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Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites

Nature Protocols volume 9, pages 1980–1996 (2014)Cite this article

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Abstract

The procedures described here are designed for engineering Saccharomyces cerevisiae to produce sesquiterpenes with an aim to either increase product titers or to simply generate a quantity of product sufficient for identification and/or downstream experimentation. Engineering high-level sesquiterpene production in S. cerevisiae often requires iterations of strain modifications and metabolite analysis. To address the latter, the methods described here were tailored for robust measurement of metabolites that we have found to be fundamental indicators of pathway flux, using only gas chromatography and mass spectrometry (GC-MS) instrumentation. Thus, by focusing on heterologous production of sesquiterpenes via the mevalonate (MEV) pathway in S. cerevisiae, we detail procedures for extraction and detection of the key pathway metabolites MEV, squalene and ergosterol, as well as the farnesyl pyrophosphate (FPP)-derived side products farnesol and nerolidol. Analysis of these compounds is important for quality control, because they are possible indicators of pathway imbalance. As many of the sesquiterpene synthase (STS) genes encountered in nature are of plant origin and often not optimal for expression in yeast, we provide guidelines for designing gene expression cassettes to enable expression in S. cerevisiae. As a case study for these protocols, we have selected the sesquiterpene amorphadiene, native to Artemisia annua and related plants. The analytical steps can be completed within 1–2 working days, and a typical experiment might take 1 week.

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Figure 1: Key metabolites of the S. cerevisiae mevalonate pathway and major farnesyl-pyrophosphate (farnesyl-PP) side products.
Figure 2: Overview of processes involved in engineering S. cerevisiae for sesquiterpene production.
Figure 3: GC-MS chromatograms (representing ions 189 and 204) relevant to quantification of the sesquiterpene amorphadiene and FPP-derived products.
Figure 4: GC-MS chromatograms (representing ions 58, 71, 189 and 204) relevant to quantification of mevalonolactone and the internal standard, caryophyllene.
Figure 5: GC-MS chromatograms (representing ions 218, 386 and 396) relevant to quantification of intracellular ergosterol and squalene.

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Acknowledgements

This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the US Department of Energy under contract no. DE-AC02-05CH11231.

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Authors and Affiliations

  1. Joint BioEnergy Institute, Emeryville, California, USA

    Sarah Rodriguez, James Kirby, Charles M Denby & Jay D Keasling

  2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Sarah Rodriguez & Jay D Keasling

  3. California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, California, USA

    James Kirby, Charles M Denby & Jay D Keasling

  4. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA

    Jay D Keasling

  5. Department of Bioengineering, University of California, Berkeley, California, USA.

    Jay D Keasling

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S.R. conducted the experiments; S.R., J.K., C.M.D. and J.D.K. wrote the manuscript.

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Correspondence to Jay D Keasling.

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J.D.K. has a financial interest in Amyris and Lygos. The remaining authors declare no competing financial interests.

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Rodriguez, S., Kirby, J., Denby, C. et al. Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites. Nat Protoc 9, 1980–1996 (2014). https://doi.org/10.1038/nprot.2014.132

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