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Growth of E. coli on formate and methanol via the reductive glycine pathway

Nature Chemical Biology volume 16, pages 538–545 (2020)Cite this article

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Abstract

Engineering a biotechnological microorganism for growth on one-carbon intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on one-carbon compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 h and growth yield of ~1.5 g cell dry weight (gCDW) per mol-formate. Short-term evolution decreased doubling time to less than 8 h and improved biomass yield to 2.3 gCDW per mol-formate. Growth on methanol and CO2 was achieved by further expression of a methanol dehydrogenase. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.

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Fig. 1: The synthetic rGlyP is similar in structure to the rAcCoAP.
Fig. 2: Modular establishment of the rGlyP.
Fig. 3: Short-term evolution improves growth on formate.
Fig. 4: Labeling pattern of proteinogenic amino acids confirms the activity of the rGlyP.
Fig. 5: Engineered growth on methanol.

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Data availability

Complete information on the experimental setup as well as detailed results are available from the corresponding author upon reasonable request.

Code availability

MATLAB code used for the analysis of the experiments is available from the corresponding author upon request.

References

  1. Blankenship, R. E. et al. Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement. Science 332, 805–809 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Scheffe, J. R. & Steinfeld, A. Oxygen exchange materials for solar thermochemical splitting of H2O and CO2: a review. Mater. Today 17, 341–348 (2014).

    Article  CAS  Google Scholar 

  3. Snoeckx, R. & Bogaerts, A. Plasma technology—a novel solution for CO2 conversion? Chem. Soc. Rev. 46, 5805–5863 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Zhang, Q., Kang, J. & Wang, Y. Development of novel catalysts for Fischer–Tropsch synthesis: tuning the product selectivity. ChemCatChem 2, 1030–1058 (2010).

    Article  CAS  Google Scholar 

  5. Jouny, M., Luc, W. & Jiao, F. General techno-economic analysis of CO2 electrolysis systems. Ind. Eng. Chem. Res. 57, 2165–2177 (2018).

    Article  CAS  Google Scholar 

  6. Yishai, O., Lindner, S. N., Gonzalez de la Cruz, J., Tenenboim, H. & Bar-Even, A. The formate bio-economy. Curr. Opin. Chem. Biol. 35, 1–9 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Szima, S. & Cormos, C. C. Improving methanol synthesis from carbon-free H2 and captured CO2: a techno-economic and environmental evaluation. J. CO2 Util. 24, 555–563 (2018).

    Article  CAS  Google Scholar 

  8. Bertsch, J. & Muller, V. Bioenergetic constraints for conversion of syngas to biofuels in acetogenic bacteria. Biotechnol. Biofuels 8, 210 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Bennett, R. K., Steinberg, L. M., Chen, W. & Papoutsakis, E. T. Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs. Curr. Opin. Biotechnol. 50, 81–93 (2017).

    Article  PubMed  CAS  Google Scholar 

  10. Muller, J. E. et al. Engineering Escherichia coli for methanol conversion. Metab. Eng. 28, 190–201 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Dai, Z. et al. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour. Technol. 245, 1407–1412 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Yu, H. & Liao, J. C. A modified serine cycle in Escherichia coli coverts methanol and CO2 to two-carbon compounds. Nat. Commun. 9, 3992 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Meyer, F. et al. Methanol-essential growth of Escherichia coli. Nat. Commun. 9, 1508 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Woolston, B. M., King, J. R., Reiter, M., Van Hove, B. & Stephanopoulos, G. Improving formaldehyde consumption drives methanol assimilation in engineered E. coli. Nat. Commun. 9, 2387 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Bennett, R. K., Gonzalez, J. E., Whitaker, W. B., Antoniewicz, M. R. & Papoutsakis, E. T. Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph. Metab. Eng. 45, 75–85 (2017).

    Article  PubMed  CAS  Google Scholar 

  16. Gonzalez, J., Bennett, R. K., Papoutsakis, E. T. & Antoniewicz, M. R. Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein. Metab. Eng. 45, 67–74 (2017).

    Article  PubMed  CAS  Google Scholar 

  17. Rohlhill, J., Sandoval, N. R. & Papoutsakis, E. T. Sort-Seq approach to engineering a formaldehyde-inducible promoter for dynamically regulated Escherichia coli growth on methanol. ACS Synth. Biol. 6, 1584–1595 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Woolston, B. M., Roth, T., Kohale, I., Liu, D. R. & Stephanopoulos, G. Development of a formaldehyde biosensor with application to synthetic methylotrophy. Biotechnol. Bioeng. 115, 206–215 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Whitaker, W. B. et al. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab. Eng. 39, 49–59 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Lu, X. et al. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nat. Commun. 10, 1378 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Wang, X. et al. Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway. Bioresour. Bioprocess. 4, 41–46 (2017).

    Article  Google Scholar 

  22. Anthony, C. The Biochemistry of Methylotrophs (Academic Press, 1982).

  23. Drake, H. L., Kirsten, K. & Matthies, C. Acetogenic Prokaryotes. in The Prokaryotes (eds., Stanley Falkow, Eugene Rosenberg, Karl-Heinz Schleifer, Erko Stackebrandt) 354–420 (Springer, 2006).

  24. Bar-Even, A., Noor, E., Flamholz, A. & Milo, R. Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes. Biochim. Biophys. Acta 1827, 1039–1047 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Bar-Even, A. Does acetogenesis really require especially low reduction potential? Biochim. Biophys. Acta 1827, 395–400 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Noor, E. et al. Pathway thermodynamics highlights kinetic obstacles in central metabolism. PLoS Comput. Biol. 10, e1003483 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Figueroa, I. A. et al. Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway. Proc. Natl Acad. Sci. USA 115, E92–E101 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Kawasaki, H., Sato, T. & Kikuchi, G. A new reaction for glycine biosynthesis. Biochem. Biophys. Res. Commun. 23, 227–233 (1966).

    Article  CAS  PubMed  Google Scholar 

  29. Motokawa, Y. & Kikuchi, G. Glycine metabolism by rat liver mitochondria. Reconstruction of the reversible glycine cleavage system with partially purified protein components. Arch. Biochem. Biophys. 164, 624–633 (1974).

    Article  CAS  PubMed  Google Scholar 

  30. Pasternack, L. B., Laude, D. A. Jr. & Appling, D. R. 13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31, 8713–8719 (1992).

    Article  CAS  PubMed  Google Scholar 

  31. Tashiro, Y., Hirano, S., Matson, M. M., Atsumi, S. & Kondo, A. Electrical-biological hybrid system for CO2 reduction. Metab. Eng. 47, 211–218 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Yishai, O., Bouzon, M., Doring, V. & Bar-Even, A. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli. ACS Synth. Biol. 7, 2023–2028 (2018).

    Article  PubMed  CAS  Google Scholar 

  33. Crowther, G. J., Kosaly, G. & Lidstrom, M. E. Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1. J. Bacteriol. 190, 5057–5062 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tishkov, V. I. & Popov, V. O. Catalytic mechanism and application of formate dehydrogenase. Biochem. (Mosc.) 69, 1252–1267 (2004).

    Article  CAS  Google Scholar 

  35. Wenk, S., Yishai, O., Lindner, S. N. & Bar-Even, A. An engineering approach for rewiring microbial metabolism. Methods Enzymol. 608, 329–367 (2018).

    Article  PubMed  CAS  Google Scholar 

  36. Bassalo, M. C. et al. Rapid and efficient one-step metabolic pathway integration in E. coli. ACS Synth. Biol. 5, 561–568 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Gleizer, S. et al. Conversion of Escherichia coli to generate all biomass carbon from CO2. Cell 179, 1255–1263.e12 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Claassens, N. J., Cotton, C. A., Kopljar, D. & Bar-Even, A. Making quantitative sense of electromicrobial production. Nat. Catal. 2, 437 (2019).

    Article  CAS  Google Scholar 

  39. Nicholls, P. Formate as an inhibitor of cytochrome c oxidase. Biochem. Biophys. Res. Commun. 67, 610–616 (1975).

    Article  CAS  PubMed  Google Scholar 

  40. Warnecke, T. & Gill, R. T. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Micro. Cell Fact. 4, 25 (2005).

    Article  CAS  Google Scholar 

  41. Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution—principles and applications for biotechnology. Micro. Cell Fact. 12, 64 (2013).

