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.

  • Letter
  • Published:

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

Nature Biotechnology volume 23, pages 1171–1176 (2005)Cite this article

Abstract

Type I polyketide synthase (PKS) genes consist of modules ∼3–6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Redesign of polyketide synthase genes.
Figure 2: Combinatorial assembly of PKS modules.
Figure 3: Predicted products of bimodular PKSs created in this work.

Similar content being viewed by others

References

  1. Walsh, C . Antibiotics: Actions, Origins, Resistance (ASM Press, Washington, DC, 2003).

    Book  Google Scholar 

  2. Cortes, J., Haydock, S.F., Roberts, G.A., Bevitt, D.J. & Leadlay, P.F. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348, 176–178 (1990).

    Article  CAS  Google Scholar 

  3. Reeves, C.D. The enzymology of combinatorial biosynthesis. Crit. Rev. Biotechnol. 23, 95–147 (2003).

    Article  CAS  Google Scholar 

  4. Tsuji, S.Y., Cane, D.E. & Khosla, C. Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules. Biochemistry 40, 2326–2331 (2001).

    Article  CAS  Google Scholar 

  5. Gokhale, R.S., Tsuji, S.Y., Cane, D.E. & Khosla, C. Dissecting and exploiting intermodular communication in polyketide synthases. Science 284, 482–485 (1999).

    Article  CAS  Google Scholar 

  6. Broadhurst, R.W., Nietlispach, D., Wheatcroft, M.P., Leadlay, P.F. & Weissman, K.J. The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 723–731 (2003).

    Article  CAS  Google Scholar 

  7. Pfeifer, B.A. & Khosla, C. Biosynthesis of polyketides in heterologous hosts. Microbiol. Mol. Biol. Rev. 65, 106–118 (2001).

    Article  CAS  Google Scholar 

  8. Kao, C.M., Katz, L. & Khosla, C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265, 509–512 (1994).

    Article  CAS  Google Scholar 

  9. McDaniel, R. et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products. Proc. Natl. Acad. Sci. USA 96, 1846–1851 (1999).

    Article  CAS  Google Scholar 

  10. Kieser, T., Bibb., M.J., Buttner, M.J., Chater, K.F. & Hopwood, D.A. . Practical Streptomyces Genetics (The John Innes Foundation, Norwich, UK, 2000).

    Google Scholar 

  11. Pfeifer, B.A., Admiraal, S.J., Gramajo, H., Cane, D.E. & Khosla, C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001).

    Article  CAS  Google Scholar 

  12. Lau, J., Tran, C., Licari, P. & Galazzo, J. Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in E. coli. J. Biotechnol. 110, 95–103 (2004).

    Article  CAS  Google Scholar 

  13. Kodumal, S.J. et al. Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc. Natl. Acad. Sci. USA 101, 15573–15578 (2004).

    Article  CAS  Google Scholar 

  14. Oliynyk, M., Brown, M.J., Cortes, J., Staunton, J. & Leadlay, P.F. A hybrid modular polyketide synthase obtained by domain swapping. Chem. Biol. 3, 833–839 (1996).

    Article  CAS  Google Scholar 

  15. Kao, C.M., Luo, G., Katz, L., Cane, D. & Khosla, C. Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase. J. Am. Chem. Soc. 117, 9105–9109 (1995).

    Article  Google Scholar 

  16. Murli, S., Kennedy, J., Dayem, L.C., Carney, J.R. & Kealey, J.T. Metabolic engineering of E. coli for improved 6-deoxyerythronolide B production. J. Ind. Microbiol. Biotechnol. 30, 500–509 (2003).

    Article  CAS  Google Scholar 

  17. Jacobsen, J.R., Cane, D.E. & Khosla, C. Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis. Biochemistry 37, 4928–4934 (1998).

    Article  CAS  Google Scholar 

  18. Beck, B.J., Aldrich, C.C., Fecik, R.A., Reynolds, K.A. & Sherman, D.H. Iterative chain elongation by a pikromycin monomodular polyketide synthase. J. Am. Chem. Soc. 125, 4682–4683 (2003).

