Citrus fruit flavor and aroma biosynthesis: Isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene

Institute of Horticulture, The Volcani Center ARO, PO Box 6, Bet-Dagan 50250, Israel.
The Plant Journal (Impact Factor: 5.97). 01/2004; 36(5):664-74. DOI: 10.1046/j.1365-313X.2003.01910.x
Source: PubMed


Citrus fruits possess unique aromas rarely found in other fruit species. While fruit flavor is composed of complex combinations of soluble and volatile compounds, several low-abundance sesquiterpenes, such as valencene, nootkatone, alpha-sinensal, and beta-sinensal, stand out in citrus as important flavor and aroma compounds. The profile of terpenoid volatiles in various citrus species and their importance as aroma compounds have been studied in detail, but much is still lacking in our understanding of the physiological, biochemical, and genetic regulation of their production. Here, we report on the isolation, functional expression, and developmental regulation of Cstps1, a sesquiterpene synthase-encoding gene, involved in citrus aroma formation. The recombinant enzyme encoded by Cstps1 was shown to convert farnesyl diphosphate to a single sesquiterpene product identified as valencene by gas chromatography-mass spectrometry (GC-MS). Phylogenetic analysis of plant terpene synthase genes localized Cstps1 to the group of angiosperm sesquiterpene synthases. Within this group, Cstps1 belongs to a subgroup of citrus sesquiterpene synthases. Cstps1 was found to be developmentally regulated: transcript was found to accumulate only towards fruit maturation, corresponding well with the timing of valencene accumulation in fruit. Although citrus fruits are non-climacteric, valencene accumulation and Cstps1 expression were found to be responsive to ethylene, providing further evidence for the role of ethylene in the final stages of citrus fruit ripening. Isolation of the gene encoding valencene synthase provides a tool for an in-depth study of the regulation of aroma compound biosynthesis in citrus and for metabolic engineering for fruit flavor characteristics.


Available from: Ahuva Frydman, Jan 20, 2015
    • "Sesquiterpene concentration varies for different species of the Citrus genus depending on the time of harvest of the fruit and its variety (Elston et al., 2005; Espina et al., 2010). The highest (þ)-valencene concentration (6 g L À1 ) has been reported for Citrus sinensis (Sharon-Asa et al., 2003). Essential oil contains a large number of sesquiterpene compounds and can be potentially used as a broad spectrum green pesticide (Koul, Walia, & Dhaliwal, 2008). "
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    ABSTRACT: The production of (+)-nootkatone, highly appreciated by fragrance and flavour industries, can be performed by whole-cell bioconversion from the sesquiterpene (+)-valencene, a compound readily available in orange essential oil. The aim of this work was to screen for microorganisms that convert (+)-valencene to (+)-nootkatone using different bioconversion systems. The screening was conducted using six different microorganisms, and bioconversion experiments were set up on surface culture using serological flasks containing PDA at 30 °C. It was observed that Botryodiplodia theobromae 1368, Yarrowia lipolytica 2.2ab, and Phanerochaete chrysosporium oxidised (+)-valencene to (+)-nootkatone, reaching (+)-nootkatone concentrations of 231.7 ± 2.1, 216.9 ± 5.8 and 100.8 ± 2.6 mg L-1, respectively. Different bioconversion conditions were also testeddaqueous, organic, and biphasicdall resulting in similar (+)-nootkatone production. Both B. theobromae 1368 and Y. lipolytica 2.2ab showed substrate inhibition above 4.2 × 10-2 and 0.13 g of (+)-valencene (g of biomass)-1, respectively, in aqueous phase experiments. Furthermore, B. theobromae 1368 and Y. lipolytica 2.2ab showed product inhibition when concentrations reached above 17.02 and 34.78 mg of (+)-nootkatone (g of biomass)-1, respectively. The experimental method presented will be useful for ongoing studies on the selection and operation of the proper bioreactor at different bioconversion conditions.
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    • "To the present day there are only a few functional (+)-valencene synthases described in literature , e.g. CnTPS1 from C. sinensis, VvVal from Vitis vinifera and CnVS from Callitropsis nootkatensis (Beekwilder et al., 2014; Lucker et al., 2004; Sharon-Asa et al., 2003). Currently, (+)-valencene is routinely "
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    ABSTRACT: The sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. Biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. Corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturally pigmented as it synthesizes the rare fifty carbon atoms (C50) containing carotenoid decaprenoxanthin. Since the carotenoid pathway of this Gram-positive bacterium has previously been engineered for efficient production of several C50 and C40 carotenoids, its potential to produce a sesquiterpene was assessed. Growth of C. glutamicum was negatively affected by (+)-valencene, but overlaying n-dodecane as organic phase for extraction of (+)-valencene was shown to be biocompatible. Heterologous expression of the (+)-valencene synthase gene from the sweet orange Citrus sinensis was not sufficient to enable (+)-valencene production, likely because provision of farnesyl pyrophosphate (FPP) by endogenous prenyltransferases was too low. However, upon deletion of two endogenous prenyltransferase genes and heterologous expression of either FPP synthase gene ispA from E. coli or ERG20 from Saccharomyces cerevisae (+)-valence production by Citrus sinensis valencene synthase was observed. Employing the valencene synthase from Nootka cypress improved (+)-valencene titers fold to 2.41±0.26mg l(-1) (+)-valencene, which is equivalent to 251±26μgg(-1) cell dry weight (CDW). This is the first report on sesquiterpene overproduction by recombinant C. glutamicum.
    Journal of Biotechnology 06/2014; 191. DOI:10.1016/j.jbiotec.2014.05.032 · 2.87 Impact Factor
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    • "Further, the Germacrene D and valencene were the most abandon sesquiterpenes (Table 3). Minor compounds including valencene have stand out in citrus as important flavour and aroma compounds [33]. "
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    ABSTRACT: The present work investigates the effect of ripening stage on the chemical composition of essential oil extracted from peel of four citrus: bitter orange (Citrus aurantium), lemon (Citrus limon), orange maltaise (Citrus sinensis), and mandarin (Citrus reticulate) and on their antibacterial activity. Essential oils yields varied during ripening from 0.46 to 2.70%, where mandarin was found to be the richest. Forty volatile compounds were identified. Limonene (67.90-90.95%) and 1,8-cineole (tr-14.72%) were the most represented compounds in bitter orange oil while limonene (37.63-69.71%), β-pinene (0.63-31.49%), γ-terpinene (0.04-9.96%), and p-cymene (0.23-9.84%) were the highest ones in lemon. In the case of mandarin, the predominant compounds were limonene (51.81-69.00%), 1,8-cineole (0.01-26.43%), and γ-terpinene (2.53-14.06%). However, results showed that orange peel oil was dominated mainly by limonene (81.52-86.43%) during ripening. The results showed that ripening stage influenced significantly the antibacterial activity of the oils against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This knowledge could help establish the optimum harvest date ensuring the maximum essential oil, limonene, as well as antibacterial compounds yields of citrus.
    The Scientific World Journal 05/2012; 2012(4):528593. DOI:10.1100/2012/528593 · 1.73 Impact Factor
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