    Article  Google Scholar 

  42. Wytock, T. P. et al. Experimental evolution of diverse Escherichia coli metabolic mutants identifies genetic loci for convergent adaptation of growth rate. PLoS Genet. 14, e1007284 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gutheil, W. G., Kasimoglu, E. & Nicholson, P. C. Induction of glutathione-dependent formaldehyde dehydrogenase activity in Escherichia coli and Hemophilus influenza. Biochem. Biophys. Res. Commun. 238, 693–696 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Kotrbova-Kozak, A., Kotrba, P., Inui, M., Sajdok, J. & Yukawa, H. Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R. Appl. Microbiol. Biotechnol. 76, 1347–1356 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Wu, T. Y. et al. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1. Appl. Microbiol. Biotechnol. 100, 4969–4983 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Roth, T. B., Woolston, B. M., Stephanopoulos, G. & Liu, D. R. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2. ACS Synth. Biol. 8, 796–806 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang, W. et al. Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa. Protein Expr. Purif. 92, 208–213 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Cotton, C. A., Claassens, N. J., Benito-Vaquerizo, S. & Bar-Even, A. Renewable methanol and formate as microbial feedstocks. Curr. Opin. Biotechnol. 62, 168–180 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Thoma, S. & Schobert, M. An improved Escherichia coli donor strain for diparental mating. FEMS Microbiol. Lett. 294, 127–132 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. Thomason, L. C., Costantino, N. & Court, D. L. E. coli genome manipulation by P1 transduction. Curr. Protoc. Mol. Biol. https://doi.org/10.1002/0471142727.mb0117s79 (2007).

  52. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006–2008 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Nyerges, A. et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc. Natl Acad. Sci. USA 113, 2502–2507 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zelcbuch, L. et al. Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res. 41, e98 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2001).

  56. Braatsch, S., Helmark, S., Kranz, H., Koebmann, B. & Jensen, P. R. Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning. Biotechniques 45, 335–337 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Giavalisco, P. et al. Elemental formula annotation of polar and lipophilic metabolites using 13C, 15N and 34S isotope labelling, in combination with high‐resolution mass spectrometry. Plant J. 68, 364–376 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Liu, A., Feng, R. & Liang, B. Microbial surface displaying formate dehydrogenase and its application in optical detection of formate. Enzym. Microb. Technol. 91, 59–65 (2016).

    Article  CAS  Google Scholar 

  59. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25, 402–408 (2001).

    CAS  PubMed  Google Scholar 

  60. Zhou, K. et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol. Biol. 12, 18 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Cotton, N. Claassens, H. He, R. Milo, E. Noor, N. Antonovsky, A. Flamholz, Y. Bar-On, W. Newell, D. Bonder, A. Satanowski, T. Erb and M. Bouzon for helpful discussions and suggestions. This work was funded by the Max Planck Society, by the German Ministry of Education and Research grant FormatPlant (part of BioEconomy 2030, Plant Breeding Research for the Bioeconomy) and by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 763911 (Project eForFuel).

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  1. Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany

    Seohyoung Kim, Steffen N. Lindner, Selçuk Aslan, Oren Yishai, Sebastian Wenk, Karin Schann & Arren Bar-Even

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Contributions

A.B.-E. designed and supervised the research and wrote the paper. S.K., S.N.L., S.A. and O.Y. genetically engineered E. coli for growth on formate and methanol, and performed the growth experiments. S.K. and S.N.L. measured biomass yield on formate and methanol. S.A. performed the qPCR experiments. S.W. and K.S. cloned the methanol dehydrogenase and formaldehyde dehydrogenase genes, and assisted in the growth experiments on methanol. S.K., S.N.L., S.A., O.Y., S.W., K.S. and A.B.-E. analyzed the data.

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Correspondence to Arren Bar-Even.

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Competing interests

A.B.-E. is cofounder of b.fab, exploring the commercialization of microbial bioproduction using formate as feedstock. The company was not involved in any way in performing or funding this study.

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Kim, S., Lindner, S.N., Aslan, S. et al. Growth of E. coli on formate and methanol via the reductive glycine pathway. Nat Chem Biol 16, 538–545 (2020). https://doi.org/10.1038/s41589-020-0473-5

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