Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues

Abstract

Structurally complex and diverse polyamines and polyamine analogues are potential therapeutics and agrochemicals that can address grand societal challenges, for example, healthy ageing and sustainable food production. However, their structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from natural resources. Here we reprogrammed the metabolism of baker’s yeast Saccharomyces cerevisiae and recruited nature’s diverse reservoir of biochemical tools to enable a complete biosynthesis of multiple polyamines and polyamine analogues. Specifically, we adopted a systematic engineering strategy to enable gram-per-litre-scale titres of spermidine, a central metabolite in polyamine metabolism. To demonstrate the potential of our polyamine platform, various polyamine synthases and ATP-dependent amide-bond-forming systems were introduced for the biosynthesis of natural and unnatural polyamine analogues. The yeast platform serves as a resource to accelerate the discovery and production of polyamines and polyamine analogues, and thereby unlocks this chemical space for further pharmacological and insecticidal studies.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Illustration of the modularized platform for the discovery and production of polyamines and polyamine analogues.
Fig. 2: Systematic engineering of yeast metabolism for the high-level production of spermidine.
Fig. 3: Biosynthesis of complex polyamines by elongating the spermidine carbon skeleton.
Fig. 4: Biocatalysis platform for amide-bond-containing natural polyamine analogues.

Data availability

Raw sequencing data is available on ArrayExpress with accession number E-MTAB- 9898. Data supporting the findings of this study are available within the article and its Supplementary Information files: the accession numbers and nucleotide sequences (codon optimized or original) of the enzymes referenced in this study are provided in this paper; source data are provided with this paper as Source Data files. All other data that support the findings of this study are available from the corresponding author upon reasonable request. All plasmids and strains used in this study are available from the corresponding author under a material transfer agreement. Source data are provided with this paper.

Code availability

All code used in the model simulations is available in GitHub (https://github.com/foodgeoff2010/Polyamine_platform).

References

  1. Gerland, P. et al. World population stabilization unlikely this century. Science 346, 234–237 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Campisi, J. et al. From discoveries in ageing research to therapeutics for healthy ageing. Nature 571, 183–192 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).

    Article  CAS  PubMed  Google Scholar 

  4. Galanie, S., Thodey, K., Trenchard, I. J., Filsinger Interrante, M. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095–1100 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lee, S. Y. et al. A comprehensive metabolic map for production of bio-based chemicals. Nat. Catal. 2, 18–33 (2019).

    Article  CAS  Google Scholar 

  6. Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Li, S., Li, Y. & Smolke, C. D. Strategies for microbial synthesis of high-value phytochemicals. Nat. Chem. 10, 395–404 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Chen, R., Yang, S., Zhang, L. & Zhou, Y. J. Advanced strategies for production of natural products in yeast. iScience 23, 100879 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Madeo, F., Eisenberg, T., Pietrocola, F. & Kroemer, G. Spermidine in health and disease. Science 359, eaan2788 (2018).

    Article  PubMed  Google Scholar 

  10. Li, Y.-Y. et al. Identification and characterization of kukoamine metabolites by multiple Ion monitoring triggered enhanced product Ion scan method with a triple-quadruple linear Ion trap mass spectrometer. J. Agric. Food Chem. 63, 10785–10790 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Mounce, B. C., Olsen, M. E., Vignuzzi, M. & Connor, J. H. Polyamines and their role in virus infection. Microbiol. Mol. Biol. Rev. 81, e00029-17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Antoniou, A. I., Pepe, D. A., Aiello, D., Siciliano, C. & Athanassopoulos, C. M. Chemoselective protection of glutathione in the preparation of bioconjugates: the case of trypanothione disulfide. J. Org. Chem. 81, 4353–4358 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Walters, D., Meurer-Grimes, B. & Rovira, I. Antifungal activity of three spermidine conjugates. FEMS Microbiol. Lett. 201, 255–258 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Jagu, E. et al. Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives. Eur. J. Med. Chem. 150, 655–666 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Bassard, J.-E., Ullmann, P., Bernier, F. & Werck-Reichhart, D. Phenolamides: bridging polyamines to the phenolic metabolism. Phytochemistry 71, 1808–1824 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Michael, A. J. Biosynthesis of polyamines and polyamine-containing molecules. Biochem. J. 473, 2315–2329 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Michael, A. J. Polyamines in eukaryotes, bacteria, and archaea. J. Biol. Chem. 291, 14896–14903 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Miller-Fleming, L., Olin-Sandoval, V., Campbell, K. & Ralser, M. Remaining mysteries of molecular biology: the role of polyamines in the cell. J. Mol. Biol. 427, 3389–3406 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Nielsen, J. & Keasling, J. D. Engineering cellular metabolism. Cell 164, 1185–1197 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Zhou, Y. J. J. et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J. Am. Chem. Soc. 134, 3234–3241 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Liu, Q. et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. Nat. Commun. 10, 4976 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Caspi, R. et al. The MetaCyc database of metabolic pathways and enzymes—a 2019 update. Nucleic Acids Res. 48, D445–D453 (2019).

