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The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2


The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin–Benson–Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h−1. The specific growth rate was further improved to 0.018 h−1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.

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Fig. 1: Scheme for engineering chemoorganoautotrophy in P. pastoris.
Fig. 2: Engineering scheme for generation of P. pastoris strains with a heterologous CBB cycle.
Fig. 3: Peroxisomal targeting of the CBB pathway leads to increased growth.
Fig. 4: Growth of engineered CBBp + RuBisCO strain depends on the supply of CO2 as a carbon source.
Fig. 5: Labeling of engineered P. pastoris cells with 13C glycerol and 12C carbon dioxide.
Fig. 6: Bioreactor cultivation of CBBp + RuBisCO and CBBpΔRuBisCO strains inoculated at low cell density and grown in the presence of CO2.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Genome sequencing reads of mutant strains are available at NCBI BioProject PRJNA586834. Sequences of codon-optimized genes are shown in the Supplementary Information. The accession numbers and locus tags of all native and heterologous sequences are shown in Supplementary Table 7 and Supplementary Table 8. Materials will be made available for non-profit research under a material-transfer agreement upon reasonable request to the corresponding author.

Code availability

Mass-spectrometry data were collected using Agilent Technologies MassHunter GCMS Acquisition Software B.07.06 SR1 combined with a post-acquisition application (Agilent SureMass). Theoretical masses were extracted from the SureMass-converted data and analyzed using the Agilent Technologies MassHunter Workstation Quantitative Analysis for TOF Build 10.0.707.0 and the Isotope Correction Toolbox v.0.04. Genome sequence analysis including calling of single-nucleotide polymorphisms was carried out using CLC Genomic Workbench 12.0 (QIAGEN). Microscopy pictures were processed using Zen 2.3 lite (blue edition) software (Carl Zeiss Microscopy). Flow cytometry data were analyzed using CytExpert (v. (Beckman Coulter). No custom code was used in this study.


  1. Steiger, M. G., Sauer, M. & Mattanovich, D. Microbial organic acid production as carbon dioxide sink. FEMS Microbiol. Lett. 364, fnx212 (2017).

    Google Scholar 

  2. Bassham, J. A. et al. The path of carbon in photosynthesis. XXI. The cyclic regeneration of carbon dioxide acceptor 1. J. Am. Chem. Soc. 76, 1760–1770 (1954).

    CAS  Google Scholar 

  3. Berg, I. A. Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Appl. Environ. Microbiol. 77, 1925–1936 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Claassens, N. J., Sousa, D. Z., dos Santos, V. A. P. M., de Vos, W. M. & van der Oost, J. Harnessing the power of microbial autotrophy. Nat. Rev. Microbiol. 14, 692–706 (2016).

    CAS  PubMed  Google Scholar 

  5. Erb, T. J. & Zarzycki, J. A short history of RubisCO: the rise and fall of nature’s predominant CO2 fixing enzyme. Curr. Opin. Biotechnol. 49, 100–107 (2018).

    CAS  PubMed  Google Scholar 

  6. Zhuang, Z.-Y. & Li, S.-Y. Rubisco-based engineered Escherichia coli for in situ carbon dioxide recycling. Bioresour. Technol. 150, 79–88 (2013).

    CAS  PubMed  Google Scholar 

  7. Antonovsky, N. et al. Sugar synthesis from CO2 in Escherichia coli. Cell 166, 115–125 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Herz, E. et al. The genetic basis for the adaptation of E. coli to sugar synthesis from CO2. Nat. Commun. 8, 1705 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. Guadalupe-Medina, V. et al. Carbon dioxide fixation by Calvin-cycle enzymes improves ethanol yield in yeast. Biotechnol. Biofuels 6, 125 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Li, Y.-J. et al. Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars. Sci. Rep. 7, 43875 (2017).

