Phenotypic difference 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 [27]. With glucose as the main carbon source, there was no significant growth difference between dissimilation pathway knockout strains and GS115. However, with methanol as the main carbon source, the biomass of dissimilation pathway knockout strains (∆fld, ∆fgh, ∆fdh) was lower (60.98%, 23.66%, 5.69%) than that of wild-type strain (GS115) (Fig. 1. A). GS115 showed concordance with ∆fdh and significant differences with ∆fld and ∆fgh at 0.01 level (p < 0.01) under YPM culture conditions. Therefore, the growth of GS115 was significantly higher than that of ∆fld and ∆fgh under methanol culture conditions. In addition, the 4% YPM plate used to monitor methanol tolerance between strains showed poor growth of dissimilation pathway knockout strains, particularly ∆fld (Fig. 1. B).

Fig. 1
figure 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

Comparative transcriptomic

Comparative transcriptomic were designed to reflect the effects of induced by gene knockout and methanol perturbations on metabolic pathways. The transcriptional profiles of ∆fld, ∆fgh, ∆fdh and GS115 incubated in methanol for 12 h were developed and designated KO_FLD, KO_FGH, KO_FDH and GS115 respectively. GS115 and KO_FDH have the smallest sample variation. Then we compared the differences between GS115 versus KO_FLD (GL), GS115 versus KO_FGH (GG) and GS115 versus KO_FDH (GD). The upward and downward DEG numbers for GL, GG, and GD were 938 and 1072, 943 and 587, and 281 and 310, respectively (Fig. 2. A). The change in the number of down-regulated genes was positively correlated with the knockout order, which is consistent with the results of the biological phenotype, demonstrating that FLD is a key enzyme gene in the dissimilation pathway of methanol metabolism. Interestingly, single knockouts did not show significant differences in transcript levels of other genes in the dissimilation pathway. The DEGs of GL and GG were analyzed by transcription factor (TF) ranking (Supplemental Table 2). Among the up-regulated genes, the zinc cluster transcriptional activator (CAY71800, CAY69410) was enriched (p < 0.05); while among the down-regulated genes, carbon source-responsive zinc-finger transcription factor (CAY71743) and proposed transcriptional activator, member of the Gal4p family of zinc cluster proteins (CAY71429) was significantly enriched among the down-regulated genes (p < 0.01).

Fig. 2
figure 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

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. B): glycolysis, TCA cycle, pentose and glucuronate interconversions, pyruvate metabolism biosynthesis of antibiotics, metabolism of various amino acids (tyrosine, phenylalanine, tryptophan, glycine, serine and threonine). A total of 357 DEGs among GL and GG were enriched in KEGG metabolic pathways (log2FC ≥ 1, q ≤ 0.05) (Fig. 2. B): most genes in the peroxisome pathway were transcriptionally upregulated, whereas a large proportion of genes in oxidative phosphorylation, glycolysis and TCA cycle were significantly downregulated. Furthermore, we were interested in genes involved in cysteine metabolism (p ≤ 0.05) and glutathione metabolism, related to formaldehyde binding.

Comparative transcriptomics and metabolomics 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 classified: organic acids and derivatives (32.561%), lipids and lipid-like molecules (19.562%), and organoheterocyclic compounds (12.227%). The first three significantly abundant differential metabolic pathways were ABC transporters, amino acid biosynthesis, and protein digestion and absorption (Additional Fig. 1). Relatively in DEGs of GL, organonitrogen compound biosynthetic process, ribosome, cellular biosynthetic process, ATP metabolic process, peptide and oxoacid metabolic process were enriched for GO term; ribosome, Biosynthesis of antibiotics, glycolysis, biosynthesis of amino acids, oxidative phosphorylation, pentose and glucuronate interconversions were enriched for KEGG pathway.

Based on univariate analysis, the differences among all metabolites (including unidentified metabolites) detected in positive and negative ion modes were analysed. In the positive ion mode (Fig. 3. A), oxidized glutathione (GSSG) (log2FC ≥ 1.17) and reduced glutathione (GSH) (log2FC ≥ 4.0) were upregulated; metabolites associated with glutathione availability were generally upregulated, such as N-methyl-L-glutamate (log2FC ≥ 4.6), homoserine (log2FC ≥ 6.0), His-Glu ( log2FC ≥ 2.8), and L-methionine (log2FC ≥ 1.7). Although the Δfld strain was unable to metabolise formylglutathione, the glutathione redox cycle was accelerated and may have reduced intracellular formaldehyde levels. Among the differential metabolites, other different species of amino acids were up-regulated, such as Ser-Tyr-Lys (log2FC ≥ 5.12), His-Asp (log2FC ≥ 2.0), Ile-Lys (log2FC ≥ 2.3), Val-Lys (log2FC ≥ 2.2), Asp-Leu (log2FC ≥ 4.0), Pro pro (log2FC ≥ 2.7), etc. And Ile-Pro was downregulated (log2FC ≤ -8.6). Among the differential metabolites, other different species of amino acids were up-regulated, such as Ser-Tyr-Lys (log2FC ≥ 5.12), His-Asp (log2FC ≥ 2.0), Ile-Lys (log2FC ≥ 2.3), Val-Lys (log2FC ≥ 2.2), Asp-Leu (log2FC ≥ 4.0), Pro-pro (log2FC ≥ 2.7), etc. In addition, acetyl coenzyme A was down-regulated (log2FC ≤ -1.4) and its branch metabolite N-acetyl histidine was significantly down-regulated (log2FC ≤ -5.1). However, diacetylchitosan was upregulated (log2FC ≥ 2.4).

Fig. 3
figure 3

Analysis of differential metabolites between Δfld and GS115 in positive (A) and negative (B) ion mode

In the negative ion mode (Fig. 3. B), the methanol assimilation pathway metabolites, D-glucose-6-phosphate (log2FC ≤ -1.3) and glyceraldehyde (log2FC ≤ -1.2) were down-regulated. Meanwhile, D-fructose (log2FC ≤ -1.5), D-gluconate (log2FC ≤ -1.7), glucitol (log2FC ≤ -1.8), and malate (log2FC ≤ -0.5) were down-regulated. D-ribose (log2FC ≤ -2.7) and thymine (log2FC ≤ -3.4) were down-regulated, indicating that Δfld is restricted during genetic replication, which may severely affect normal cell growth.

Through the interaction of DEGs of amino acid metabolism, aldehyde dehydrogenase (NAD +) (PAS_chr4_0043), glutamate dehydrogenase (NADP +) (PAS_chr1-1_0107), ornithine decarboxylase (PAS_chr3_0417) were screened out as important genes. The assimilation pathway and central carbon metabolites were down-regulated in Δfld, which may affect intracellular organic carbon fixation. The up-regulation of chitinose may be due to the resistance of chitin to oxidative stress. In contrast, most species of amino acids in the differential metabolites are up-regulated when synthesized from scratch. Therefore, knockdown of FLD, although not efficient in carbon utilization, facilitates amino acid metabolism and product synthesis.

Oxidative phosphorylation

The 60 DEGs from the three groups in the oxidative phosphorylation pathways were clustered. The degree of oxidative phosphorylation of GD was similar, while GL and GG were significantly down-regulated (Fig. 4).

Fig. 4
figure 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

F-type H + -transporting ATPase subunit beta (ATP1 and ATP2, PAS_chr3_0576 and PAS_chr2-2_0165) were conspicuous downregulated in GL and GG (log2FC ≤ -1.5, q ≤ 0.05). The difference in fold change between RNA-seq and qRT-PCR of Oxidative phosphorylation is demonstrated in Fig. 5. ATP1 and ATP2 are proton-transporting ATP synthase complexes with nucleoside phosphatase activity that bind to purine nucleotides and act in nucleotide biosynthetic processes and nitrogen compound metabolism. In comparative metabolomics, differences in purine metabolism and pyrimidine metabolism of nucleotides may be associated with transcriptional down-regulation of mitochondria-related enzyme subunits. This leaves ATP in limited supply.

Fig. 5
figure 5

Fold change between the RNA-seq and qRT-PCR of DEGs

Cytochrome c oxidase subunit (COX5B and COX6B, PAS_chr2-1_0361 and PAS_chr4_0422) were downregulated in GL (log2FC ≤ -2.5, q ≤ 0.05) (Fig. 5). This severely diminished the mitochondrial-cytoplasmic proton transmembrane transporter protein activity. The transcript levels of the ubiquitin-cytochrome c reductase core subunit (e.g. UQCRC2, PAS_chr2-2_0430), which has catalytic activity for binding cations and metal ions, were down-regulated (log2FC ≤ -1.9, q ≤ 0.01), reducing the catalytic efficiency of the enzyme during oxidative phosphorylation. Transcript levels of NADH dehydrogenase complex subunits (e.g. NDUFB7, PAS_chr1-1_0172) were significantly down-regulated in GL (log2FC ≤ -2.4) and GG (log2FC ≤ -1.8, Q ≤ 0.02). It uses NAD(P)H, quinone or similar compounds as receptors and has oxidoreductase activity. Down-regulated genes in the oxidative phosphorylation pathway, such as the NADH dehydrogenase complex subunit, are associated with metabolic pathways in human Alzheimer’s disease, cardiovascular disease and non-alcoholic fatty liver disease. The dissection of the mechanisms underlying strains defective in the dissimilatory pathway provides a theoretical basis for human pathology.

Screened 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 (not shown in the picture).

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. Others were downregulated. 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 might compensate for the absence of FDH.

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 dissimilation pathway genes (Fig. 6). 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 induced by methanol, which may lead to an increase in formaldehyde content in the peroxisome.

Fig. 6
figure 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)

Knockout of the dissimilation pathway gene down-regulates the assimilation pathway of formaldehyde. 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 ∆fld (Fig. 5). Correspondingly, D-glucose 6-phosphate was downregulated (log2FC ≤ -1.3,VIP ≥ 1.8). This again validates the prominence of FLD in the dissimilation pathway.

When FGH was knocked out, catalase (CAT, PAS_chr2-2_0131, log2FC ≥ 1) was upregulated, which means reactive oxygen species (ROS) induced a pronounced oxidative stress response (Fig. 5). 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).

Previous studies have shown that under methanol culture conditions, genes encoding methanol metabolism are upregulated, and glycolysis and TCA cycle transcription are downregulated [29]. 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, transcription of acetyl-coenzyme A synthetase (ACAS1, PAS_chr3_0403) and alcohol dehydrogenase (ADH, PAS_chr1-3_0153) were upregulated (log2FC ≥ 1, q ≤ 0.05) (Fig. 5). Aldehyde dehydrogenase (NAD +) (ALDH, PAS_chr3_0987) (log2FC ≥ 3.7, q ≤ 6.61E-07) was significantly upregulated in GG (Fig. 5). 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 ACAS2 is more affected by methanol induction.

Glutathione redox cycling and amino acid metabolism

Glutathione (GSH, L-γ-glutamyl-L-cysteinylglycine) is the main sulfur compound and appears as the major nonprotein thiol compound in yeasts. In cells, glutathione mainly exists in the reduced form GSH, as oxidized glutathione (GSSG) is converted rapidly by glutathione reductase. In the process of glutathione reduction and oxidation (Fig. 7), 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 positive ion mode, GSH and GSSG were upregulated (log2FC ≥ 4.0 and log2FC ≥ 1.1, VIP ≥ 1) when FLD was knockout. 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 in GG (log2FC ≥ 10, q ≤ 2.7E-54).

Fig. 7
figure 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)

We clustered the 149 DEGs in the amino acid metabolism. The knockout of each dissimilation gene produced a comparable up and down regulation trend, which may reveal the effect on the amino acid pathway such as arginine, proline, valine, leucine, isoleucine, lysine, glycine, serine and threonine metabolism. 5-Methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (metE, PAS_chr2-1_0160) was upregulated in GG (log2FC ≥ 1.6, q ≤ 0.05). 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). After FLD knockouted, L-homoserine was significantly upregulated (log2FC ≥ 6.0, VIP ≥ 4.9). Some amino acidsm, like Ser-Tyr-Lys, His-Asp, His-Glu, Ile-Lys, Val-Lys were up-regulated (VIP ≥ 1). And 1-methylhistidine was downregulated (log2FC ≤ -5.5, VIP ≥ 1). 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.

Upregulation of proteasomes, peroxisomes and autophagy

The knockout of genes in the dissimilation pathway caused upregulation of peroxisomes and autophagy. 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). The protein component of DPCs is targeted for repair by proteases of the Wss1/SPRTN family. This indicated that protease digestion of DPC was a stress response to formaldehyde. 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; the difference was not significant in GD.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.


This article is autogenerated using RSS feeds and has not been created or edited by OA JF.

Click here for Source link (