    Article  PubMed Central  Google Scholar 

  23. Orth, J. D., Thiele, I. & Palsson, B. Ø. What is flux balance analysis? Nat. Biotechnol. 28, 245–248 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kurian, L., Palanimurugan, R., Gödderz, D. & Dohmen, R. J. Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature 477, 490–494 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Phillips, M. A., Coffino, P. & Wang, C. C. Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. J. Biol. Chem. 262, 8721–8727 (1987).

    Article  CAS  PubMed  Google Scholar 

  26. Qin, J. et al. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of l-ornithine. Nat. Commun. 6, 8224 (2015).

    Article  PubMed  Google Scholar 

  27. Hamasaki-Katagiri, N., Tabor, C. W. & Tabor, H. Spermidine biosynthesis in Saccharomyces cerevisiae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35–43 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Pegg, A. E. Toxicity of polyamines and their metabolic products. Chem. Res. Toxicol. 26, 1782–1800 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Albers, E. Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 5′-methylthioadenosine. IUBMB Life 61, 1132–1142 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Toms, A. V., Kinsland, C., McCloskey, D. E., Pegg, A. E. & Ealick, S. E. Evolutionary links as revealed by the structure of Thermotoga maritima S-adenosylmethionine decarboxylase. J. Biol. Chem. 279, 33837–33846 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Yerlikaya, A. & Stanley, B. A. S-adenosylmethionine decarboxylase degradation by the 26S proteasome is accelerated by substrate-mediated transamination. J. Biol. Chem. 279, 12469–12478 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Becker, M. A., Smith, P. R., Taylor, W., Mustafi, R. & Switzer, R. L. The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J. Clin. Investig. 96, 2133–2141 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hu, H. et al. DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-l-methionine production in Pichia pastoris. J. Biotechnol. 141, 97–103 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Roje, S. et al. Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo. J. Biol. Chem. 277, 4056–4061 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Darbani, B., Stovicek, V., van der Hoek, S. A. & Borodina, I. Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 116, 19415–19420 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Uemura, T., Tachihara, K., Tomitori, H., Kashiwagi, K. & Igarashi, K. Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation. J. Biol. Chem. 280, 9646–9652 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Tachihara, K., Uemura, T., Kashiwagi, K. & Igarashi, K. Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae. J. Biol. Chem. 280, 12637–12642 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Panicot, M. et al. A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell 14, 2539–2551 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ober, D. & Hartmann, T. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proc. Natl Acad. Sci. USA 96, 14777–14782 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Knott, J. M., Römer, P. & Sumper, M. Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine. FEBS Lett. 581, 3081–3086 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Petchey, M. R. & Grogan, G. Enzyme-catalysed synthesis of secondary and tertiary amides. Adv. Synth. Catal. 361, 3895–3914 (2019).

    Article  CAS  Google Scholar 

  42. Eudes, A. et al. Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast. Microb. Cell Factories 15, 198 (2016).

    Article  Google Scholar 

  43. Costa, M. A. et al. Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: syringyl lignin and sinapate/sinapyl alcohol derivative formation. Phytochemistry 66, 2072–2091 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Eichenberger, M. et al. Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties. Metab. Eng. 39, 80–89 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhou, K., Qiao, K., Edgar, S. & Stephanopoulos, G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat. Biotechnol. 33, 377–383 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Müller, K., Faeh, C. & Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 317, 1881–1886 (2007).

    Article  PubMed  Google Scholar 

  47. Kim, S.-K., Jin, Y.-S., Choi, I.-G., Park, Y.-C. & Seo, J.-H. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents. Metab. Eng. 29, 46–55 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Ljungdahl, P. O. & Daignan-Fornier, B. Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae. Genetics 190, 885–929 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gumulya, Y. et al. Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nat. Catal. 1, 878–888 (2018).

    Article  CAS  Google Scholar 

  50. Li, C., Zhang, R., Wang, J., Wilson, L. M. & Yan, Y. Protein engineering for improving and diversifying natural product biosynthesis. Trends Biotechnol. 38, 729–744 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Narayan, P., Ehsani, S. & Lindquist, S. Combating neurodegenerative disease with chemical probes and model systems. Nat. Chem. Biol. 10, 911–920 (2014).

    Article  CAS  PubMed  Google Scholar 

  52. Zhang, J., van den Herik, B. M. & Wahl, S. A. Alpha-ketoglutarate utilization in Saccharomyces cerevisiae: transport, compartmentation and catabolism. Sci. Rep. 10, 12838 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Mans, R. et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res. 15, fov004 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Luo, H. et al. Coupling S-adenosylmethionine-dependent methylation to growth: design and uses. PLoS Biol. 17, e2007050 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mikkelsen, M. D. et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab. Eng. 14, 104–111 (2012).

    Article  CAS  PubMed  Google Scholar 

  56. Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Canelas, A. B. et al. Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Anal. Chem. 81, 7379–7389 (2009).

    Article  CAS  PubMed  Google Scholar 

  58. Heirendt, L. et al. Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0. Nat. Protoc. 14, 639–702 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Deatherage, D. E. & Barrick, J. E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151, 165–188 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Shi, L. et al. Evolution of bacterial protein-tyrosine kinases and their relaxed specificity toward substrates. Genome Biol. Evol. 6, 800–817 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498–2504 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Nakamura, T., Yamada, K. D., Tomii, K. & Katoh, K. Parallelization of MAFFT for large-scale multiple sequence alignments. Bioinformatics 34, 2490–2492 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Goodstein, D. M. et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 40, D1178–D1186 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was funded by the Novo Nordisk Foundation (NNF10CC1016517), the Swedish Foundation for Strategic Research and the Knut and Alice Wallenberg Foundation. We thank J. Zhang, I. Borodina and Q-L. Liu for helpful discussions, J. Zhang for kindly providing plasmids from the CRISPR/Cas9 genome editing system, Q-L. Liu and Y. Chen for kindly providing aromatic chemical overproducing strains, A. Hoffmeyer for genome sequencing, M. Gossing, D. Romero-Suarez, the Chalmers Mass Spectrometry Infrastructure and the Analytical Core Facility of the Novo Nordisk Foundation Center for Biosustainability at Technical University of Denmark for assistance with metabolite analysis.

Author information

Authors and Affiliations

Authors

Contributions

J.Q. and J.N. conceived the study with input from A.K. (spermidine section). J.Q. designed and performed the experiments, analysed the data and drafted the manuscript. M.K. assisted with the MS-based metabolite analysis. B.J., Y.C. and E.Ö. assisted with the computational and bioinformatic analysis. M.K.J., J.D.K., A.K. and B.J. assisted with the data analysis and interpretation. J.N., M.K.J. and J.D.K. supervised the study. All the authors revised and approved the manuscript.

Corresponding author

Correspondence to Jens Nielsen.

Ethics declarations

Competing interests

J.Q., J.N. and A.K. are listed as inventors on patent applications related to microbial production of polyamines and/or polyamine analogues. J.Q. and J.N. are scientific co-founders of Chrysea Ltd. A.K. and J.N. are shareholders in Biopetrolia AB. J.D.K. has interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Apertor Labs, Ansa Biosiences and Berkeley Brewing Sciences. All the other authors declare no competing interests.

Additional information

Peer review information Nature Catalysis thanks Rajib Saha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, references and Figs. 1–.45.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–15.

Supplementary Data

Source Data for Supplementary Figs. 2–8.

Source data

Source Data Fig. 2

Statistical Source Data for Fig. 2.

Source Data Fig. 3

Statistical Source Data for Fig. 3.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qin, J., Krivoruchko, A., Ji, B. et al. Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues. Nat Catal 4, 498–509 (2021). https://doi.org/10.1038/s41929-021-00631-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-021-00631-z

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research