title: Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites.
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.
authors: 1] Joint BioEnergy Institute, Emeryville, California, USA. [2] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors: Sarah
authors: S
authors: Rodriguez
authors: 1] Joint BioEnergy Institute, Emeryville, California, USA. [2] California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, California, USA.
authors: James
authors: J
authors: Kirby
authors: 1] Joint BioEnergy Institute, Emeryville, California, USA. [2] California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, California, USA.
authors: Charles M
authors: CM
authors: Denby
authors: 1] Joint BioEnergy Institute, Emeryville, California, USA. [2] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. [3] California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, California, USA. [4] Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA. [5] Department of Bioengineering, University of California, Berkeley, California, USA.
authors: Jay D
authors: JD
authors: Keasling
mesh_terms: D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D000081223:Polycyclic Sesquiterpenes; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene