Patent Application: US-48976204-A

Abstract:
the invention provides the use of enzymes derived from plants in biocatalysis . the regio - and stercoselective introduction of an oxygen group into an unactivated organic compound is still a largely unresolved challenge to organic chemistry . we have shown that enzymes of asteraceae species are capable of converting with high regio - and stereospecificity for example sesquiterpene olefins to commercially interesting products .

Description:
the ability of the germacrene a hydroxylase present in chicory roots to hydroxylate other substrates in addition to germacrene a was investigated . fresh roots of cultivatedchicory ( cichorium intybus l ., cv focus ) harvested during late summer were obtained from a grower in veenendaal , the netherlands . the chicory roots were cut into small pieces , frozen in liquid nitrogen , and stored at − 80 ° c . (+)- germacrene a was isolated from costus roots ( de kraker et al , 2001b ). alloisolongifolene , (−)- α - gurjunene , (+)- γ - gurjunene , and (+)- ledene were purchased from fluka . icn biomedicals furnished (−)- α - cubebene . the compounds (−)- limonene and (+)- limonene were purchased from merck and janssen , respectively . amorpha - 4 , 11 - diene wassynthesised by dr . b . j . m . jansen ( bouwmeester et al ., 1999a ). germacrone was isolated from the natural oil of geranium macrorrhizum and germacrene b was synthesised by dr . d . p . piet ( piet et al ., 1995 ). (−)- α - trans - bergamotene and (−)- β - elemene were a gift from prof . w . a könig ( hamburg university ). neointermedeol was synthesised by dr . r . p . w . kesselmans ( kesselmans , 1992 ). (+)- β - selinene was isolated from celery oil and was a gift from dr . t . a . van beek . substrates were dissolved at 10 mm concentrations in ethanol . alloisolongifolene alcohol was prepared from alloisolongifolene via its corresponding aldehyde by seo 2 , according to the protocol of umbreit and sharpless ( 1977 ). to a solution of 77 mg seo 2 and 68 mg salicylic acid in 30 ml ch 2 cl 2 , 100 mg alloisolongifolene was added . stirring at room temperature turned the reaction mixture yellow and a red solid precipitated in the first few hours . the reaction was monitored by gc - ms along with tlc and was stoppedby the addition of 60 ml demineralised water after 2 . 5 days . the reaction mixture was extracted with 45 ml ether , and the organic phase was subsequently washed with 30 ml brine . the red precipitate remained in the aqueous phase during extraction . after drying and evaporation , the organic phase yielded 214 mg of a crude solid that after flash chromatography on silica with pentane - ch 2 cl 2 ( 3 : 1 ) yielded 31 mg of alloisolongifolene aldehyde , a strong odorous compound ( cedar - wood like ). 1 h nmr ( 200 mhz , c 6 d 6 ) δ 0 . 71 ( s , 3h , me ) δ 0 . 90 ( s , 3h , me ), δ 0 . 93 - 2 . 04 ( m , 13h ), δ 5 . 42 ( s , 1h , ch 2 ═ c ), δ 5 . 73 ( s , 1h , ch 2 ═ c ), 3 9 . 35 ( s , 1h , ch ═ o ). 13 c nmr ( 50 mhz , c 6 d 6 ) δ15 . 0 ( q ), δ 19 . 6 ( q ), δ 20 . 5 ( t ), δ 22 . 2 ( t ), δ 32 . 3 ( t ), δ 36 . 3 ( t ), δ 38 . 7 ( t ), δ 45 . 1 ( t ), δ 46 . 1 ( s ), δ 47 . 1 ( s ), δ 47 . 7 ( s ), δ 50 . 8 ( d ), δ 133 . 5 ( t ), δ 157 . 0 ( s ), δ 193 . 9 ( d ). eims ( 70 ev ) m / z : 218 [ m ] + ( 41 ), 203 ( 48 ), 189 ( 32 ), 185 ( 30 ), 175 ( 42 ), 161 ( 68 ), 147 ( 53 ), 145 ( 37 ), 133 ( 35 ), 119 ( 46 ), 107 ( 42 ), 105 ( 80 ), 95 ( 33 ), 93 ( 46 ), 91 ( 100 ), 81 ( 37 ), 79 ( 65 ), 77 ( 64 ), 55 ( 43 ), 53 ( 34 ), 41 ( 74 ), 39 ( 45 ). fifteen milligrams of the aldehyde was added to a solution of 1 . 8 mg lialh 4 in 0 . 5 ml ether . the grey suspension was stirred for 17 . 5 hours at room temperature and for an additional half our after the careful addition of one spatula na 2 so 4 . 10h 2 o . one and a half millilitre of demineralised water was added to the mixture that was subsequently 3 times extracted with 1 ml of ether . the ether was passed through glasswool - plugged pasteur pipette filled with silica and a spatula tip of mgso 4 . the solvent was evaporated yielding 17 mg of alloisolongifolene alcohol . 1 h nmr ( 200 mhz , c 6 d 6 ) δ 0 . 77 ( s , 3h , me ), δ 0 . 91 ( s , 3h , me ), 0 . 96 - 1 . 63 ( m , 13h ), δ 3 . 96 ( m , 2h ), δ 4 . 95 ( d , 1h , j = 1 . 3 hz ) δ 5 . 30 ( dd , 1h , j = 3 . 26 hz , 1 . 55 hz ). 13 c nmr ( 50 mhz , c 6 d 6 ) δ 15 . 1 ( q ), δ 19 . 7 ( q ), δ 20 . 7 ( t ), δ 22 . 3 ( t ), δ 32 . 4 ( t ), 37 . 5 ( t ), 38 . 7 ( t ), 44 . 0 ( t ), δ 46 . 1 ( s ), δ 47 . 8 ( s ), 6 48 . 2 ( s ), δ 51 . 6 ( d ), δ 63 . 4 ( t ), δ 107 . 9 ( t ), δ 155 . 9 ( s ). eims ( 70 ev ) m / z : 220 [ m ] + ( 5 ), 189 ( 35 ), 187 ( 36 ), 163 ( 30 ), 161 ( 56 ), 160 ( 37 ), 159 ( 30 ), 147 ( 36 ), 145 ( 38 ), 133 ( 32 ), 131 ( 37 ), 119 ( 44 ), 107 ( 61 ), 105 ( 91 ), 95 ( 94 ), 93 ( 57 ), 91 ( 100 ), 81 ( 53 ), 79 ( 60 ), 77 ( 52 ), 67 ( 39 ), 55 ( 45 ), 41 ( 62 ). amorpha - 4 , 11 - diene - 12 - ol was prepared from 100 mg artemisinic acid . the acid was dissolved in 6 ml of dry ether and esterified by adding diazomethane and mixing for 1 hour . gc - ms analyses of the reaction mixture showed the methyl ester and another compound ( 3 : 1 ), probably the methyl ester that was not connected to a cyclopropyl group . after removal of ether , 8 . 6 mg of the mixture was dissolved in 20 ml of ether - tbf ( 1 : 1 ) and cooled at − 60 ° c . under a flow of dry nitrogen for 2 hours . the mixture was subsequently stirred for 1 hour at − 30 ° c . with an excess of lialh 4 , after which the reaction was stopped by the addition of na 2 so 4 . 