–proteasome pathway, was significantly upregulated, in GD the difference was not significant.
To understand the common impact of knockouts in the dissimilation pathway, we used Venn diagrams to show genes in different comparison groups. A total of 137 DEGs among the three groups were enriched in KEGG metabolic pathways (log2FC≥0.5, q≤0.05) (Fig. 2. C): glycolysis, TCA cycle, pentose and glucuronate interconversions, pyruvate metabolism biosynthesis of antibiotics, metabolism of various amino acids (tyrosine, phenylalanine, tyrosine and tryptophan, glycine, serine and threonine). A total of 137 DEGs among GL and GG were enriched in KEGG metabolic pathways (log2FC≥1, q≤0.05) (Fig. 2. C): most genes in the peroxisome pathway were transcriptionally upregulated, while oxidative phosphorylation, glycolysis and TCA cycle harbored a large proportion of genes that were significantly downregulated. Furthermore, we were interested in genes involved in cysteine metabolism (p≤0.05) and glutathione metabolism due to formaldehyde binding.
Comparison transcriptome of ∆fld and Wild-type strain
Formaldehyde dehydrogenase is the first enzyme in the dissimilation pathway, and its knockout can severely compromise strain robustness. We used BMM medium with methanol as the sole carbon source for 24 h to evaluate the metabolomic differences between ∆fld and wild-type strain.
All identified metabolites were clustered according to chemical classification, and the proportions of each type of metabolite are shown (Fig. 2. D): organic acids and derivatives (32.561%), (lipids and lipid-like molecules (19.562%), and organoheterocyclic compounds (12.227%). Based on univariate analysis, the differences among all metabolites (including unidentified metabolites) detected in positive and negative ion modes were analysed. There were significantly increased nucleosides, nucleotides, analogues, and organic oxygen compounds in positive ion mode and organic heterocyclic compounds, organic nitrogen compounds and benzenoids in negative ion mode. DL-threonine, meperidine, L-homoserine, and several organic acids and derivatives were reduced. A comparative analysis of differences between GL showed 138 differential metabolic pathways, with the top three significantly enriched differential metabolic pathways being ABC transporters, amino acid biosynthesis, and protein digestion and absorption (Additional Figure. 1).
Methanol metabolism pathway
The main carbon metabolism pathways in P. pastoris include methanol metabolism, glycolysis, the TCA cycle, the pentose phosphate pathway and ethanol metabolism. By comparing transcriptomes, we attempted to explain the variation in the methanol metabolism pathway after knockout of the heterotrimeric pathway genes (Fig. 1).
Previous studies have shown that under methanol culture conditions, genes encoding methanol metabolism are upregulated, and glycolysis and TCA cycle transcription are downregulated . Significant transcriptional downregulation of genes involved in glycolysis and the TCA cycle was observed in GL and GG. Interestingly, malate dehydrogenase (MDH2, PAS_chr4_0815) was transcriptionally upregulated in GL, GG (log2FC≥2.7, q≤7.93E-06) and GD. Genes involved in alcohol metabolism were significantly transcriptionally upregulated (q≤0.05). Among them, acetyl-coenzyme A synthetase transcription (ACAS1, PAS_chr3_0403) and alcohol dehydrogenase (ADH, PAS_chr1-3_0153) transcription were upregulated (log2FC≥1, q≤0.05). Aldehyde dehydrogenase (NAD+) (ALDH, PAS_chr3_0987) (log2FC≥3.7, q≤6.61E-07) was significantly upregulated in GG. However, ACAS2 (PAS_chr2-1_0767) was downregulated in GL and GG (log2FC≤-1.4, q≤6.61E-07). This may mean that the two ACASs are responsible for different metabolic pathways in Pichia pastoris and that ACAS2 is more affected by methanol induction.
With the exception of FLD, knockdown of FGH and FDH genes had little effect on the assimilation and dissimilation pathways of methanol. In contrast, the dihydroxyacetone synthase (DAS, PAS_chr3_0832 and PAS_chr3_0834, log2FC≤-0.8) and fructose-bisphosphate aldolase (FBA, PAS_chr1-1_0072, log2FC≤-1.7) of the assimilation pathway were significantly downregulated when FLD was knocked out, which may be one of the reasons for the low biomass of KO_FLD. This again validates the prominence of FLD in the dissimilation pathway. The FLD1 promoter is strongly and independently induced by either methanol as the sole carbon source (with ammonium sulfate as the nitrogen source) or methylamine as the sole nitrogen source (with glucose as the carbon source). FLD coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth . Furthermore, in peroxisomes, alcohol oxidase 2 (AOX2, PAS_chr4_0152) was upregulated in GL (log2FC≥1.3, q≤4.96E-12) and GD (log2FC≥0.9, q≤1E-04). AOX2 is strongly induced by methanol, which may lead to an increase in formaldehyde content in the peroxisome.
