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.

Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli

Abstract

The development of non-petrochemical sources for the plastics industry continues to progress as large multinationals focus on renewable resources to replace fossil carbon1. Many bacteria are known to accumulate polyoxoesters as water-insoluble granules in the cytoplasm. The thermoplastic and/or elastomeric behaviour of these biodegradable polymers holds promise for the development of various technological applications2,3. Here, we report the synthesis and characterization of microbial polythioesters (PTEs), a novel class of biopolymers of general technological relevance. Biosynthesis of PTE homopolymers was achieved using a recombinant strain of Escherichia coli that expressed a non-natural pathway consisting of a butyrate kinase, a phosphotransbutyrylase, and a PHA synthase. Different homopolymers were produced, consisting of either 3-mercaptopropionate, 3-mercaptobutyrate, or 3-mercaptovalerate repeating units, if the respective mercaptoalkanoic acids were provided as precursor substrates to the fermentative process. The PTEs contributed up to 30% (w/w) of the cellular dry weight and were identified as hydrophobic inclusions in the cytoplasm. The chemical and stereochemical homogeneity of the purified PTEs were identified by different methods, and the estimated physical properties were compared to the oxypolyester equivalents, revealing low crystalline order and, for the poly(3-mercaptopropionate) improved thermal stability. The ability to produce PTEs through a biosynthetic route opens up new avenues in the field of biomaterials.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Metabolic pathways for the biosynthesis of PTEs.
Figure 2
Figure 3: 13C-CP/MAS-NMR spectra of different purification steps of PMP isolated from cells of E. coli JM109 pBPP1.

References

  1. Eissen, M., Metzger, J.O., Schmidt, E. & Schneiwind, U. 10 years after Rio-concepts on the contribution of chemistry to a sustainable development. Angew. Chem. Int. Ed. 41, 414–436 (2002).

    CAS  Google Scholar 

  2. Anderson, A.J. & Dawes, E.A. Ocurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450–472 (1990).

    CAS  Google Scholar 

  3. Hocking, P.J. & Marchessault, R.H. in Chemistry and Technology of Biodegradable Polymers (ed. Griffin, G.J.L.) 48–96 (Chapman and Hall, London, 1994).

    Google Scholar 

  4. Steinbüchel, A. & Valentin, H.E. Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol. Lett. 128, 219–228 (1995).

    Google Scholar 

  5. Takagi, Y., Hashii, M., Maehara, A. & Yamane, T. Biosynthesis of polyhydroxyalkanoate with a thiophenoxy side group obtained from Pseudomonas putida. Macromolecules 32, 8315–8318 (1999).

    CAS  Google Scholar 

  6. Ewering Ewering, C., Lütke-Eversloh, T., Luftmann, H. & Steinbüchel, A. Identification of novel sulphur-containing bacterial polyesters: biosynthesis of poly(3-hydroxy-S-propyl-ω-thioalkanoates) containing thioether linkages in the side chains. Microbiology 148, 1397–1406 (2002).

    Google Scholar 

  7. Lütke-Eversloh, T., Bergander, K., Luftmann, H. & Steinbüchel, A. Identification of a new class of biopolymer: bacterial synthesis of a sulphur containing polymer with thioester linkages. Microbiology 147, 11–19 (2001).

    Google Scholar 

  8. Lütke-Eversloh, T., Bergander, K., Luftmann, H. & Steinbüchel, A. Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulphur analogue to poly(3-hydroxybutyrate) (PHB). Biomacromolecules 2, 1061–1065 (2001).

    Google Scholar 

  9. Lütke-Eversloh, T., Kawada, J., Marchessault, R.H. & Steinbüchel, A. Characterization of biological polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha. Biomacromolecules 3, 159–166 (2002).

    Google Scholar 

  10. Liu, S.-J. & Steinbüchel, A. A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli. Appl. Environ. Microbiol. 66, 739–743 (2000).

    CAS  Google Scholar 

  11. Gorenflo, V., Schmack, G., Vogel, R. & Steinbüchel, A. Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties. Biomacromolecules 2, 45–57 (2001).