10h 2 o . the temperature was raised to room temperature , and the reaction mixture was stirred an additional half hour . after drying with mgso 4 overnight , the mixture was filtered through a buichner funnel and the solvent removed . the desired alcohol was isolated and purified by preparative thin layer chromatography performed on silica gel plates , using a mixture of petroleum ether - ethyl acetate ( 3 : 1 ) as an eluent ( r f 0 . 7 ). amorpha - 4 , 11 - diene - 12 - ol was obtained in an overall yield of 14 % as a mixture with 4 - amorphene - 12 - ol ( 9 : 1 ). 1 h nmr ( 400 mhz , cdcl 3 ): δ 0 . 87 ( d , 3h , mel5 , j = 6 . 0 hz ), δ 1 . 50 ( br s , 3h , me 14 ), δ 1 . 0 - 2 . 2 ( m , 11h ), δ 2 . 47 ( m , 1h , h 6 ), δ 4 . 10 ( s , 2h , h 12 ), δ 4 . 83 ( br s , 1h , h 13 ′ ), δ 5 . 04 ( br s , 1h , h 13 ), δ 5 . 18 ( br s , 1h , h 4 ). eims ( 70 ev ) m / z : 220 [ m ] + ( 6 ), 202 ( 35 ), 189 ( 66 ), 187 ( 34 ), 145 ( 40 ), 132 ( 52 ), 131 ( 36 ), 121 ( 100 ), 119 ( 81 ), 105 ( 51 ), 93 ( 74 ), 91 ( 70 ), 81 ( 47 ), 79 ( 77 ), 77 ( 50 ), 55 ( 41 ), 41 ( 47 ). [ heading - 0062 ] extraction of the (+)- germacrene a hydroxylase from chicory roots . to prepare a crude enzyme extract that contains the cytochrome p450 enzymes including the (+)- germacrene a hydroxylase ( de kraker et al ., 2001a ), 25 grams of deep frozen (− 80 ° c .) cubes of chicory roots were mixed by means of a sorvall mixer together with 2 . 5 grams pvpp and 40 ml extraction buffer . during the whole procedure the plant material was kept on ice as much as possible . after 5 times several seconds of mixing , the slurry was transferred to the cheesecloth with an additional 10 ml of extraction buffer . the cheesecloth was squeezed out and the filtrate was centrifuged ( sigma 2k15 ) for 20 minutes at 20 , 000 g and 4 ° c . the supernatant was poured over a funnel filled with glass wool . the filtrate was divided over 8 ml centrifuge tubes and subsequently centrifuged ( centrikon t - 2070 , kontron instruments ) for 90 minutes at 150 , 000 g and 4 ° c . the resulting microsomal pellets ( about 6 ) were stored under argon at − 80 ° c . before incubation , 10 pellets were pottered in 30 ml of assay buffer consisting of 25 mm tris ph 7 . 5 ), 2 mm dtt , 1 mm ascorbic acid , 5 μm fad , 5 μm fmn and 10 % ( v / v ) glycerol . the enzyme suspension was divided in 1 ml aliquots and incubated with 5 μl substrate solution in the presence of a 1 mm nadph - regenerating system that consisted of 1 mm nadph , 5 mm glucose - 6 - phosphate , and 1 . 2 iu glucose - 6 - phosphate dehydrogenase ( all from sigma ). the initial concentration of substrate in each assay was 45 μm and all were done in duplicate . to the blank assays no nadph regenerating system was added , so that cytochrome p450 enzymes ( including the (+)- germacrene a hydroxylase ) were not active . after 60 minutes the incubations were stopped by storing them at − 20 ° c . in a freezer . the enzyme assays were extracted 2 times with 1 ml 20 % ( v / v ) ether in pentane , after the addition of 5 μm cis - nerolidol ( fluka ). the organic phase was filtered through a glasswool - plugged ( dimethyl chlorosilane - treated , chrompack ) pasteur pipette that contained 0 . 4 gram of silica and a little anhydrous mgso 4 . the pipette was rinsed with 1 . 5 ml ether and the extract was concentrated to approximately 50 μl under a stream of nitrogen . the concentrated extracts were analysed by gc - ms as described by de kraker et al . ( 2001a ). to investigate whether enzymatic hydroxylations of the substrates were catalysed by the (+)- germacrene a hydroxylase , standard incubations of the various substrates ( 50 μm were carried out in the presence of 50 μm (+)- germacrene a , or 50 μm (−)-( α )- cubebene to exclude any possible general negative effect of sesquiterpene olefins on enzyme activity . to the control incubations 5 μl of ethanol was added , instead of the ethanol solution of either (+)- germacrene a or (−)-( α )- cubebene . most of the tested substrates were hydroxylated ( see table 1 ) and their molecular mass was correspondingly raised with 16 amu . (−)-( α )- cubebene and (−)-( α )- gurjunene which do not contain an isopropenyl or isopropylidene side chain were not converted ( fig6 ). also germacrone was not accepted as a substrate . some conversion of (+)- limonene and (−)- limonene occurred , but in such small amounts that it was not further investigated . in table i the sesquiterpenes are depicted that were converted into sesquiterpene alcohols in the presence of nadph and the microsomal pellet that contains the (+)- germacrene a hydroxylase . the conversion rates of the various reactions are expressed relatively to the hydroxylation of β - selinene that is set at 100 % and corresponds to a β - costol peak - size of 6 . 3 × the internal standard ( 2 . 5 nmol cis - nerolidol in each assay ). approximately 30 % of the added 50 nmol β - selinene was hydroxylated . not all formed hydroxylation products could be identified , since reference samples or mass spectra of the expected sesquiterpene alcohols were not available . incubation of amorpha - 4 , 11 - diene yielded two alcohols . the major product was identified as amorpha - 4 , 11 - diene - 12 - ol that results from hydroxylation of the isopropenyl side chain . the structure of the earlier eluting alcohol is unknown ; though assuming an allylic hydroxylation that does not take place at a bridgehead atom , it is either amorpha - 4 , 11 - diene - 2 - ol or amorpha - 4 , 11 - diene - 14 - ol . eims ( 70 ev ) m / z : 220 m + ( 12 ), 189 ( 77 ), 162 ( 46 ), 147 ( 38 ), 121 ( 45 ), 119 ( 60 ), 107 ( 58 ), 105 ( 68 ), 95 ( 41 ) 93 ( 75 ), 91 ( 72 ), 81 ( 100 ), 79 ( 70 ), 77 ( 48 ), 55 ( 52 ), 43 ( 49 ), 41 ( 52 ). incubation of (−)- α - trans - bergamotene yielded ( e )- trans - bergamota - 2 , 12 - dien - 14 - ol , eims ( 70 ev ) m / z : 220 [ m ] + (& lt ; 1 ), 145 ( 10 ), 132 ( 28 ), 131 ( 10 ), 121 ( 11 ), 120 ( 14 ), 119 ( 75 ), 107 ( 29 ), 105 ( 22 ), 95 ( 10 ), 94 ( 15 ), 93 ( 100 ), 81 ( 14 ), 79 ( 33 ), 77 ( 31 ), 68 ( 17 ), 67 ( 11 ), 55 ( 29 ), 53 ( 11 ), 43 ( 36 ), 41 ( 28 ), 39 ( 14 ). shaking the pentane - ether extract of the incubation with mno 2 overnight yielded a compound that has the same retention time and mass spectrum as ( e )- trans - bergamota - 2 , 12 - dien - 14 - al , present in costus root oil ( maurer and grieder 1977 ). incubation of (+)- γ - gurjunene yielded an unknown sesquiterpene alcohol . eims ( 70 ev ) m / z : 220 [ m ] + ( 35 ), 189 ( 36 ), 187 ( 37 ), 161 ( 39 ), 147 ( 31 ), 146 ( 35 ), 145 ( 55 ), 133 ( 33 ), 131 ( 64 ), 121 ( 41 ), 119 ( 54 ), 117 ( 31 ), 105 ( 81 ), 95 ( 41 ), 93 ( 58 ), 91 ( 100 ), 81 ( 92 ), 79 ( 71 ), 77 ( 54 ), 67 ( 38 ), 55 ( 49 ), 41 ( 59 ). stirring the pentane - ether extract of the enzyme assay overnight with a spatula of mno 2 gave complete conversion into an aldehyde or ketone ([ m + ] 218 ). because this reagent is specific for α , β - unsaturated alcohols ( march , 1992 ) and the only other available allylic position are at a tertiary carbon - atom , the biochemical hydroxylation must have occurred in the isopropenyl side chain of (+)- γ - gurjunene yielding 1s , 4s , 7r , 10r )- 5 , 11 ( 13 )- guaiadiene - 12 - ol . (+)- germacrene a was , according to previous results , converted into germacra - 1 ( 10 ), 4 , 11 - trien - 12 - ol , but somewhat unexpected the subsequent oxidations ( approx . 15 % of the produced germacrene alcohol ) into germacra - 1 ( 10 ), 4 , 11 - trien - 12 - al and germacra - 1 ( 10 ), 4 , 11 - trien - 12 - oic were also clearly detected . this indicated that the microsomal pellet is not completely free of active dehydrogenases . apparently the amount of produced germacrene alcohol is sufficient to enable these subsequent reactions . moreover the dehydrogenases are active despite the used ph of 7 . 5 and the presence of the nadph - regenerating system that reduces any nadp + present . in the incubations with β - selinene and β - elemene only minute quantities of aldehyde and / or acid were measured . for this reason the conversion of β - selinene was set at 100 % and not that of (+)- germacrene a gc - ms analyses of the incubation of germacrene b yielded two products with an almost identical mass spectrum and retention time . kovats &# 39 ; index 1694 , eims ( 70 ev ) m / z : 220 [ m ] + (& lt ; 1 ), 202 ( 15 ), 187 ( 24 ), 159 ( 20 ), 147 ( 20 ), 145 ( 24 ), 134 ( 22 ), 133 ( 26 ), 131 ( 22 ), 123 ( 19 ), 121 ( 75 ), 120 ( 27 ), 119 ( 100 ), 109 ( 34 ), 108 ( 15 ), 107 ( 48 ), 106 ( 22 ), 105 ( 66 ), 95 ( 38 ), 94 ( 20 ), 93 ( 65 ), 92 ( 17 ), 91 ( 73 ), 81 ( 50 ), 79 ( 52 ), 77 ( 44 ), 71 ( 17 ), 69 ( 28 ), 68 ( 15 ), 67 ( 48 ), 65 ( 17 ), 57 ( 19 ), 55 ( 94 ), 53 ( 34 ), 43 ( 46 ), 41 ( 73 ), 39 ( 32 ). kovats &# 39 ; index 1700 , eims ( 70 ev ) m / z : 220 [ m ] + (& lt ; 1 ), 202 ( 15 ), 189 ( 15 ), 187 ( 21 ), 159 ( 20 ), 147 ( 22 ), 145 ( 23 ), 137 ( 17 ), 134 ( 21 ), 133 ( 28 ), 131 ( 26 ), 123 ( 22 ), 122 ( 15 ), 121 ( 100 ), 120 ( 28 ), 119 ( 91 ), 117 ( 15 ), 109 ( 36 ), 108 ( 18 ), 107 ( 52 ), 106 ( 23 ), 105 ( 77 ), 95 ( 45 ), 94 ( 24 ), 93 ( 86 ), 92 ( 21 ), 91 ( 80 ), 81 ( 61 ), 79 ( 66 ), 77 ( 53 ), 71 ( 21 ), 69 ( 32 ), 68 ( 18 ), 67 ( 55 ), 65 ( 21 ), 57 ( 32 ), 55 ( 69 ), 53 ( 43 ), 43 ( 62 ), 41 ( 91 ), 39 ( 42 ). presumably both methyl groups in the isopropenyl side chain of germacrene b are hydroxylated yielding almost identical products . hydroxylation at any other position is unlikely , because they would probably also have been observed for (+)- germacrene a the products are measured as their cope - rearrangement products and lowering the injection port temperature from 250 ° c . to 150 ° c . yielded faint broadened peaks due to on - column cope - rearrangement . incubation of (+)- ledene yielded an unknown sesquiterpene alcohol . eims ( 70 ev ) in / z : 220 [ m ] + ( 12 ), 187 ( 25 ), 159 ( 42 ), 151 ( 29 ), 147 ( 32 ), 145 ( 32 ), 133 ( 26 ), 131 ( 25 ), 121 ( 31 ), 119 ( 62 ), 107 ( 86 ), 105 ( 100 ), 95 ( 40 ), 93 ( 74 ), 91 ( 82 ), 81 ( 53 ), 79 ( 54 ), 77 ( 39 ), 55 ( 38 ), 43 ( 33 ), 41 ( 47 ). neointermedeol is presumably hydroxylated in the isopropenyl side chain like β - selinene , yielding 4β - h - eudesm - 11 ( 13 )- ene , 4 , 12 - diol . eims ( 70 ev ) m / z : 238 [ m ] + (& lt ; 1 ) 223 ( 46 ), 135 ( 76 ), 93 ( 47 ), 81 ( 39 ), 79 ( 47 ), 71 ( 47 ), 55 ( 39 ), 43 ( 100 ), 41 ( 40 ). the microsomal pellets that were used in the experiments do not exclusively contain the (+)- germacrene a hydroxylase , but also other membrane bound enzymes that are present in chicory roots . hence , some of the conversion described in table i might as well be catalysed by other oxidising enzymes than the (+)- germacrene a hydroxylase . to investigate this , incubations of the different substrates were done in the presence of 50 μm (+)- germacrene a . incubations were also carried out with 50 μm (−)- α - cubebene instead , a sesquiterpene that is not hydroxylated ( fig6 ), to test the effect of the addition of any arbitrary sesquiterpene olefin on enzyme activity ( see table ii ). the results presented in table ii show that all enzymatic hydroxylations were inhibited to 90 % by the addition of (+)- germacrene a , except for β - selinene and neointermedeol whose hydroxylation was inhibited by 60 and 68 %, respectively . the relatively small inhibition of β - costol formation agrees