    PubMed  PubMed Central  Google Scholar 

  11. Xia, P.-F. et al. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae. ACS Synth. Biol. 6, 276–283 (2017).

    CAS  PubMed  Google Scholar 

  12. Papapetridis, I. et al. Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield. Biotechnol. Biofuels 11, 17 (2018).

    PubMed  PubMed Central  Google Scholar 

  13. Schada von Borzyskowski, L. et al. An engineered Calvin–Benson–Bassham cycle for carbon dioxide fixation in Methylobacterium extorquens AM1. Metab. Eng. 47, 423–433 (2018).

    CAS  PubMed  Google Scholar 

  14. Phaff, H. J., Miller, M. W. & Shifrine, M. The taxonomy of yeasts isolated from Drosophila in the Yosemite region of California. Antonie Van Leeuwenhoek 22, 145–161 (1956).

    CAS  PubMed  Google Scholar 

  15. Kurtzman, C. P. Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. Int. J. Syst. Evol. Microbiol. 55, 973–976 (2005).

    CAS  PubMed  Google Scholar 

  16. Gasser, B. et al. Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol. 8, 191–208 (2013).

    CAS  PubMed  Google Scholar 

  17. Liu, L. et al. How to achieve high-level expression of microbial enzymes. Bioengineered 4, 212–223 (2013).

    PubMed  PubMed Central  Google Scholar 

  18. Peña, D. A., Gasser, B., Zanghellini, J., Steiger, M. G. & Mattanovich, D. Metabolic engineering of Pichia pastoris. Metab. Eng. 50, 2–15 (2018).

    PubMed  Google Scholar 

  19. Agrawal, G., Shang, H. H., Xia, Z.-J. & Subramani, S. Functional regions of the peroxin Pex19 necessary for peroxisome biogenesis. J. Biol. Chem. 292, 11547–11560 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Ma, C., Agrawal, G. & Subramani, S. Peroxisome assembly: matrix and membrane protein biogenesis. J. Cell Biol. 193, 7–16 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rußmayer, H. et al. Systems-level organization of yeast methylotrophic lifestyle. BMC Biol. 13, 80 (2015).

    PubMed  PubMed Central  Google Scholar 

  22. Weninger, A., Hatzl, A.-M., Schmid, C., Vogl, T. & Glieder, A. Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J. Biotechnol. 235, 139–149 (2016).

    CAS  PubMed  Google Scholar 

  23. Prielhofer, R. et al. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. BMC Syst. Biol. 11, 123 (2017).

    PubMed  PubMed Central  Google Scholar 

  24. Gassler, T., Heistinger, L., Mattanovich, D., Gasser, B. & Prielhofer, R. CRISPR/Cas9-mediated homology-directed genome editing in Pichia pastoris. Methods Mol. Biol. 1923, 211–225 (2019).

    CAS  PubMed  Google Scholar 

  25. Lee, P. C. Peroxisome targeting of lycopene pathway enzymes in Pichia pastoris. Methods Mol. Biol. 898, 161–169 (2012).

    CAS  PubMed  Google Scholar 

  26. Raines, C. A., Lloyd, J. C. & Dyer, T. A. New insights into the structure and function of sedoheptulose-1,7-bisphosphatase; an important but neglected Calvin cycle enzyme. J. Exp. Bot. 50, 1–8 (1999).

    CAS  Google Scholar 

  27. Waites, M. J. & Quayle, D. J. R. Dihydroxyacetone synthase: a special transketolase for formaldehyde fixation from the methylotrophic yeast Candida boidinii CBS 5777. J. Gen. Microbiol. 129, 935–944 (1983).

    CAS  Google Scholar 

  28. Visser, W. F., van Roermund, C. W. T., Ijlst, L., Waterham, H. R. & Wanders, R. J. A. Metabolite transport across the peroxisomal membrane. Biochem. J. 401, 365–375 (2007).

    CAS  PubMed  Google Scholar 

  29. de Koning, W., Gleeson, M. A. G., Harder, W. & Dijkhuizen, L. Regulation of methanol metabolism in the yeast Hansenula polymorpha. Arch. Microbiol. 147, 375–382 (1987).

    Google Scholar 

  30. Siu, K.-H. et al. Synthetic scaffolds for pathway enhancement. Curr. Opin. Biotechnol. 36, 98–106 (2015).

    CAS  PubMed  Google Scholar 

  31. Hammer, S. K. & Avalos, J. L. Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832 (2017).

    CAS  PubMed  Google Scholar 

  32. Bar-Even, A., Noor, E., Lewis, N. E. & Milo, R. Design and analysis of synthetic carbon fixation pathways. Proc. Natl Acad. Sci. USA 107, 8889–8894 (2010).

    CAS  PubMed  Google Scholar 

  33. Schwander, T., Schada von Borzyskowski, L., Burgener, S., Cortina, N. S. & Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354, 900–904 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Barenholz, U. et al. Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points. Elife 6, e20667 (2017).

    PubMed  PubMed Central  Google Scholar 

  35. Anderson, R. M. et al. Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J. Biol. Chem. 277, 18881–18890 (2002).

    CAS  PubMed  Google Scholar 

  36. Rouzeau, C. et al. Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase. Res. Microbiol. 169, 335–342 (2018).

    CAS  PubMed  Google Scholar 

  37. Yang, Z. & Zhang, Z. Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: a review. Biotechnol. Adv. 36, 182–195 (2018).

    CAS  PubMed  Google Scholar 

  38. Olah, G. A. Towards oil independence through renewable methanol chemistry. Angew. Chem. Int. Ed. 52, 104–107 (2013).

    CAS  Google Scholar 

  39. Werpy, T. & Petersen, G. Value Added Chemicals from Biomass: Volume I—Results of Screening for Potential Candidates from Sugars and Synthesis Gas (U.S. Department of Energy, 2004).

  40. Mattanovich, D., Jungo, C., Wenger, J., Dabros, M. & Maurer, M. In Industrial Scale Suspension Culture of Living Cells (eds. Meyer, H.-P. & Schmidhalter D.) 94–129 (Wiley-Blackwell, 2014).

  41. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).

    PubMed  PubMed Central  Google Scholar 

  42. Sarkari, P., Marx, H., Blumhoff, M. L., Mattanovich, D. & Steiger, M. G. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour. Technol. 245, 1327–1333 (2017).

    CAS  PubMed  Google Scholar 

  43. Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

    CAS  PubMed  Google Scholar 

  44. Küberl, A. et al. High-quality genome sequence of Pichia pastoris CBS7435. J. Biotechnol. 154, 312–320 (2011).

    PubMed  Google Scholar 

  45. Valli, M. et al. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function. FEMS Yeast Res. 16, fow051 (2016).

    PubMed  Google Scholar 

  46. Cregg, J. M. In Pichia Protocols (ed. Cregg, J. M.) 27–40 (Humana Press, 1998).

  47. Blumhoff, M. L., Steiger, M. G., Mattanovich, D. & Sauer, M. Targeting enzymes to the right compartment: metabolic engineering for itaconic acid production by Aspergillus niger. Metab. Eng. 19, 26–32 (2013).

    CAS  PubMed  Google Scholar 

  48. Rußmayer, H. et al. Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching. FEMS Yeast Res. 15, fov049 (2015).

    PubMed  Google Scholar 

  49. Mairinger, T. et al. Gas chromatography-quadrupole time-of-flight mass spectrometry-based determination of isotopologue and tandem mass isotopomer fractions of primary metabolites for 13C-metabolic flux analysis. Anal. Chem. 87, 11792–11802 (2015).

    CAS  PubMed  Google Scholar 

  50. Zamboni, N., Fendt, S.-M., Rühl, M. & Sauer, U. 13C-based metabolic flux analysis. Nat. Protoc. 4, 878–892 (2009).

    CAS  PubMed  Google Scholar 

  51. Jungreuthmayer, C., Neubauer, S., Mairinger, T., Zanghellini, J. & Hann, S. ICT: isotope correction toolbox. Bioinformatics 32, btv514 (2015).

    Google Scholar 

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This work has been supported by the Federal Ministry for Digital and Economic Affairs, the Federal Ministry for Transport, Innovation and Technology, the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and ZIT - Technology Agency of the City of Vienna through the COMET Funding Program managed by the Austrian Research Promotion Agency FFG (grant to D.M. and M.G.S.). We also thank the Austrian Science Fund for support to D.M. and T.G. (FWF W1224, Doctoral Program on Biomolecular Technology of Proteins (BioToP)). The funding agencies had no influence on the conduct of this research. EQ BOKU VIBT is acknowledged for providing fermentation and mass-spectrometry equipment. We kindly acknowledge technical support from S. Heinzl and P. Tondl. We also kindly thank H. Rußmayer for help during labeling experiments.

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Authors and Affiliations



D.M. conceived and initiated the project. D.M., M.G.S., T.G., M.S. and B.G. designed the experiments. T.G. and M.E. carried out the experiments. S.H., C.T. and T.C. performed mass spectrometry. T.G., M.G.S., M.S. and D.M. analyzed the data. T.G., M.G.S. and D.M. wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Diethard Mattanovich.

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Competing interests

D.M., T.G., M.G.S., M.S. and B.G. are inventors of a patent application (application number PCT/EP2018/064158) that is based on the results reported in this publication.

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Integrated supplementary information

Supplementary Figure 1 Strategy for engineering chemoorganoautotrophy in Pichia pastoris.

(a) Wild type P. pastoris is able to use methanol (MeOH) for biomass in the assimilatory branch of the MeOH utilization by fixation of formaldehyde (FA) to xylulose-5-phosphate (Xu5P) which is regenerated in the xylulose monophosphate (XuMP) cycle (purple pathway) and as an energy source in the dissimilatory branch (pink pathway) by oxidation to carbon dioxide (CO2) under formation of NADH. (b) In an engineered strain MeOH assimilation is blocked by deletion of dihydroxyacetone synthase (DAS1 and DAS2) and a CO2 fixation pathway similar to a Calvin-Benson-Bassham (CBB) cycle is integrated (integration of RuBisCO, PRK, TDH3, PGK1, TKL1, TPI1, groEL and groES). RuBisCO carboxylates ribulose-1,5-bisphosphate which is regenerated in the synthetic CBB cycle; Additionally, the alcohol oxidase 1 gene AOX1 was deleted to avoid accumulation of formaldehyde. The enzymatic steps shown in magenta are present in both pathways (a and b). Abbreviations: alcohol oxidase (Aox), dihydroxyacetone synthase (Das), fructose-1,6-bisphosphate aldolase (Fba1-2), fructose-1,6-bisphosphatase (Fbp1), sedoheptulose-1,7-bisphosphatase (Shb17), ribose-5-phosphate ketol-isomerase (Rki1-2), D-ribulose-5-phosphate 3-epimerase (Rpe1-2), formaldehyde dehydrogenase (Fld1), S-formylglutathione hydrolase (Fgh1), formate dehydrogenase (Fdh1), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), dihydroxyacetone (DHA), glyceraldehyde-3-phosphate (GAP), 3-phosphoglycerate (3PGA), glutathione (GSH), ribose-5-phosphate (R5P), ribulose-1,5-bisphosphate (RuBP).

Supplementary Figure 2 Re-integration of AOX1 does not lead to higher growth rates.

CBBp+RuBisCO (black line) and CBBp+RuBisCO (purple line) were cultivated under chemoorganoautotrophic conditions and the mean of at least three biological replicates (independent transformations) with single values (black and purple signs) is shown.

Supplementary Figure 3 Transketolase 1 (Tkl1) does not substitute for dihydroxyacetone synthase (Das1/2).

Cells were cultivated in 3 g L-1 glycerol containing minimal medium and then fed with methanol (MeOH) shots as indicated (first shot was for induction while glycerol was still present). Strains: Δaox1 (red dashed line), Δaox1,Δdas1: Δaox1::TDH3p,PGK1p and Δdas1 (yellow line), Δaox1,Δdas1das2::TKL1p: Δaox1::TDH3p,PGK1p, Δdas1 and Δdas2::TKL1p (dark blue line), Δaox1,Δdas1das2: Δaox1::TDH3p,PGK1p, Δdas1 and Δdas2 (blue line) and CBBΔRuBisCO: Δaox1::TDH3p,PRKp,PGK1p, Δdas1 and Δdas2::TPI1p,TKL1p, Each clone was cultivated in duplicates (error bars indicate s.e.m.) and the Δaox1 control was cultivated as triplicate (error bars show s.d. of three independent cultivations, n=3).

Supplementary Figure 4 Engineered CBBp+RuBisCO grow in presence of methanol and CO2.

(a) Engineered CBBp+RuBisCO (a and b) are able to grow in presence of methanol and CO2 while CBBpΔRuBisCO are not. CBS7435 wt cells grow well in presence of both substrates, since methanol can be utilized for biomass and energy generation. Cells were cultivated in batch phase (16.0 g L-1 glycerol) until 10 g L-1 cell dry weight (CDW) and then fed with 0.5 – 1.0% (v/v) methanol pulses and a constant inflow of 5% CO2. CDW values are calculated from OD measurements (correlation: 1 OD600 unit = 0.191 g L-1 CDW) and standard error bars indicate s.e.m.; (b) Methanol consumption. Methanol uptake rates were determined during cultivation (time frame of 24 h indicated by arrows in (a) and showed the highest methanol utilization by CBS7435 wt cells followed by the engineered CBBp+RuBisCO (a and b – experiment repeated twice). The strain lacking RuBisCO (CBBpΔRuBisCO) showed slow methanol utilization.

Supplementary Figure 5 Labeling of engineered P. pastoris cells shows complete biomass formation from 12C carbon dioxide.

(ac) Bioreactor cultivation of 13C labelled biomass from two CBBp+RuBisCO clones (a, b – individual transformations) and the reinoculated biomass from one clone (c) with 12CO2 as a carbon source. The 13C biomass content of the inoculum was enriched by cultivation on fully labelled 13C glycerol. During cultivation, cells were fed with 5 % 12CO2 and methanol. (df) Logarithmic CDW value indicates the specific growth rate for the strains in all three reactors. (g, h) Mean cell size (error bars show cell size s.d. of yeast population – 50000 events recorded) and cell viability (error bars indicate s.e.m.) during the cultivation of CBBp+RuBisCO a and b. (i) Characteristic bright field image from day 1 (upper panel) and day 21 (lower panel) of cultivation (bar = 10µm). (k, l) 13C content of the biomass measured by EA-IRMS for CBBp+RuBisCO a and CBBp+RuBisCO b (k) and its reinoculation (l) empty bars indicate calculated values and full bars show measured values ± s.e.m.

Supplementary Figure 6 Adaptive Laboratory Evolution (ALE) leads to higher growth rates.

Cultivation of strains after ALE in serial batch cultivations for 27 to 29 generations; Cell Dry Weight (CDW) values are calculated from OD600 measurements (correlation: 1 OD600 unit = 0.191 g L-1 CDW, error bars indicate s.e.m.), (a) The pools (full lines) from evolved cells were compared for growth on CO2 and methanol to each parental strain cultivated under the same conditions (dashed lines); (b) from one Pool (CBBp+RuBisCO b) 12 single colonies were further characterized under the same conditions showing better growth for 3 single colonies derived from the evolved population. This experiment was repeated twice with similar results.

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Supplementary Figs. 1–6, Supplementary Tables 1–10 and Supplementary Sequences 1–4.

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Gassler, T., Sauer, M., Gasser, B. et al. The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2. Nat Biotechnol 38, 210–216 (2020).

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