    Article  CAS  Google Scholar 

  19. Wilkinson, B. et al. Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase. Chem. Biol. 7, 111–117 (2000).

    Article  CAS  Google Scholar 

  20. Moss, S.J., Martin, C.J. & Wilkinson, B. Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity. Nat. Prod. Rep. 21, 575–593 (2004).

    Article  CAS  Google Scholar 

  21. Ranganathan, A. et al. Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues. Chem. Biol. 6, 731–741 (1999).

    Article  CAS  Google Scholar 

  22. Wu, N., Cane, D.E. & Khosla, C. Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases. Biochemistry 41, 5056–5066 (2002).

    Article  CAS  Google Scholar 

  23. Watanabe, K., Wang, C.C., Boddy, C.N., Cane, D.E. & Khosla, C. Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J. Biol. Chem. 278, 42020–42026 (2003).

    Article  CAS  Google Scholar 

  24. McDaniel, R., Kao, C.M., Hwang, J.S. & Khosla, C. Engineered intermodular and intramodular polyketide synthases fusions. Chem. Biol. 4, 667–674 (1997).

    Article  CAS  Google Scholar 

  25. Kim, B.S. et al. An unexpected interaction between the modular polyketide synthases, erythromycin DEBS1 and pikromycin PikAIV, leads to efficient triketide lactone synthesis. Biochemistry 41, 10827–10833 (2002).

    Article  CAS  Google Scholar 

  26. Ashley, G.W. & Carney, J.R. API-mass spectrometry of polyketides. I. A study on the fragmentation of triketide lactones. J. Antibiot. (Tokyo) 57, 224–234 (2004).

    Article  CAS  Google Scholar 

  27. Beck, B.J., Aldrich, C.C., Fecik, R.A., Reynolds, K.A. & Sherman, D.H. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J. Am. Chem. Soc. 125, 12551–12557 (2003).

    Article  CAS  Google Scholar 

  28. Chuck, J. et al. Molecular recognition of diketide substrates by a beta-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase. Chem. Biol. 4, 757–766 (1997).

    Article  CAS  Google Scholar 

  29. Hinterding, K., Singhanat, S. & Oberer, L. Stereoselective synthesis of polyketide fragments using a novel intramolecular Claisen-like condensation/reduction sequence. Tetrahedr. Lett. 42, 8463–8465 (2001).

    Article  CAS  Google Scholar 

  30. Wu, N., Tsuji, S.Y., Cane, D.E. & Khosla, C. Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J. Am. Chem. Soc. 122, 6465–6474 (2001).

    Article  Google Scholar 

Download references

Acknowledgements

We thank Jim Kealey, Sumati Murli and Jonathan Kennedy for providing strains, vectors and procedures for polyketide production in E. coli and Gary Ashley and Leonard Katz for the critical review of the manuscript. This work was supported in part by National Institute of Standards and Technology Advanced Program Grant 70NANB2H3014.

Author information

Author notes

  1. Hugo G Menzella and Ralph Reid: These authors contributed equally to this study.

Authors and Affiliations

  1. Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, 94545, California, USA

    Hugo G Menzella, Ralph Reid, John R Carney, Sunil S Chandran, Sarah J Reisinger, Kedar G Patel, David A Hopwood & Daniel V Santi

  2. Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK

    David A Hopwood

Authors
  1. Hugo G Menzella
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ralph Reid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John R Carney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sunil S Chandran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sarah J Reisinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kedar G Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David A Hopwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Daniel V Santi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel V Santi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Product ion scan of unlabeled (a) and 13C labeled (b) TKLs. (PDF 25 kb)

Supplementary Table 1

Sources of 140 modules analyzed. (PDF 24 kb)

Supplementary Table 2

Restriction sites introduced in PKS building blocks. (PDF 18 kb)

Supplementary Table 3

Substrate specificities of PKS modules used in this study. (PDF 24 kb)

Supplementary Table 4

Expression plasmids used in the bimodular tests. (PDF 47 kb)

Supplementary Table 5

Characterization of triketide lactones obtained in this study by mass spectrometry. (PDF 22 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Menzella, H., Reid, R., Carney, J. et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 23, 1171–1176 (2005). https://doi.org/10.1038/nbt1128

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1128

This article is cited by

Search

Advanced 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