When FGH was knocked out, catalase (CAT, PAS_chr2-2_0131, log2FC≥1) was upregulated, which means increased oxidative stress. Glutathione peroxidase (GPX, PAS_chr2-1_0033，log2FC≥0.6) was upregulated, and glutathione reductase (GSR, PAS_chr3_1011, log2FC≤-0.8) was downregulated. The distribution of FGH between peroxisomes and the cytosol was demonstrated . Knockdown of FGH may affect the binding of formaldehyde to glutathione.
The majority of methanol is metabolized via an energy-generating dissimilation pathway, leading to a corresponding increase in mitochondrial size and number . The 60 DEGs from the three groups in the oxidative phosphorylation pathways were clustered, and the GL and GG genes were significantly downregulated (Fig. 3). The oxidative phosphorylation of the wild type was equivalent to that of KO_FDH. The physiological role of FDH was revealed to be mainly detoxification of formate rather than stimulated energy generation . In GD, only NADH dehydrogenase (NDH, PAS_chr3_0792), a mitochondrial external NADH dehydrogenase or type II NAD(P)H: quinone oxidoreductase, was upregulated (log2FC≥1.2, q≤0.01). It may make up for the lack of FDH.
Our transcripts validate the importance of FLD for energy supply. FLD knockout reduces NADP supply. Therefore, FLD activity was identified as the main bottleneck of effective recovery of NADH through methanol dissimilation. In the engineered strain modification, butanediol productivity was improved by increasing FLD activity . The same was true for the knockout of FGH. However, the knockout of FGH affected the recovery of GSH. In GG, cytochrome c oxidase subunit 7c (COX7C, PAS_chr2-2_0266) (log2FC≥2.9, q≤0.01) and haem o synthase (COX10, PAS_chr1-3_0194) (log2FC≥2.0, q≤0.01) were significantly upregulated. However, downregulation of other genes affects ATP production.
Adsorption of formaldehyde by glutathione and amino acids
The methanol metabolism-related generation of reactive oxygen species (ROS) induced a pronounced oxidative stress response. CAT upregulation after FGH knockdown increased ROS. Methanol oxidation stress results in the accumulation of formaldehyde and hydrogen peroxide [32, 33]. Superoxide dismutase [Cu-Zn] (PAS_chr4_0786), which destroys radicals normally produced within cells that are toxic to biological systems, was upregulated significantly in GL (log2FC≥1.0, q≤0.01) and GG (log2FC≥2.5, q≤0.01).
Glutathione (GSH, L-γ-glutamyl-L-cysteinylglycine) is the main sulfur compound and appears as the major nonprotein thiol compound in yeasts. An important reaction by formaldehyde is the formation of compounds with the tripeptide glutathione, between 50% and 80% of endogenous formaldehyde occurs in the form of compounds that include glutathione [16,34]. GSH scavenges cytotoxic H2O2 and maintains a redox balance in the cellular compartments . GSH seems to be involved in the response of yeasts to different nutritional and oxidative stresses . The highest overall glutathione levels correlated with their high viability. In cells, glutathione mainly exists in the reduced form GSH, as oxidized glutathione (GSSG) is converted rapidly by glutathione reductase. GSH/GSSG ratio rose, suggesting stronger protection against oxidative stress, and was also correlated with high glutathione reductase activity [37, 38]. In the process of glutathione reduction and oxidation, glutathione peroxidase (GPX, PAS_chr2-2_0382) was upregulated (log2FC≥0.6, q≤0.05), while glutathione reductase (NADPH) (GSR, PAS_chr3_1011) was downregulated (log2FC≤-0.6, q≤0.05) in GG. In the metabolome, knockdown of fld increased the amount of GSSG compared to that in the original strain. Knockdown of FLD and FGH may accelerate the GSH redox cycle.
The formation of S-[1-(N2-deoxyguanosinyl) methyl] glutathione between glutathione and DNA was induced by formaldehyde. The involvement of the cysteine residue of GSH in coupling suggests that other thiols may participate in the formation of this type of DNA damage from formaldehyde . Formaldehyde also reacts easily with proteins and decreases the number of free amino groups. When mammalian cells are exposed to formaldehyde, the levels of the reaction products of formaldehyde with the amino acids cysteine and histidine, namely, timonacic and spinacine, are increased. These reactions take place spontaneously, and the formation of timonacic is reversible. The reactions of formaldehyde with cysteine and histidine are alternative routes of formaldehyde metabolism . Methanol-grown cells have a higher protein content but lower free amino acid content. In the context of the upregulation of many amino acid biosynthesis genes or proteins, this suggests an increased flux towards amino acid and protein synthesis, which is also reflected in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation . With FLD knockout, genes for ribosomal and amino acid metabolism are downregulated, affecting amino acid synthesis and thus weakening growth.
We clustered the 149 DEGs in the amino acid metabolism. 5-Methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (metE, PAS_chr2-1_0160) had zero expression in KO_FLD and was upregulated in GG (log2FC≥1.6, q≤0.05). Cystathionine gamma-lyase (CTH, PAS_chr1-4_0489) had zero expression in GS115_M. CTH, an enzyme involved in sulfur compound metabolism and cysteine metabolism, showed significantly upregulated transcription (log2FC≥10, q≤2.7E-54). This may verify the possible adsorption of formaldehyde by glutathione and amino acids. Afterwards, we performed a differential clustering analysis using the remaining 147 genes (Additional Figure. 2). The knockout of each dissimilation gene produced a comparable up- and down-regulation trend, which may reveal the effect of knocking out the dissimilation pathway on the amino acid pathway. GO enrichment analysis revealed that in GL and GG, genes involved in the metabolic and biosynthetic processes of organic acids and carboxylic acids were transcriptionally downregulated, whereas in GD, they were transcriptionally upregulated (q≤0.01). Another difference is that primary amine oxidase (AOC3, PAS_chr1-4_0441, PAS_chr2-1_0307 and PAS_chr4_0621) and amidase (amiE, PAS_chr3_0283) are upregulated in GL and GD. The knockout of two dehydrogenases in the dissimilation pathway may have affected the metabolism of organic nitrogen compounds.
Proline dehydrogenase (PRODH, PAS_chr1-3_0269) is upregulated in order of knockout by sequence (log2FC≥1.4, 2.8, 3.4, q≤0.01), which facilitates the process of proline catabolism to glutamate. The glutamate-cysteine ligase catalytic subunit (GCLC, PAS_chr1-1_0184) is transcriptionally upregulated in GG and GD (log2FC≥0.5, q≤0.01). Glutathione S-transferase (GST, PAS_chr2-1_0490) was significantly upregulated in GG (log2FC≥1.6, q≤0.01) and downregulated in GL (log2FC≤-1.3, q≤0.05). Cys-Gly metallodipeptidase (DUG1, PAS_chr3_0353) was significantly upregulated in GL (log2FC≥2.1, q≤1.5E-07).
Effect of DNA cross-linking on proteasome and peroxisome
Excessive ROS are formed from peroxisome metabolism when methanol-grown wild-type cells are exposed to excess methanol . Upregulation of the peroxisome in the dissimilation pathway knockout strains pathway may be closely related to the concentration of formaldehyde. The upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes . Accumulating evidence indicates that damaged components of eukaryotic cells are removed by autophagic degradation . The knockdown of genes involved in the dissimilation pathway caused upregulation of peroxisomes and autophagy.
Mutations in the gene encoding the putative human homologue of a yeast DPC protease cause premature ageing and cancer predisposition syndrome in humans . Proteolytic cleavage of the protein moiety of a DPC is a general strategy for removing the lesion and can be accomplished through a DPC-specific protease and/or proteasome-mediated degradation. Nucleotide excision repair and homologous recombination are each involved in repairing DPCs, with their respective roles likely dependent on the nature and size of the adduct . The transcription level of the DNA-dependent metalloprotease WSS1 (PAS_chr3_0200) increased significantly (log2FC≥1.1(GG), log2FC≥1.9(GL), q≤0.01), and the proteasome gene was significantly upregulated after the dissimilation pathway was knocked out in methanol culture. This indicated that protease digestion of DPC was a stress response to formaldehyde. The protein component of DPCs is targeted for repair by proteases of the Wss1/SPRTN family. Formaldehyde exposure triggers widespread ubiquitylation events in cells [47, 48]. The proteasome may eliminate DNA conjugates caused by excessive formaldehyde. The proteasome is a large protein complex responsible for the degradation of intracellular proteins. The polymerization of ubiquitin, a key molecule known to work in concert with the proteasome, serves as a degradation signal for numerous target proteins, the destruction of a protein is initiated by covalent attachment of a chain consisting of several copies of ubiquitin. In eukaryotes, the autophagy–lysosome system and the ubiquitin–proteasome system (UPS) are the two major quality control pathways responsible for maintaining proteome homeostasis and directing recycling to meet nutrient demand. In contrast, autophagy can eliminate larger protein complexes, insoluble protein aggregates, and even entire organelles and pathogens in toto due to the sheer size of the engulfing autophagic vesicles . The ubiquitin–proteasome system controls almost all basic cellular processes—such as progression through the cell cycle, signal transduction, cell death, immune responses, metabolism, protein quality control and development-by degrading short-lived regulatory or structurally aberrant proteins, connecting ubiquitylation and autophagy as key regulatory events in proteasome quality control [49, 50]. In Saccharomyces cerevisiae, several genes involved in DNA repair (eight RAD genes) that have been identified as specific for methanol toxicity were previously reported as determinants of tolerance for formaldehyde, a methanol detoxification pathway intermediate . Knockdown of FLD and FGH allows the accumulation of formaldehyde, leading to upregulation of DNA repair genes. It is also of interest that genes related to DPCs in P. pastoris could be mining.