The biosynthesis of sesquiterpene lactones in chicory (Cichorium intybus L.) roots

Wild chicory(Cichorium intybusL.) is a blue-flowered composite plant that has spread all over the world from the Mediterranean. Sprouts of chicory var.foliosumHegi that are grown in the dark became popular as a vegetable (Belgian endive) halfway through the nineteenth century. Nowadays it is a common crop in Belgium, northern France, and the Netherlands. The well-known bitter taste of chicory is associated with the presence of sesquiterpene lactones of which the three major ones are the guaianolides lactucin, 8-deoxylactucin, and lactucopicrin (Fig. 1). Additionally, smaller amounts of eudesmanolides and germacranolides are present. The average sesquiterpene lactone content of the wild variety(sylvestre) is 0.42% dry weight in the roots and 0.26% in the leaves. The sesquiterpene lactones in chicory act as feeding deterrent toward insects, but may have an antifungal and antibacterial function as well(Chapter1). Sesquiterpene lactones are considered as a major class of plant secondary products, which mainly occur in the Asteraceae. Over 4000 different structures are known, but the majority of them has a guaiane, eudesmane, or germacrene framework (i.e. guaianolides, eudesmanolides, germacranolides). Sesquiterpene lactones with such a framework arethought to originate from (+)-costunolide, the most elementary structure of a germacrene sesquiterpene lactone. In this thesis a pathway for the biosynthesis of (+)-costunolide in chicory roots has been established (Fig. 2). Figure2. Established pathway for the biosynthesis of(+)-costunolide from farnesyl diphosphate (FPP) in chicory.The committed step in the biosynthesis of (+)-costunolide is the cyclisation of farnesyl diphosphate (FPP) into (+)-germacrene A(Chapter2). The involved (+)-germacrene A synthase was isolated from chicory roots and purified 200-fold by a combination of anion exchange and dye-ligand chromatography. The isolated enzyme belongs to the group of sesquiterpene synthases, has aKm-value of 6.6_M, an estimated molecular weight of 54 kD, and a (broad) pH optimum around 6.7. The recent isolation of genes encoding the (+)-germacrene A synthase of chicory makes it possibly to block this crucial step in sesquiterpene lactone biosynthesis, which may result in new less bitter tasting varieties of Belgian endive. Formation of the lactone ring involves the introduction of a carboxylic acid function in the isopropenyl group of (+)-germacrene A(Chapter3). It starts with the hydroxylation of (+)-germacrene A to germacra-1(10),4,11(13)-trien-12-ol by the (+)-germacrene A hydroxylase. This cytochrome P450 enzyme is NADPH-dependent, has a pH optimum at 8.0, and is blue-light reversibly inhibited by CO. Germacra-1(10),4,11(13)-trien-12-ol is subsequently oxidised to germacra-1(10),4,11(13)-trien-12-oic acid via germacra-1( 10),4,11(13)-trien-12-al by pyridine nucleotide dependent dehydrogenases. Some questions about the exact cofactor dependence of the dehydrogenase catalysed reactions remain, but on the whole the best results were obtained with NADP+. Conversion of germacra-1(10),4,11(13)-trien-12-oic acid into (+)-costunolide is catalysed by the (+)-costunolide synthase(Chapter5). This enzyme is also a cytochrome P450 enzyme, since it depends upon NADPH and is blue-light reversibly inhibited by CO. Biosynthesis of (+)-costunolide in the presence of18 O2resulted in the incorporation of one atom of18O. This supports the concept that the lactone ring is formed via a hydroxylation at theC6-position of the germacrene acid, after which the hydroxyl group attacks the carboxyl group atC12. It is not clear whether the final lactonisation is also mediated by the (+)-costunolide synthase or occurs spontaneously (outside the enzyme). (+)-Costunolide is converted into 11(S),13-dihydrocostunolide and leucodin by an enzyme extract from chicory roots in the presence of NADPH andO2(Fig. 3). It is to be expected that other sesquiterpene lactones are formed as well in these incubations, but it is unlikely thatthey can be detected by the GC-MS method which was used to analyse the enzyme assays. The formation of 11(S),13-dihydrocostunolide is catalysed by a stereoselective enoate reductase and does also occur in the absence ofO2. The formation of leucodin involves a cytochrome P450 enzyme and presumably also a dehydrogenase, but it is unclear how cyclisation into the guaiane framework takes place. 11(S),13-Dihydrocostunolide is a reasonable intermediate in the biosynthesis of all 11,13-dihydro-sesquiterpene lactones present in chicory, but its involvement in leucodin biosynthesis was not investigated. Notably, leucodin is only one hydroxylation step away from 11(S),13-dihydro-8- deoxylactucin, a minor bitter sesquiterpene lactone of chicory. The germacrene intermediates of sesquiterpene lactone biosynthesis can be isolated from fresh roots ofSaussurea lappa(costus roots)(Chapter4). The occurrence of these germacrene intermediates along with high amounts of (+)-costunolide and dehydrocostus lactone within one and the same plant is an additional proof for the established pathway depicted in Figure 2. The germacrene compounds are susceptible to proton-induced cyclisations and to heat induced Cope rearrangement yielding eudesmanes and elemenes respectively. However, the isolated germacrenes are not that unstable as often suggested by literature. They remain for instance intact during the enzyme incubations at 30°C. The best way to analyse the oxygenated germacrenes is to let them undergo a Cope rearrangement to their corresponding elemenes immediately at the start of the GC-run. This can be achieved by the use of injection port temperatures of at least 250°C; if this is not done they will generally yield very broad peaks(Chapter4). Cope rearrangement into (-)-_-elemene was used to determine the absolute configuration of the enzymatically produced germacrene A on an enantioselective GC-column(Chapter2). In the presence of NADPH, a microsomal pellet from chicory roots is able to hydroxylate various sesquiterpene olefins, which are exogenous to the plant(Chapter6). Most of these hydroxylations take place at the allylic position of an isopropenyl or isopropylidene group (Fig. 4). The number of products obtained from a certain substrate is confined to one or in, a few cases, two sesquiterpene alcohols. Although the microsomal pellet contains various membrane bound enzymes, the majority of hydroxylations is ascribed to the (+)-germacrene A synthase since (+)-germacrene A competitively inhibits their biotransformation. This disputes the common idea that cytochrome P450 enzymes of plant secondary metabolism have a narrow substratespecificity. The unforeseen hydroxylation of (+)-valencene into_-nootkatol is presumably catalysed by a different cytochrome P450 enzyme, possibly the same that is involved in biosynthesis of leucodin from (+)-costunolide. During incubation_-nootkatol is rapidly oxidised further by NAD(P)+-dependent dehydrogenases into nootkatone, a much sought-after component with a distinctive flavour of grapefruit.The achieved regioselective, and in the case of_-nootkatol also stereoselective, introduction of a hydroxyl group into sesquiterpene olefins is often difficult to achieve by organic chemical methods. Nonetheless, the small quantities of oxygenated products obtained are a major drawback in the application of the isolated oxidising enzymes from chicory roots. It would be worthwhile to isolate the genes encoding the involved cytochrome P450 enzymes and to functionally overexpress them in yeast. In this way higher enzymatic activities and larger amounts of possibly interesting products may be expected(Chapter7).</font

Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a ( )-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. ( )-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP -dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The ( )-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylatesβ-elemene with a modest degree of enantioselectivity