with the observation that it is nearly as well hydroxylated as (+)- germacrene a ( table i ); the structural correlation between β - selinene and neointermedeol possibly also explains the small effect of (+)- germacrene a upon the formation of 4β - h - eudesm - 11 ( 13 )- ene - 4 , 12 - diol . more surprisingly is that (+)- germacrene a also inhibitsthe formation of sesquiterpene alcohols in which no isopropenyl or isopropylidene side chain is involved , like ledene alcohol . hydroxylation of the sesquiterpenes is not dramatically influenced by the addition of (−)- α - cubebene , except for the formation of alloisolongifolene alcohol , a product that already under normal assay conditions is only formed in small quantities . before experiments as described in this example were started , it was tested which organic solventcould be best used to dissolve the substrate . stock solutions of 10 mm γ - gurjunene were prepared in hexane , pentane , iso - propanol ethanol and dmso , and 5 μl of these solutions was added to the incubation - mixture . on the basis of the results of table iii ethanol was chosen as solvent for the substrates in all experiments , instead of the commonly used pentane ( e . g . karp et al ., 1990 ). a microsomal enzyme preparation from chicory roots can hydroxylate sesquiterpene olefins that are not present in chicory in the presence of nadph ( table i ). most of these hydroxylations occur at the isopropenyl or isopropylidene side chain , yielding in some cases sesquiterpene alcohols that have not previously been described , e . g . amorpha - 4 , 11 - dien - 12 - ol and alloisolongifolene alcohol . the novelty of the formed sesquiterpene alcohols in some cases hampered their identification , and the structure assignment of the hydroxylation products of germacrene b , (+)- γ - guriunene and neointermedeol is tentative or , like for (+)- ledene , not possible at all . unfortunately , these compounds were not yet produced in sufficient amount to isolate them for structure elucidation by 1 h nmr . the substrates for hydroxylation preferably do not contain any polar group , according the observation that neointermedeol is 15 - fold less efficiently hydroxylated than β - selinene and the observation that germacrone is not hydroxylated whereas germacrene b is . the size of the substrate is also of importance as bydroxylation of limonene , a smaller monoterpene , did hardly occur . in the case of amorpha - 4 , 11 - diene two distinct sesquiterpene alcohol products were formed . hydroxylations occurring at isopropenyl side chains and — less efficiently — at isopropylidene side chains are catalysed by the (+)- germacrene a hydroxylase that is present in the microsomal pellet of chicory roots . accordingly , these hydroxylations could be competitively inhibited by (+)- germacrene a ( table ii ). hydroxylations of various non - natural substrates by a cytochrome p450 enzyme of plant secondary metabolism contradicts the common belief that enzymes of plant secondary metabolism have a narrow substrate specificity . ( donaldson and luster , 1991 ; halkier , 1996 ; schuler , 1996 ; mihaliak et al ., 1993 ; karp et al ., 1990 ). formation of the unknown ledene alcohol and the unknown amorpha - 4 , 11 - diene alcohol ( n 1720 ) cannot have occurred in an isopropenyl / isopropylidene side chain , nevertheless these reactions are also inhibited by (+)- germacrene a . although hard to understand , it suggests the involvement of the (+)- germacrene a hydroxylase in these reaction as well . notably , the rate of competitive inhibition by (+)- germacrene a of the formation of amorpha - 4 , 11 - diene - 12 - ol and the unknown amorpha - 4 , 11 - diene alcohol is the same . the hydroxylation of amorphadiene - 4 , 11 - diene in the isopropenyl side chain is interesting as well . it was hypothesised to be an important step in the formation of the anti - malarial drug artemisinin in artemisia annua but has not yet been reported for this plant ( bouwmeester et al ., 1999a ). generally , the production of new materials for the flavor and fragrance industry has been a powerful driving force in the research on the hydroxylation of terpenes by micro - organisms . although in some cases successful , these microbiological conversions often yield a broad spectrum of products , including epoxides and diols ( lamare and furstoss , 1990 ; drauz and waldmann , 1995 ; faber , 2000 ) and the oxidations often occur at double bonds . in contrast , the hydroxylations catalysed by the microsomal pellet of chicory yield one , sometimes two products , and occur more regiospecifically . illustrative is the case of (+)- γ - gurjunene : incubation with the plant pathogenic fungus glomerella cingulata yields ( 1s , 4s , 7r , 10r )- 5 - guaien - 11 , 13 - diol and ( 1s , 4s , 7r , 10s )- 5 - guaien - 10 , 11 , 13 - triol ( miyazawa et al ., 1998 ), whereas incubation with the microsomal pellet of chicory yielded ( 1s , 4s , 7r , 10r )- 5 , 11 ( 13 )- guaiadien - 12 - ol ( fig7 ). identification and characterisation of enzymes involved in the conversion of germacrene acid to sesquiterpene lactones (+)- costunolide is the most rudimentary structure of a germacranolide and considered to be the parent - compound of all germacrene - derived sesquiterpene lactones , among them the guaianolides , eudesmanolides and germacranolides of chicory . it is very likely that this compound is formed from fpp through (+)- germacrene a , germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - ol , and germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - oic acid ( 4 ) ( fig2 ) ( de kraker et al ., 1998 , 2001a ). this germacrane acid 