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

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.

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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.

Data availability

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

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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.).

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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.

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Correspondence to Sotirios C. Kampranis.

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

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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

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