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Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (–)-β-pinene and γ-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2+ as bivalent metal ion cofactor resulted in higher activity than Mg2+, with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 µm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.
Lemon, Citrus limon (L.) Burm. f., is a member of the large Rutaceae family containing 130 genera in seven subfamilies, with many important fruit and essential oil producers. Lemon essential oil has the highest import value of all essential oils imported to the USA and is widely used as flavouring agent in bakery, as fragrance in perfumery and also for pharmaceutical applications . The essential oil is produced from the peel or flavedo of the fruit. This layer consists of the epidermis covering the exocarp consisting of irregular parenchymatous cells, which are completely enclosing numerous glands or oil sacs. Below this green layer in maturing fruits is the albedo layer (mesocarp), a thick spongy white mass of tissue, rich in pectins, surrounding the fleshy, juicy interior of the fruit. Aldehydes, such as citral are minor components present in the C. limon essential oil. However, they contribute more to the characteristic flavour than the bulk components which are the olefinic monoterpenes . Monoterpenes are the C10 branch of the terpene family and consist of two head to tail coupled isoprene units (C5). They are beneficial for plants as they function in the defence against herbivores and plant pathogens or as attractants for pollinators. Sites for biogenesis of monoterpenes have been investigated extensively. In gymnosperms, such as grand fir, terpenes are produced in resin ducts [2,3]. Their biosynthesis is induced upon wounding [4–6], indicating their role in the defence against bark beetle infestation. For angiosperms many investigations have been carried out on Labiatae, especially on Mentha species, where monoterpenes are formed in the glandular trichomes, and on the umbelliferous caraway, where monoterpenes are produced in essential oil ducts of the fruits [7–12]. In Citrus, the specialized structures for the storage and accumulation of large amounts of terpenes are the glands in the flavedo, the so-called secretory cavities. Research on lemon showed that these cavities develop schizogenously on most aerial plant parts [3,13]. The cells lining these secretory cavities are thought to be responsible for the production of the terpenoids . In cold pressed lemon peel oil from different origins, around 61% of the total monoterpene content consists of limonene together with lower levels of β-pinene (17%) and γ-terpinene (9%) . Recently, the enantiomeric composition of some of the chiral terpene olefins present in the lemon oil was determined using a multidimensional tandem GC-MS system (MDGC-MS) . The main chiral components of the cold pressed lemon oil were 4R-(+)-limonene with 96.6% enantiomeric excess (e.e.), and (–)-(1S,5S)-β-pinene with 88% e.e. .
The main monoterpenes of lemon can be obtained by heterologous expression of enzymes from several plant species that were isolated using a number of different strategies. cDNAs encoding (–)-limonene synthase were previously isolated from several Mentha species, Abies grandis and Perilla frutescens, using a PCR based approach, with sequence information obtained by protein sequencing of the purified enzyme , or by using the first cloned Mentha spicata cDNA as a probe . For A. grandis homology-based cloning, degenerate PCR primers based on conserved domains of a number of terpene synthase genes were used . So far only one cDNA encoding a (+)-limonene synthase has been isolated from Schizonepeta tenuifolia, a member of the Labiatae family .
(–)-(1S,5S)-β-Pinene was the major product of a β-pinene synthase cDNA from Artemisia annua submitted to GenBank (accession no. AF276072), and of a (–)-(1S,5S)-pinene synthase that was previously isolated from A. grandis. This enzyme produces 58% (–)-(1S,5S)-β-pinene, but also 42% (–)-(1S,5S)-α-pinene. A cDNA encoding γ-terpinene synthase as its main activity has not been reported on yet.
Although the composition of lemon essential oil has had considerable attention and enzymes responsible for the production of monoterpenes in the peel of lemon have been partially purified , their corresponding cDNAs have never been isolated and characterized. So far only the cDNA of a sesquiterpene synthase producing (E)-β-farnesene as main product has been described from Citrus junos. Here we report on the isolation of four new monoterpene synthase cDNAs by random sequencing of a flavedo-derived cDNA library of C. limon and their characterization by functional expression in Escherichia coli.
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- Materials and methods
The four monoterpene synthase cDNAs that have been isolated and characterized here account for the formation of more than 90% of the content of lemon essential oil. Most of the monoterpenoids that were found in the young lemon peel are either main or side products of the monoterpene synthases isolated and characterized in the present paper. Only the origin of the trace amounts of linalool, α-terpineol and (E)-β-ocimene that are also present in the lemon extract remain unexplained, as they are not a product of any of the synthases presented in this paper.
To isolate these monoterpene synthases from lemon, we used a random sequencing approach on a cDNA library from young lemon flavedo. This method has previously been proven to be successful for the isolation of full length cDNAs, particularly if the source tissue of the library is highly specialized with regard to the process to be studied [39–41]. The levels of identity of the lemon monoterpene synthases indicate that they should be grouped within the tpsb clade of the angiosperm monoterpene synthases (Fig. 1, and Table 1) . Although the four lemon cDNAs cluster in the same clade, they clearly form two distinct classes, one containing B93 and D85 and the other C62 and M34, because there are large differences both in the putative plastid targeting signals (only 16–18% identity) and the coding sequences (only 48–51% identity), suggesting that they have evolved separately.
This is confirmed by the phylogenetic analysis (Fig. 2). The separate clustering of the lemon genes B93, D85, Q. ilex myrcene synthase and the A. annua monoterpene synthases from the limonene synthases C62 and M34, suggests that the two groups of lemon synthases diverged in ancient times, even before Quercus and Artemisia separated from Citrus.
Monoterpene biosynthesis has been shown to be localized in the plastids in plants [9,42], and this is in accordance with the fact that all monoterpene synthases published to date bear an N-terminal transit peptide [10,15–17,28,33,35, 36,43,44]. Monoterpene synthases are nuclear encoded preproteins that are destined to be imported in the plastids, where they are proteolytically processed into their mature forms. Plastid targeting signals are typically rich in serines and threonines and low in acidic and basic amino acids and about 45–70 amino acids long. Usually they show only little homology.
The predictions using mnastarr and mnastarr indicate that all the four putative monoterpene synthases contain plastid targeting sequences. The lengths of the predicted targeting signals are rather short but the distance to the RRX8W motif, common to monoterpene synthases of the tpsb clade, from where significant homology starts with other monoterpene synthases is 52 or 55 amino acids long. The RRX8W motif is supposed to be required to give a functional mature protein and could have a function in the diphosphate migration step accompanying formation of the intermediate linalyl diphosphate before the final cyclization step catalysed by the monoterpene synthases . The DDXXD motif, present in all terpene synthases, is supposed to bind the bivalent metal ion cofactor, usually Mn2+ or Mg2+ and is responsible for the ionization of the diphosphate group of geranyl diphosphate [34,45,46]. The active site domain of sesquiterpene synthases and probably also other terpene synthases is located on the C-terminal part of these proteins starting shortly before the DDXXD motif . Therefore it was suggested that the C-terminal part of the terpene synthase proteins determines the final specific product outcome . Less than 10% overall sequence divergence has been shown to result in a significantly different product composition . Table 1 shows that the identity level before the DDXXD motif between the B93 and D85 proteins (ClγTS and Cl(–)βPINS) is higher (89%) than after the DDXXD motif (78%), suggesting that these two enzymes, although they are very homologous, are likely to catalyse the formation of two different products.
For the other two homologous protein sequences encoded by C62 and M34 (Cl(+)LIMS1 and Cl(+)LIMS2), the identity before the DDXXD motif was almost the same as from the DDXXD motif onwards. This makes it likely that these proteins catalyse the formation of identical products.
The characterization of product specificity by functional expression in E. coli of the monoterpene synthases of lemon confirmed that both C62 and M34 (Cl(+)LIMS1 and Cl(+)LIMS2) encode enzymes that specifically form a single product (+)-limonene, with only small traces of myrcene and (+)-α-pinene. Myrcene and α-pinene are trace products that were also described for (–)-limonene synthase from spearmint, but with undetermined stereochemistry . Although both limonene synthase enzymes produce exclusively (+)-limonene as a main product, the stereoselectivity for the trace coproduct α-pinene is less strong.
The other two monoterpene synthases encoded by B93 and D85, which show less sequence identity, indeed produce different main products, γ-terpinene and (–)-β-pinene, respectively. Furthermore these are much less specific in their product formation, leading to formation of a number of side products (up to 11% of total). It is a common feature of many monoterpene synthases that they are able to form multiple products from geranyl diphosphate as was shown by functional expression of synthases from several species such as spearmint, sage and grand fir [10,16,35,43]. The (–)-β-pinene synthase produces almost exclusively the (–)-enantiomer, and its side products show a similar enantiomeric composition, but with less stereoselectivity than the main product.