    CAS  Google Scholar 

  12. Hocking, P.J. & Marchessault, R.H. Microstructure of poly[(R,S)-β-hydroxybutyrate] by 13C NMR. Macromolecules 28, 6401–6409 (1995).

    CAS  Google Scholar 

  13. Morgan, K.H., Furneaux, R.H. & Larsen, N.G. Solid state NMR studies on the structure of starch granules. Carbohyd. Res. 276, 387–399 (1995).

    CAS  Google Scholar 

  14. Horii, F, Yamamoto, A., Hirai, A. & Kitamaru, R. Structural study of amylose polymorphs by crosspolarization magic-angle-spinning NMR spectroscopy. Carbohyd. Res. 160, 29–40 (1987).

    CAS  Google Scholar 

  15. Suehiro, K., Chatani, Y. & Tadokoro, H. Structural studies of polyesters. VI. Disordered crystal structure (Form II) of poly(β-propiolactone). Polym. J. 7, 352–358 (1975).

    CAS  Google Scholar 

  16. Okamura, K. & Marchessault, R.H. Conformation of Biopolymers Vol. 2 (ed. Ramachandran, G.N.) 709–720 (Academic, London, 1967).

    Google Scholar 

  17. Marchessault, R.H., Morikawa, H., Revol, J.F. & Bluhm, T.L. Physical properties of a naturally occurring polyester: Poly(β-hydroxyvalerate)/poly(β-hydroxybutyrate) Macromolecules 17, 1882–1884 (1984).

    CAS  Google Scholar 

  18. Cornibert, J. & Marchessault, R.H. Physical properties of poly-β-hydoroxybutyrate IV. Conformational analysis and crystalline structure. J. Mol. Biol. 71, 735–756 (1972).

    CAS  Google Scholar 

  19. Marvel, C.S. & Kotch, A. Polythiolesters. J. Am. Chem. Soc. 73, 1100–1102 (1951).

    CAS  Google Scholar 

  20. Sandler, S.R. in Organic Chemistry Vol. 29-III (ed. Wasserman, H.H.) 46–72 (Academic, San Diego, USA, 1996).

    Google Scholar 

  21. Lütke-Eversloh, T., Steinbüchel, A. & Ewering, C. Sulfur containing polyhydroxyalkanoate compositions and method of production. US patent 60,226,139 (2000).

  22. Jones, M. Microbial alternative to plastics. Microbiol. Today 28, 36 (2001).

    Google Scholar 

  23. Wood, F. Antibacterial biopolymer may aid healing. Chem. Ind. 3, 64 (2001).

    Google Scholar 

  24. Sambrock, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

    Google Scholar 

  25. Schjånberg, E. Pentensäure und Thioessigsäure. Chem. Ber. 74, 1751–1759 (1941).

    Google Scholar 

  26. Arnold, M. Histochemie. Einführung in die Grundlagen und Prinzipien der Methoden (Springer, Berlin, 1968).

    Google Scholar 

  27. Spurr, A.R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct. Res. 26, 31–43 (1969).

    CAS  Google Scholar 

  28. Brandrup, J., Immergut, E.H. & Grulke, E.A. (eds) Polymer Handbook 4th edn (Wiley, New York 1999).

    Google Scholar 

Download references

Acknowledgements

We thank Ursula Malkus for expert electron microscopic preparation of the specimens and for taking the TEM micrographs, and Gudrun Kiefermann (Institut für Medizinische Physik und Biophysik, WWU Münster) for photographic work. Further spectroscopic analysis of PTE samples by Heinrich Luftmann and Klaus Bergander (Institut für Organische Chemie, WWU Münster) is gratefully acknowledged. Paul Xia performed the 13C high-resolution NMR experiments.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Alexander Steinbüchel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lütke-Eversloh, T., Fischer, A., Remminghorst, U. et al. Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli. Nature Mater 1, 236–240 (2002). https://doi.org/10.1038/nmat773

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat773

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing