Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2015
Yeast 32 (2015)1. - ISSN 0749-503X - p. 159 - 171.
monoterpene biosynthesis - escherichia-coli - synthase - precursor
Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization
Dong, L. ; Miettinen, K. ; Verstappen, F.W.A. ; Voster, A. ; Jongsma, M.A. ; Memelink, J. ; Krol, S. van der; Bouwmeester, H.J. - \ 2013
Metabolic Engineering 20 (2013). - ISSN 1096-7176 - p. 198 - 211.
indole alkaloid pathway - gland secretory-cells - roseus hairy roots - catharanthus-roseus - monoterpene biosynthesis - isoprenoid biosynthesis - plant transformation - essential oils - grape juice - metabolism
Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.
Volatile science? Metabolic engineering of terpenoids in plants
Aharoni, A. ; Jongsma, M.A. ; Bouwmeester, H.J. - \ 2005
Trends in Plant Science 10 (2005)12. - ISSN 1360-1385 - p. 594 - 602.
transgenic arabidopsis plants - linalool synthase gene - isoprenoid biosynthesis - essential oil - monoterpene biosynthesis - diphosphate synthase - s-linalool - functional expression - menthofuran synthase - plastidial pathways
Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites
Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants
Aharoni, A. ; Giri, A.P. ; Deuerlein, S. ; Griepink, F.C. ; Kogel, W.J. de; Verstappen, F.W.A. ; Verhoeven, H.A. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2003
The Plant Cell 15 (2003)12. - ISSN 1040-4651 - p. 2866 - 2884.
sesquiterpene cyclase gene - linalool synthase gene - functional expression - monoterpene biosynthesis - isoprenoid biosynthesis - diphosphate synthase - cdna isolation - s-linalool - glandular trichomes - thaliana
Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants