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.

  • Article
  • Published:

Synthesis of 11-carbon terpenoids in yeast using protein and metabolic engineering

Nature Chemical Biology volume 14, pages 1090–1098 (2018)Cite this article

Subjects

Abstract

One application of synthetic biology is the redesign of existing biological systems to acquire new functions. In this context, expanding the chemical code underlying key biosynthetic pathways will lead to the synthesis of compounds with new structures and potentially new biological activities. Terpenoids are a large group of specialized metabolites with numerous applications. Yet, being synthesized from five-carbon units, they are restricted to distinct classes that differ by five carbon atoms (C10, C15, C20, etc.). To expand the diversity of terpenoid structures, we engineered yeast cells to synthesize a noncanonical building block with 11 carbons, and produced 40 C11 terpene scaffolds that can form the basis for an entire terpenoid class. By identifying a single-residue switch that converts C10 plant monoterpene synthases to C11-specific enzymes, we engineered dedicated synthases for C11 terpene production. This approach will enable the systematic expansion of the chemical space accessed by terpenoids.

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

Fig. 1: Terpenoid biosynthetic code.
Fig. 2: Production of 2meGPP in yeast.
Fig. 3: Percentage of C11 and C10 terpene scaffolds produced by the different wild-type terpene synthases in yeast.
Fig. 4: Production of C11 terpenoids in yeast by plant monoterpene synthases.
Fig. 5: Proposed mechanism and structure of SfCinS1.
Fig. 6: Engineering dedicated C11 terpene synthases.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

References

  1. Dewick, P. M. Medicinal natural products: a biosynthetic approach. (Wiley, Chichester, UK, 2009).

    Book  Google Scholar 

  2. Pichersky, E., Noel, J. P. & Dudareva, N. Biosynthesis of plant volatiles: nature’s diversity and ingenuity. Science 311, 808–811 (2006).

    Article  CAS  Google Scholar 

  3. Gershenzon, J. & Dudareva, N. The function of terpene natural products in the natural world. Nat. Chem. Biol. 3, 408–414 (2007).

    Article  CAS  Google Scholar 

  4. Ignea, C. et al. Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast. Metab. Eng. 28, 91–103 (2015).

    Article  CAS  Google Scholar 

  5. Jiang, J., He, X. & Cane, D. E. Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme. Nat. Chem. Biol. 3, 711–715 (2007).

    Article  CAS  Google Scholar 

  6. Herde, M. et al. Identification and regulation of TPS04/GES, an Arabidopsis geranyllinalool synthase catalyzing the first step in the formation of the insect-induced volatile C16-homoterpene TMTT. Plant Cell 20, 1152–1168 (2008).

    Article  CAS  Google Scholar 

  7. Lee, S. et al. Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis. Proc. Natl Acad. Sci. USA 107, 21205–21210 (2010).

    Article  CAS  Google Scholar 

  8. Magnard, J. L. et al. PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses. Science 349, 81–83 (2015).

    Article  CAS  Google Scholar 

  9. Richter, A. et al. Characterization of biosynthetic pathways for the production of the volatile homoterpenes DMNT and TMTT in Zea mays. Plant Cell 28, 2651–2665 (2016).

    Article  CAS  Google Scholar 

  10. Vedula, L. S. et al. Exploring biosynthetic diversity with trichodiene synthase. Arch. Biochem. Biophys. 466, 260–266 (2007).

    Article  CAS  Google Scholar 

  11. Rising, K. A. et al. Formation of a novel macrocyclic alkaloid from the unnatural farnesyl diphosphate analogue anilinogeranyl diphosphate by 5-epi-aristolochene synthase. ACS. Chem. Biol. 10, 1729–1736 (2015).

    Article  CAS  Google Scholar 

  12. Karahadian, C., Josephson, D. B. & Lindsay, R. C. Volatile compounds from Penicillium sp. contributing musty-earthy notes to Brie and Camembert cheese flavors. J. Agric. Food. Chem. 33, 339–343 (1985).

    Article  CAS  Google Scholar 

  13. Komatsu, M., Tsuda, M., Omura, S., Oikawa, H. & Ikeda, H. Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc. Natl Acad. Sci. USA 105, 7422–7427 (2008).

    Article  CAS  Google Scholar 

  14. Wang, C. M. & Cane, D. E. Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor. J. Am. Chem. Soc. 130, 8908–8909 (2008).

    Article  CAS  Google Scholar 

  15. Dickschat, J. S. et al. Biosynthesis of the off-flavor 2-methylisoborneol by the myxobacterium Nannocystis exedens. Angew. Chem. Int. Edn Engl. 46, 8287–8290 (2007).

    Article  CAS  Google Scholar 

  16. Brock, N. L., Ravella, S. R., Schulz, S. & Dickschat, J. S. A detailed view of 2-methylisoborneol biosynthesis. Angew. Chem. Int. Edn Engl. 52, 2100–2104 (2013).

    Article  CAS  Google Scholar 

  17. Dickschat, J. S. Bacterial terpene cyclases. Nat. Prod. Rep. 33, 87–110 (2016).

    Article  CAS  Google Scholar 

  18. Chou, W. K., Ikeda, H. & Cane, D. E. Cloning and characterization of Pfl_1841, a 2-methylenebornane synthase in Pseudomonas fluorescens PfO-1. Tetrahedron 67, 6627–6632 (2011).

    Article  CAS  Google Scholar 

  19. Giglio, S., Chou, W. K., Ikeda, H., Cane, D. E. & Monis, P. T. Biosynthesis of 2-methylisoborneol in cyanobacteria. Environ. Sci. Technol. 45, 992–998 (2011).

    Article  CAS  Google Scholar 

  20. Yamada, Y. et al. Terpene synthases are widely distributed in bacteria. Proc. Natl Acad. Sci. USA 112, 857–862 (2015).

    Article  CAS  Google Scholar 

  21. Schumann, R. & Pendleton, P. Dehydration products of 2-methylisoborneol. Water Res. 31, 1243–1246 (1997).

    Article  CAS  Google Scholar 

  22. Zhou, Y. 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  Google Scholar 

  23. Ignea, C., Pontini, M., Maffei, M. E., Makris, A. M. & Kampranis, S. C. Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synth. Biol. 3, 298–306 (2014).

    Article  CAS  Google Scholar 

  24. Stanley Fernandez, S. M., Kellogg, B. A. & Poulter, C. D. Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity. Biochemistry 39, 15316–15321 (2000).

    Article  CAS  Google Scholar 

  25. Jiang, Z., Kempinski, C., Bush, C. J., Nybo, S. E. & Chappell, J. Engineering triterpene and methylated triterpene production in plants provides biochemical and physiological insights into terpene metabolism. Plant Physiol. 170, 702–716 (2016).

    Article  CAS  Google Scholar 

  26. Springer, M., Weissman, J. S. & Kirschner, M. W. A general lack of compensation for gene dosage in yeast. Mol. Syst. Biol. 6, 368 (2010).

    Article  Google Scholar 

  27. Köksal, M., Chou, W. K., Cane, D. E. & Christianson, D. W. Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate. Biochemistry 51, 3011–3020 (2012).

    Article  Google Scholar 

  28. Chou, W. K., Gould, C. A. & Cane, D. E. Incubation of 2-methylisoborneol synthase with the intermediate analog 2-methylneryl diphosphate. J. Antibiot. (Tokyo) 70, 625–631 (2017).

    Article  CAS  Google Scholar 

  29. Citron, C. A., Barra, L., Wink, J. & Dickschat, J. S. Volatiles from nineteen recently genome sequenced actinomycetes. Org. Biomol. Chem. 13, 2673–2683 (2015).

    Article  CAS  Google Scholar 

  30. Kampranis, S. C. et al. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function. Plant Cell 19, 1994–2005 (2007).

    Article  CAS  Google Scholar 

  31. Phillips, M. A., Wildung, M. R., Williams, D. C., Hyatt, D. C. & Croteau, R. cDNA isolation, functional expression, and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis. Arch. Biochem. Biophys. 411, 267–276 (2003).

    Article  CAS  Google Scholar 

  32. Iijima, Y. et al. The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil. Plant Physiol. 136, 3724–3736 (2004).

    Article  CAS  Google Scholar 

  33. Iijima, Y., Gang, D. R., Fridman, E., Lewinsohn, E. & Pichersky, E. Characterization of geraniol synthase from the peltate glands of sweet basil. Plant Physiol. 134, 370–379 (2004).

    Article  CAS  Google Scholar 

  34. Lücker, J. et al. Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases. Eur. J. Biochem. 269, 3160–3171 (2002).

    Article  Google Scholar 

  35. Tsaballa, A. et al. Use of the de novo transcriptome analysis of silver-leaf nightshade (Solanum elaeagnifolium) to identify gene expression changes associated with wounding and terpene biosynthesis. BMC Genomics 16, 504 (2015).

    Article  Google Scholar 

  36. Hyatt, D. C. et al. Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc. Natl Acad. Sci. USA 104, 5360–5365 (2007).

    Article  CAS  Google Scholar 

  37. Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  38. Ignea, C. et al. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microb. Cell. Fact. 10, 4 (2011).

    Article  CAS  Google Scholar 

  39. Genee, H. J. et al. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly. ACS Synth. Biol. 4, 342–349 (2015).

    Article  CAS  Google Scholar 

  40. Ignea, C. et al. Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae. Microb. Cell. Fact. 11, 162 (2012).

    Article  CAS  Google Scholar 

  41. Trikka, F. A. et al. Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes. BMC Genomics 16, 935 (2015).

    Article  Google Scholar 

  42. Finkelstein, J., Antony, E., Hingorani, M. M. & O’Donnell, M. Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy. Anal. Biochem. 319, 78–87 (2003).

    Article  CAS  Google Scholar 

  43. Ignea, C. et al. Carnosic acid biosynthesis elucidated by a synthetic biology platform. Proc. Natl Acad. Sci. USA 113, 3681–3686 (2016).

    Article  CAS  Google Scholar 

  44. Zaed, A. M. & Sutherland, A. Stereoselective synthesis of the bicyclic guanidine alkaloid (+)-monanchorin. Org. Biomol. Chem. 8, 4394–4399 (2010).

    Article  CAS  Google Scholar 

  45. Winterfeldt, E. Applications of diisobutylaluminium hydride (DIBAL) and triisobulyaluminium (TIBA) as reducing agents in organic synthesis. 7, Synthesis 617–630 (1975).

  46. Dixit, V. M., Laskovics, F. M., Noall, W. I. & Poulter, C. D. Tris(tetrabutylammonium) hydrogen pyrophosphate. A new reagent for the preparation of allylic pyrophosphate esters. J. Org. Chem. 46, 1967–1969 (1981).

    Article  CAS  Google Scholar 

  47. Miyata, O., Ozawa, Y., Ninomiya, I. & Naito, T. Radical cyclization in heterocycle synthesis. Part 10:1A concise synthesis of (-)-kainic acid via sulfanyl radical addition-cyclization-elimination reaction. Tetrahedron 56, 6199–6207 (2000).

    Article  CAS  Google Scholar 

  48. Peitsch, M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem. Soc. Trans. 24, 274–279 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank D. Cane and W. Chou (Brown University, USA) for providing the bacterial construct pET28a(+)/PlGPPMT, C.E. Vickers (University of Queensland, Australia) for providing construct pCEV-G2-Ph/ClLimS, F. Geu-Flores (University of Copenhagen, Denmark) for critical reading of the manuscript, and M. Raadam for technical assistance. We are grateful to D.I. Pattison and E. Lazaridi from the PLEN metabolomics platform for their support. This work was supported by the Greek General Secretariat of Research and Technology (GSRT) grant 11ΣΥΝ_3_770 (to A.M.M. and S.C.K.) and the Novo Nordisk Foundation grants NNF16OC0019554 (to C.I.) and NNF16OC0021760 (to S.C.K.).

Author information

Author notes

  1. Codruta Ignea

    Present address: Department of Chemistry, University of Copenhagen, Copenhagen, Denmark

  2. These authors contributed equally: Codruta Ignea, Marianna Pontini.

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark

    Codruta Ignea, Mohammed S. Motawia & Sotirios C. Kampranis

  2. Mediterranean Agronomic Institute of Chania, Chania, Greece

    Codruta Ignea, Marianna Pontini & Sotirios C. Kampranis

  3. Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy

    Marianna Pontini & Massimo E. Maffei

  4. Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece

    Antonios M. Makris

Authors
  1. Codruta Ignea
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marianna Pontini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammed S. Motawia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Massimo E. Maffei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Antonios M. Makris
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sotirios C. Kampranis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.C.K. conceived the project and designed experiments; C.I. designed experiments, engineered MIC1 strain, expressed and analyzed C10 and C11 wild-type and mutant synthases in yeast and bacteria, performed mutagenesis of PlGPPMT, PtPinS, SpSabS, ObMyrS, ClLimS, SeCamS, produced the purified proteins, and conducted in vitro enzymatic assays for the determination of the kinetic parameters; M.P. performed expression and analysis of PlGPPMT, SfCinS1 wild-type and mutants, wild-type SpSabS, and conducted yeast optimization for 2meGPP production, M.S.M. performed the chemical synthesis of 2meGPP; M.E.M. and A.M.M. assisted in data analysis; S.C.K. and C.I. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Sotirios C. Kampranis.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Tables 1–9, Supplementary Figures 1–11, Supplementary Notes 1–2

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ignea, C., Pontini, M., Motawia, M.S. et al. Synthesis of 11-carbon terpenoids in yeast using protein and metabolic engineering. Nat Chem Biol 14, 1090–1098 (2018). https://doi.org/10.1038/s41589-018-0166-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0166-5

This article is cited by

Search

Advanced 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