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. 2023 Nov 27;14(1):7754.
doi: 10.1038/s41467-023-43610-7.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii

Bernd M Mitic  1   2 , Christina Troyer  2 , Lisa Lutz  1   3 , Michael Baumschabl  1   3 , Stephan Hann  2   3 , Diethard Mattanovich  4   5
Affiliations

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii

Bernd M Mitic et al. Nat Commun. .

Abstract

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Scheme describing methanol and formate assimilation pathways.
The natural main methanol assimilation pathway in K. phaffii, the xylulose 5-phosphate pathway, and the ribulose 5-phosphate pathway (B. methanolicus) are methanol assimilation pathways that fix formaldehyde. Glyceraldehyde phosphate is produced via pentose phosphate pathway reactions and is then the key metabolite for all biomass production. As methanol is dissimilated, the reductive glycine pathway and the serine cycle are methanol and formate assimilation pathways. Formate fixation occurs in the tetrahydrofolate cycle, and both co-assimilate CO2. The O2-tolerant reductive glycine pathway leads to pyruvate, while the O2-sensitive reductive glycine pathway (D. desulfuricans) and the serine cycle (M. extroquens) culminate in acetyl-CoA as a subsequent metabolite for biomass production. Reactions focus on those involving carbon only. For the other compounds, enzymes and gene names involved, see Supplementary Fig. 1 & 2. Dashed reactions are not encoded in K. phaffii. Abbreviations: THF tetrahydrofolate, R5P ribose 5-phosphate, S7P sedoheptulose 7-phosphate, E4P erythrose 4-phosphate, Xu5P xylulose 5-phosphate, Ru5P ribulose 5-phosphate, FBP fructose bis-phosphate, DHAP dihydroxyacetone phosphate, GAP glyceraldehyde phosphate, BPG bis-phosphoglycerate, 2- & 3-PG 2-& 3-phosphoglycerate, PEP phosphoenolpyruvate, H-Pyr hydroxyl-pyruvate, Acetyl-P acetyl-phosphate, Adh alcohol dehydrogenase, Aox alcohol oxidase, Das dihydroxyacetone synthase, Hps 3-hexulose-6-phosphate synthase, Phi phosphohexose isomerase, Gck glycerate 2-kinase, Pdc phosphoenolpyruvate carboxylase, Mtk malate-CoA ligase, Mcl malyl-CoA lyase.
Fig. 2
Fig. 2. Carbon isotopologue distribution analysis via GC-CI/EI-TOFMS.
a–d K. phaffii strains (see Table 1) labelled with different carbon sources (n = 2 biological replicates for labelled strains, number of replicates (n) of the natC controls is indicated in the bar). “BB” in the metabolite name refers to the amino acid backbone, i.e., C1 and C2 only; “DC” refers to the decarboxylated amino acid, i.e., all carbon atoms excluding C1 (see Supplementary Data 3), the molecular structures of these fragments are shown in Supplementary Fig. 3 and for serine as an example in Fig. 3f. The number x in”M + x” indicates the number of 13C-carbon atoms and thus specifies the respective isotopologue. As shown in a–c for the forward labelling experiments, pathway routes were assessed by tracing the relative 13C abundance in the metabolites. Generally, an upstream metabolite of any active pathway must contain more 13C than the corresponding downstream metabolite. An increase in 13C-content resulted in a decrease in the isotopologue M + 0 that contains 12C only. For all forward labelling approaches, the isotopologue fraction of M + 0 is also indicated as a number in the corresponding bar, see (a–c). For reverse labelling (2d), 12C incorporation was traced. Therefore, any decrease in abundance of isotopologues containing 13C indicated CO2 incorporation. (a) das1Δdas2Δ strain labelled with 13C-methanol for 72 h without any other carbon source; (b) aox1Δaox2Δ strain labelled with 13C-methanol for 52 h without any other carbon source; (c) wildtype strain labelled with 13C-sodium formate for 72 h without any other carbon source; (d) 13C-labeled das1Δdas2Δ strain reverse labelled with 5% natC-CO2 (12C tracing) and fed with 13C-methanol; (e) reductive glycine pathway and native xylulose 5-phosphate pathway illustrated to the TCA cycle, all measured metabolites are highlighted with bold letters; (f) illustration of labelling and reverse labelling workflow. Labelling data of additional metabolites, see Supplementary Fig. 7; Abbreviations: MeOH methanol, FA formic acid, THF tetrahydrofolate, TCA tricarboxylic acid cycle, R5P ribose 5-phosphate, S7P sedoheptulose 7-phosphate, 2-PG 2-phosphoglycerate, 3-PG 3-phosphoglycerate, PEP phosphoenolpyruvate, AKG α-ketoglutarate, I-Cit isocitrate. Error bars in a–d represent corrected standard deviations of mean values. Icons in (f) were obtained from Freepik (Fuels) at https://www.flaticon.com/. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Carbon isotopologue distribution analysis of additional knockout strains.
As in Fig. 2, K. phaffii strains (see Table 1) are labelled with different carbon sources (n = 2 biological replicates for labelled strains, number of replicates (n) of the natC controls is indicated in the bar). “BB” in the metabolite name refers to the amino acid backbone, i.e., C1 and C2 only, “DC” refers to the decarboxylated amino acid, i.e., all carbon atoms except C1 (molecular structure of serine see (f)). (a) das1Δdas2 Δmis1-1Δmis1-2&3Δ strain labelled with 13C-methanol for 24 h; (b) das1Δdas2Δ mis1-1Δmis1-2&3Δ strain labelled with 13C-sodium formate for 24 h; (c) das1Δdas2Δ shm1Δshm2Δ strain labelled with 13C-methanol for 24 h; (d) das1Δdas2Δ gcv1Δgcv2Δ strain labelled with 13C-methanol for 24 h; (e) das1Δdas2Δ PstrongGCV1&2&3&LPD1 strain labelled with 13C-methanol for 24 h; (f) the reductive glycine pathway with molecular structures, compartments and all overexpressed or deleted genes. Carbon derived from methanol or formate is marked with a purple circle, carbon from CO2 with a green circle; (g) structures of the fragments of serine “BB” and “DC” as described above, for derivatized structures see Supplementary Fig. 3. Labelling data of additional metabolites, see Supplementary Fig. 7 & 12. Error bars in a-d represent corrected standard deviations of mean values. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Results of growth analysis.
Cultures were inoculated at an OD600 of approximately 0.5. To aid comparison, all data was normalised to an initial value of 0.5. All strains assessed have DAS1&2 knockouts, some strains have additional knockouts or overexpressed genes as indicated (genotypes are listed in Table 1). Data are average values from biological duplicates with the standard deviation indicated by the shaded area. Long-term cultivation (up to 46 days), repeatability studies, non-normalised data and further cultivation of overexpressed strains are shown in Supplementary Fig. 13 & 14. (a) DAS1&2 knockout strains cultivated on methanol or formate, with elevated CO2, or the addition of glycine or serine, and the overexpression of the reductive glycine pathway. (b) SHM double knockout strains grown on glycerol with supplementation of methanol, formate and CO2, as indicated, and of glycine as a control. (c) SHM1 knockout strain both with and without overexpression of M. extorquens MIS genes, grown with methanol and CO2 at two different temperatures, and the parental DasKO strain as a negative control. (d) Same strains as in Fig. 4c grown on formate and CO2. Since cultures started to flocculate in cultivation of Shm1KO and Shm1KO MeMisOE on formate additional cell counting was performed to verify growth. Data is shown in Supplementary Fig. 14c for cultures on methanol (as a non-flocculating control) and in Supplementary Fig. 14d for cultures on formate. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Carbon isotopologue distribution analysis of growing Shm1KO strain.
K. phaffii strains das1Δdas2 shm1Δ and das1Δdas2 as control were labelled with 13C-methanol for 24 h with 5% CO2 (with natural isotope distribution) added in the atmosphere. Data display is the same as in Fig. 2 & 3 (n = 3 biological replicates for labelled strains, number of replicates (n) of the natC controls is indicated in the bar). Error bars represent corrected standard deviations of mean values. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. The growth supporting oxygen-tolerant reductive glycine pathway in K. phaffii.
The oxygen-tolerant reductive glycine pathway is the sole, natively-active alternative methanol assimilation pathway in the yeast K. phaffii; and is also the sole, native formate and native CO2 assimilation pathway. It is initiated via methanol or formate assimilation and continues via methylene-THF, glycine and serine towards the formation of pyruvate and even further towards the formation of oxaloacetate. With the deletion of SHM1 DAS1&2 double knockout strains can grow via the displayed and compartmentalized pathway without any overexpressions. Abbreviations: THF tetrahydrofolate; TCA tricarboxylic acid cycle; XuMP xylulose 5-phosphate pathway; PPP pentose phosphate pathway.
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