Considering the high sequence homology of the γ-terpinene synthase, producing an achiral product, to the (–)-β-pinene synthase, it would be expected that all side products would give similar enantiomers. However, the data show that although the most prevalent side products above 5% have an e.e. for the (–)-enantiomer, there is also a side product with an e.e. of the opposite enantiomer [(+)-β-pinene]. Furthermore, the stereoselectivity for most of the side products is even weaker than for the other lemon clones. Remarkably, the (+)-enantiomer of the β-pinene side product is formed in very high e.e. (96%). Other monoterpene synthases have been described that have low stereoselectivity for some of their side products, such as 1,8-cineole synthase and bornyl diphosphate synthase from common sage. The 1,8-cineole synthase produces for most side products an e.e. of the (+)-enantiomers, but for β-pinene an e.e. of the (–)-enantiomer . As an explanation, Croteau and coworkers suggested that the E. coli host could proteolytically process the enzyme to a form that could compromise substrate and intermediate binding conformations.
In an investigation where monoterpene synthase activity from lemon was partially purified, the preference for Mn2+ as a cofactor instead of Mg2+ was reported . The heterologously expressed enzymes from lemon show the same cofactor preference.
Lemon monoterpene synthases apparently do not prefer Mg2+ as the other cloned angiosperm synthases, but Mn2+ like the gymnosperm synthases . These latter enzymes also require a monovalent ion, preferably K+ for activity [34,37], while the lemon enzymes are inhibited by potassium ions. The pH optimum of the lemon synthases is close to pH 7 like other angiosperm synthases, while the gymnosperm synthases show a pH optimum that is generally higher, such as pH 7.8 for the grand fir and lodgepole pine synthases [34,37,48].
The enzyme activity curves show that the activity decreases dramatically when the substrate concentration increases above 10–50 µmnastarr as shown for Cl(–)βPINS (Fig. 5). This cannot be caused by product inhibition as the products of the synthases will migrate to the hexane phase used in the assays and are therefore not expected to be interfering with the enzyme. The enzymes show substrate inhibition characteristics, a feature not previously reported for other cloned monoterpene synthases. The observation that the partially purified native monoterpene synthase enzyme fraction from lemon flavedo also showed substrate inhibition at higher substrate concentrations than five times the Km rules out the possibility that this phenomenon is the consequence of changes to the protein due to cloning artefacts . An explanation could be that at higher concentrations, the allylic diphosphates start forming enzymatically inactive 2 : 1 complexes with metal ions, bound to the enzyme. Recent crystallographic work has shown that both epi-aristolochene and trichodiene synthase contain three Mg2+ ions in their active site, two of which are chelated by the DDXXD motif of the active site and a third which is liganded by a triad of active site residues [47,49].
The Km values determined for the monoterpene synthases from C. limon as determined by Michaelis–Menten kinetics are in a similar range as the values for other monoterpene synthases cloned thus far. The limonene synthases have a lower Km value than the β-pinene and the γ-terpinene synthase. Although no data are available about relative expression ratios of the four genes, the difference in Km may explain in part why the level of limonene compared to the other main products in the lemon peel is so much higher.
This report describes the first cloned monoterpene synthase that forms γ-terpinene as a major product. A homodimeric γ-terpinene synthase enzyme, purified from T. vulgaris produced in addition to the main product also small amounts of α-thujene and lesser quantities of myrcene, α-terpinene, limonene, linalool, terpinen-4-ol, and α-terpineol . However the gene encoding this enzyme has so far not been isolated. In addition this is the first report on a (–)-β-pinene synthase cDNA.
Limonene is widely used in beverages and the cosmetics industry, and (+)-limonene also has anticarcinogenic properties . The previously isolated (+)-limonene synthase from S. tenuifolia produces, apart from (+)-limonene, also a substantial amount of a nonidentified monoterpene side product . The lemon cDNA encoding (+)-limonene synthase however, produces more than 99% pure and exclusively (+)-limonene. Such a pure compound synthesized by a heterologously expressed enzyme could perhaps be a more natural alternative than chemical synthesis and possibly a cheaper alternative than purification from plants.