4 is thought to be hydroxylated at the c 6 - position by a cytochrome p450 enzyme after which lactonisation yields (+)- costunolide ( 5 ) ( fig8 ) germacrene acid was isolated from fresh costus roots ( de kraker et al ., 2001b ), which makes it possible to investigate this last step in formation of the lactone ring of sesquiterpene lactones ( geissman , 1973 ; fischer et al ., 1979 ; fischer , 1990 ). (+)- costunolide is considered to be a branching point in the biosynthesis of sesquiterpene lactones from where pathways for the formation of guaianolides , eudlesmanolides and germ acranolides divides it has been postulated by various authors that cyclisation of (+)- costunolide to either guaanolides or eudesmanolides is mediated by respectively c 4 - c 5 epoxidation or c 1 - c 10 epoxidation , respectively ( brown et al ., 1975 ; fischer 1990 ; teisseire , 1994 ; piet et al ., 1995 ). alternatively , the necessity of a c 3 - hydroxylation of the germacrenolide for formation of a guaianolide has been proposed ( piet et al ., 1996 ). fresh roots of cultivated chicory ( cichorium intybus l ., cv focus ) were harvested during late summer and obtained from a grower ( j . de mik ) in veenendaal , the netherlands . the chicory roots were cut into small pieces , frozen in liquid nitrogen and stored at − 80 ° c . germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - oic acid ( 4 ), (+)- costunolide ( 5 ), * and dehydrocostus lactone ( fig9 , 18 ) were isolated from costus roots ( d ) e kraker et al ., 2001b ). synthesis of a mixture of α - and γ - costic acid is described by de kraker et al . ( 2001b ), whereas the synthesis of elema - 1 , 3 , 11 ( 13 )- trien - 12 - oic acid ( 9 ) is described in ( de kraker et al ., 2001a ). a sample of leucodin ( 22 ) was kindly provided both by prof . m . ando ( niigata university , japan ; ando et , al ., 1994 ) and dr . shi yong ryu ( korea research institute of chemical technology , yusung , taej on , korea ), who also provided its c 11 - epimer achillin ( ho et al ., 1998 ). 11 , 13 - dihydro - dehydrocostus lactone ( mokko lactone ) was a gift of prof . y . asakawa c ( okushima bunri university , japan ). parthenolide ( 17 ) was purchased from sigma , cis - nerolidol from fluka . ether ( diethyl ether ) and pentane were redistilled before use . a gc - standard of 11 ( s ), 13 - dihydro - costunolide ( 20 ) was prepared from 2 mg of (+)- costunolide ( 5 ) that was dissolved in 1 . 5 ml ethyl acetate and stirred with 1 . 5 mg of nabh 4 at 0 ° c ., a procedure described for the reduction of the c 11 - c 13 exocyclic double bond of various sesquiterpene lactones ( asakawa et al ., 1980 ; asakawa , 1982 ; seto et al ., 1988 ). after 45 minutes the reaction was stopped by the addition of 1 % hac and 2 ml extra ethyl acetate . the organic phase was filtered through a glass - wool plugged pasteur pipette that contained 0 . 45 g of silica and a little anhydrous mgso 4 . gc - ms analysis of the filtrate showed that half of the (+)- costunolide was converted into 11 ( s ), 13 - dihydro - costunolide , whereas no trace of its c 11 - epimer was detected . a cell free extract of chicory roots was prepared from the frozen material in the same way as described for isolation of the germacrene a hydroxylase ( example 1 ), but mgcl 2 was not added to the extraction buffer since the accompanying (+)- germacrene a synthase activity was not essential for the detection of (+)- costunolide synthase activity . the prepared 20 , 000 g supernatant was desalted with an econo - pac 10 dg column ( biorad ) to an assay buffer consisting of 25 mm tris ( ph 7 . 5 ), 1 mm ascorbic acid , 5 μm fad 5 μm fmn and 10 %( v / v ) glycerol . dtt was omitted from the assay buffer , because sh - groups present in dtt might undergo an “ michael - type addition ” to the c 11 - c 13 exocyclic double bond of (+)- costunolide kupchan et al ., 1970 ). a 1 - ml aliquot of the desalted supernatant was incubated with 3 μl of a 15 mm solution of germacrene acid ( 4 ) in tert - butylmethylether and a 1 mm nadph - regenerating system , which consists of 1 mm nadph , 5 mm glucose - 6 - phosphate , and 1 . 2 iu glucose - 6 - phosphate dehydrogenase ( all from sigma ). incubations were also done with boiled desalted supernatant and in the absence of nadph . the experiments were repeated with elema - 1 , 3 , 11 ( 13 )- trien - 12 - oic acid ( fig2 , 9 ) and a mixture of α - and γ - costic acid ( 14 ) that might serve as substrate analogues for the germacrene acid ( 4 ). after 1 h of incubation at 30 ° c ., the reactions were stored in the freezer . the incubations were extracted three times with 1 ml of 20 % ( v / v ) ether in pentane , after the addition of a 0 . 2 mm cis - nerolidol solution in ethanol that serves as internal standard . the organic phase was filtered through a glass - wool plugged ( dimethyl chlorosilane - treated glasswool ; chrompack ) pasteur pipette that contained 0 . 45 g of silica and a little anhydrous mgso 4 . the column was washed with 1 . 5 ml of ether and the extract was carefully concentrated to approximately 50 μl under a stream of nitrogen . samples of 2 μl were analysed by gc - ms using an injection port temperature of 320 ° c . to provoke cope rearrangement of (+)- costunolide ( 5 ). mass spectra were recorded at 70 ev scanning from 35 to 300 atomic mass units ; the gc oven temperature was programmed as described before ( de kraker et al ., 1998 ). to determine whether the formation of (+)- costunolide ( 5 ) from germacrene acid ( 4 ) was catalysed by a cytochrome p450 - enzyme , the effect of various established cytochrome p450 - inhibitors ( cytochrome c , metyrapone , clotrimazole , micozanole , and amino - benzotriazole ) on this reaction were tested , as well as the effect of co or an argon atmosphere . the cofactor dependence was also investigated , i . e . nad ( p ) + or nadh was added instead of nadph . experiments were carried out in a similar manner as described for the germacrene a hydroxylase in example i using a 20 , 000 g supernatant and 5 μl of 0 . 2 mm cis - nerolidol as internal standard . blue - light reversal of co - inhibition was investigated with gas - mixtures of 10 % o 2 plus 90 % n 2 ( blank ) and 10 % o 2 plus 90 % co . incorporation of oxygen was investigated with 18 o 2 ( 99 % pure ; icon services , mt . marlon , n . y ., usa ). one ml of incubation mixture , placed in a ( vented ) septum - capped 4 . 5 - ml vial , was first bubbled with nitrogen to remove air and next with oxygen - 18 . the mass spectra of the formed compounds were compared with those formed in the standard enzyme assays with air . incubations as described for the germacrene acid were also done with (+)- costunolide ( 30 μm ) to test whether any further enzymatic conversion of this compound would occur during the incubation of germacrene acid . parthenolide ( 20 μm ) ( fig9 , 17 ) was incubated as well since it might be an intermediate in the formation of guaianolides . dehydrocostus lactone ( 20 μm ) ( 18 ) was incubated as a model compound for reductions that occurred at the c 11 - c 13 exocyclic double bond of (+)- costunolide . the effect of established cytochrome p450 inhibitors and co on the conversion of (+)- costunolide was studied , just as the effect of various pyridine nucleotide cofactors and an argon atmosphere . gc - ms analyses of the pentane - ether extract from the incubation of a 20 , 000 g chicory root supernatant with germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - oic acid ( 4 ) in the presence of an nadph - regenerating system showed three products that were not detected in incubations without nadph or in incubations with boiled supernatant ( fig1 a + b ): the major peak co - eluted with a standard of (+)- costunolide ( 5 ) that at an injection - port temperature of 320 ° c . is detected as its cope - rearrangement product dehydrosaussurea lactone ( 10 ) ( fig3 c ). the substrate germacrene acid was also measured as it cope - rearrangement product ( s ), i . e . elemene acid ( 9 ) and diastereomeric elemene acid ( de kraker et al ., 2001b ). the two other products were identified as 11 ( s ), 13 - dihydrocostunolide ( 20 ) that is cope - rearranged into saussurea lactone ( 21 ), and leucodin ( 22 ) ( fig1 ). both 11 ( s ). 13 - dihydrocostunolide and leucodin are enzymatically formed from (+)- costunolide and accordingly they also appeared in incubations of (+)- costunolide with the 20 , 000 g supernatant and nadph . it cannot be excluded that even more products are formed during the incubation of germacrene acid , because higher oxygenated sesquiterpene lactones are likely not volatile enough for detection by gas chromatography . furthermore , the presence of fatty acids in chicory extracts ( sannai et al ., 1982 ) complicates the gc - analysis , because they yield big peaks (●) under which smaller product peaks might “ disappear ”. there was no detectable conversion of the substrate analogues α - and γ - costic acid ( 14 ) and elema - 1 , 3 , 11 ( 13 )- trien - 12 - oic acid ( 9 ). for characterisation of the (+)- costunolide synthase the response of the gc - ms to different concentrations of (+)- costunolide should preferably be linear . yet , at the used injection port temperature of 320 ° c ., the gc - trace of costunolide ( fig1 c ) does not show a sharp peak of dehydrosaussurea lactone ( 19 )— but a tailing peak that contains minor peaks of costunolide - related products like α - and β - cyclocostunolide . apparently (+)- costunolide is more resistant to cope - rearrangement than for instance germacrene acid ( 4 ). in literature it has also been noticed that a lactone ring has a strong influence on the thermal stability of germacrenes , and that cope - rearrangement of (+)- costunolide is reversible ( jain et al ., 1970 ; minnaard , 1997 ). therefore , on a gc - column dehydrosaussurea lactone ( 19 ) might even undergo to some extent the reverse reaction towards (+)- costunolide ( grieco and nishazwa , 1977 ). avoiding cope - rearrangement in the injection port by lowering the injection port temperature would nevertheless result in cope - rearrangement of (+)- costunolide during its migration through the column and yielded a broad hump ( similar to the one shown for germacrene aldehyde in example 1 ) instead of a clear (+)- costunolide peak . despite the poor quality of the dehydrosaussurea lactone gc - peak , it is linear with injected (+)- costunolide concentrations in the range of 5 μm to 50 μm . the response factor relative to cis - nerolidol is 0 . 15 , and concentrations below 5 μm are not measurable which means that (+)- costunolide concentrations below 0 . 25 μm in the incubation mixture , i . e . 0 . 25 nmol , are not detected . more of a problem for characterisation of the (+)- costunolide synthase is that almost certainly not all subsequent conversion products of costunolide are detected . hence , the given enzyme activities are a summation of the peaks of dehydrosaussurea lactone ( 19 ), saussurea lactone ( 21 ) and leucodin ( 22 ) and are consequently more an indication than an absolute value of costunolide synthase activity . quantitative measurement of the elemene acid peak ( substrate peak ) was not an option , because it is not linear with concentration and , more generally , acid peaks on gc are of poor quality . table iv shows that the costunolide synthase is dependent on oxygen and nadph , whereas nadh was much less effective as a reductant . this suggests the involvement of a cytochrome p450 enzyme , which was confirmed by the effect of various established cytochrome p450 inhibitors ( west , 1980 ; mihaliak et al ., 1993 ). in the presence of cytochrome c ( 100 μm ), no enzymatic products of germacrene acid ( 4 ) were measured ; miconazole ( 100 μm ) reduced the amount of measurable products with 71 %, aminobenzotriazole ( 100 μm ) with 44 %, metyrapone ( 1 mm ) with 78 %, and clotrimazole ( 100 μm ) with 97 %. the strongest proof for the involvement of a cytochrome p450 enzyme is blue - light reversible inhibition of enzyme activity by co ( west 1980 ; mihaliak et al ., 1993 ). the results depicted in fig1 show an inhibitory effect of co on the produced sum of (+)- costunolide , 11 ( s ), 13 - dihydrocostunolide and leucodin that could be reversed by blue light , to some extent . incubation of (+)- costunolide with a 20 , 000 g supernatant chicory root supernatant in the presence of nadph yielded 11 ( s ), 13 - dihydrocostunolide ( 20 ) and leucodin ( 22 ). no other products were detected upon gc - ms analyses of the incubation , but comparison of the decrease in peak height of dehydrosaussurea lactone with the intensity of the products peaks products strongly suggested the formation of other products that are not detected on gc . the enzyme activity that catalyses the reduction of the 11 ( s ), 13 - exocyclic double bond of costunolide was not capable of doing the same with dehydrocostus lactone ( 18 ). incubation of parthenblide ( 17 ) did not give leucodin ( 22 ), but conversion of parthenolide into other not - measurable sesquiterpene lactones cannot be excluded . parthenolide itself disintegrates upon gc - analyses in a number of peaks and hence no quantitative determination was done on the amount of parthenolide present in after incubation . to test which type ( s ) of enzymes might catalyse the formation of 11 ( s ), 13 - dihydrocostunolide ( 20 ) and leucodin ( 22 ) the pyridine nucleotide cofactors were varied ( table v ). formation of both compounds is dependent upon nadph , but a part of the (+)- costunolide reductase activity is retained in the absence of any cofactor . table vi shows that formation of leucodin from (+)- costunolide is dependent upon oxygen , whereas the formation of 11 , 13 - dihydrocostunolide is not . formation of leucodin is strongly inhibited by co which suggests the involvemenit of a cytochrome p450 enzyme . accordingly it is also inhibited by all of the tested cytochrome p450 inhibitors except amino - benzotriazole . incubation of germacrene acid ( 4 ) in the presence oxygen - 18 gave the incorporation of one atom of 18 o into (+)- costunolide ( 5 ). the molecular ion peak in the mass spectrum of dehydrosaussurea lactone ( 19 ) was raised from 232 to 234 amu ( fig1 ). similar changes were observed in the mass spectrum of saussurea lactone ( 21 ), the cope rearrangement product of 11 ( s ), 13 - dihydrocostunolide ( 20 ). its ion peak was shifted from 234 to 236 amu whereas the [ m - me ] + peak was shifted from 219 to 222 amu . bubbling of the enzyme assay with oxygen - 18 ( 99 %) had not any measurable negative effect on enzyme activity . the mass spectra of leucodin showed the incorporation of two atoms of 18 o and the mass of the ion peak was clearly raised with 4 units from 246 to 250 amu ( fig1 ). unfortunately , in the enzyme assay the gc - peak of leucodin is superpositioned on the tailing peak of linoleic acid ( fig1 a ) which contaminates the mass spectrum of leucodin particular in the lower mass range . the present results show that chicory contains an enzyme that converts germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - oic acid ( 4 ) into (+)- costunolide ( 5 ), yielding the lactone ring present in sesquiterpene lactones . this step is the final proof for the postulated pathway from fpp through (+)- germacrene a , germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - ol , and germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - oic acid ( 4 ) to the germacrene - derived sesquiterpene lactones ( geissman , 1973 ; fischer et al ., 1979 ; fischer , 1990 ). the (+)- costunolide synthase is a cytochrome p450 enzyme that is dependent upon nadph and o 2 and is accordingly inhibited by various established cytochrome p450 inhibitors ( west , 1980 ; mihaliak et al ., 1993 ). blue light reversible co - inhibition of enzyme activity could be demonstrated as well , although the results were somewhat disturbed by subsequent enzymatic conversions of (+)- costunolide . incubation in the presence of oxygen - 18 showed the incorporation of one atom of 18 o into (+)- costunolide , another typical feature of cytochrome p - 450 enzymes ( west , 1980 ; mihaliak et al ., 1993 ). it also confirms the mechanism in which germacrene acid is first hydroxylated at the c 6 - position ( fischer et al ., 1979 ) after which this hydroxyl group attacks , presumably enzyme - mediated , the carboxyl group at c 12 . during the resulting lactonisation it so happens that the oxygen isotope is incorporated in the lactone ring ( fig1 ). the (+)- costunolide synthase is not capable of converting the substrate analogues α - costic acid ( fig2 , 14 ), or elema - 1 , 3 , 11 ( 13 )- trien - 12 - oic acid ( 9 ) which is not unexpected , because the c 6 - position is not allylic . however , γ - costic acid ( 13 ), in which the c 6 - position is allylic , was not converted either , so apparently the geometry of the cyclodecadiene ring system is necessary for reaction . (+)- costunolide ( 5 ) is in incubations with nadph and the 20 , 000 g supernatant of chicory roots subsequently converted into 11 ( s ), 13 - dihydrocostunolide ( 20 ) and leucodin ( 22 ) ( fig1 ). formation of 11 ( s ), 13 - dihydrocostunolide is not dependent upon oxygen , but is strongly enhanced in the presence of nadph whereas a small enzyme activity of 15 % is retained in its absence . the reduction of the c 11 - c 13 exocyclic double bond issimilar to the type of reactions catalysed by enoate reductases , a group of iron - sulphur flavoproteins that has been isolated from clostridium and catalyses the reduction of the olefinic bond of α , β - unsaturated carboxylic acids under anaerobic conditions in the presence of reducing agents ( holland , 1992 ). the reduction of the c 11 - c 13 exocyclic double bond in costunolide occurs stereoselectively and yields only the 11 ( s ), 13 - stereoisomer is formed . the stereochemistry at c11 is identical to that of the 11 ( s ), 13 - dihydro sesquiterpene lactones present in chicory ( seto et al ., 1988 ; van beek et al ., 1990 ). in contrast to chicory , some other plant species contain c 11 - epimers like achillin which is the 11 ( r )- epimer of leucodin ( martinez et al ., 1988 ; ho et al ., 1998 ), indicating that an enantioselective enzyme that synthesises these c 11 - epimers should exist in these other species . the enzyme exhibits at least some substrate specificity because the c 11 - c 13 exocyclic double bond of dehydrocostuslacton ( 18 ) was not reduced . the formation of leucodin ( 22 ) proves that guaianolides originate from (+)- costunolide . their formation likely involves more than one enzyme . it was not investigated whether leucodin originates from 11 ( s ), 13 - dihydrocostunolide ( 20 ). parthenolide ( 17 ) is not involved in leucodin biosynthesis , but it cannot be excluded that 11 ( s ), 13 - dihydroparthenolide is . various authors have suggested that either 4 , 5 - epoxide or c 3 - hydroxylation is necessary to direct cyclisation of (+)- costunolide towards a guaiane framework ( brown et al ., 1975 ; fischer 1990 ; teisseire , 1994 ; piet et al ., 1995 , piet et al ., 1996 ). formation of leucodin from (+)- costunolide could be inhibited by the addition of established cytochrome p450 inhibitors or co , whereas formation of 11 ( s ), 13 - dihydrocostunolide ( 20 ) could not . furthermore experiments with oxygen - 18 demonstrated that the oxygen atom of the keto group also originates from molecular oxygen , which at least suggest the involvement of a cytochrome p450 - enzyme in leucodin biosynthesis ( west , 1980 ; mihaliak et al ., 1993 ). leucodin can be regarded as a precursor of 11 ( s ), 13 - dihydro - 8 - deoxylactucin , a minor sesquiterpene lactone of chicory ( van beek et al , 1990 ). possibly this sesquiterpene lactone is also formed during the executed incubations , but it is not detected in gc - ms measurement due to its polarity / lesser volatility . (+)- costunolide and possibly 11 ( s ), 13 - dihydrocostunolide ( 20 ) are likely to be involved in the biosynthesis of the other bitter tasting sesquiterpene lactones of chicory . to detect and analyse these compounds in enzymatic reaction mixtures will involve derivatisation and / or the use of other chromatographic techniques like hplc . bioconversion of valencene to nootkatone using enzymes isolated from chicory roots because of the presence of a cytochrome p450 hydroxylating a sesquiterpenoid skeleton at the c2 position and a dehydrogenase activity that could further oxidize this hydroxy group to form a ketone as demonstrated in example 2 , we decided to investigate the oxidation of the sesquiterpene valencene to study whether nootkatone would be formed . β - nootkatol was prepared by reduction of 190 mg nootkatone ( fluka ) with 20 mg of lialh 4 in in 5 ml dry ether ( diethyl - ether ) ( shoji et al ., 1984 ). after stirring the grey suspension for one night , the reaction was stopped by careful addition of na 2 so 4 . 10h 2 o . the mlxture was stirred for an additional 30 minutes , and dried by the addition of mgso 4 . the solids were filtered off and the ether was washed with distilled water . after drying and evaporation of the solvent , 140 mg of a crude oil was obtained that besides β - nootkatol contained 4 % of α - nootkatol , eims ( 70 ev ) m / z : 220 [ m ] + ( 33 ), 177 ( 77 ), 161 ( 40 ), 145 ( 52 ), 131 ( 77 ), 119 ( 100 ), 109 ( 41 ), 107 ( 50 ), 105 ( 59 ), 95 ( 46 ), 93 ( 60 ), 91 ( 43 ), 81 ( 45 ), 79 ( 80 ), 77 ( 64 ), 69 ( 47 ), 67 ( 52 ), 55 ( 58 ), 43 ( 55 ), 41 ( 94 ), 39 ( 54 ). after flash chromatography of 50 mg from this crude oil on silica with ether - pentane ( 2 : 1 ), fractions devoid of any trace of α :- nootkatol were pooled yielding 3 . 6 mg of β - nootkatol . 1 h nmr ( 200 mhz , c 6 d 6 ) δ 0 . 83 ( d , 3h , j = 3 hz ), δ 0 . 95 ( s , 3h ), δ 0 . 96 - 1 . 47 ( m , 7h ), δ 1 . 75 ( m , 3h ), δ 1 . 93 ( dt , 1h , j = 12 . 7 and 2 . 7 hz ), δ 2 . 07 ( m , 1h ), 6 2 . 20 - 2 . 33 ( m , 21 ), δ 4 . 19 - 4 . 26 ( m , 1h ), 6 4 . 88 ( br s , 2h ), 5 . 44 ( br d , 1h ). 13 c nmr ( 100 mhz , dept , cdcl 3 ) δ 15 . 7 ( q ), δ 18 . 4 ( q ), δ 21 . 1 ( q ), δ 32 . 8 ( t ), 6 33 . 4 ( t ), δ37 . 8 ( t ), δ 38 . 5 ( s ), δ 39 . 8 ( d ), & amp ; 41 . 3 ( d ), 8 45 . 1 ( t ), δ 68 . 1 ( d ), δ 109 . 2 ( t ), 125 . 9 ( d ), δ144 . 9 ( s ), δ 150 . 2 ( s ). eims ( 70 ev ) m / z : 220 [ m ] + ( 56 ), 177 ( 100 ), 145 ( 31 ), 135 ( 51 ), 131 ( 43 ), 123 ( 38 ), 121 ( 91 ), 119 ( 81 ), 109 ( 42 ), 107 ( 66 ), 105 ( 62 ), 95 ( 49 ), 93 ( 69 ), 91 ( 87 ), 81 ( 39 ), 79 ( 60 ), 77 ( 56 ), 69 ( 45 ), 67 ( 51 ): 55 ( 62 ), 53 ( 42 ), 43 ( 52 ), 41 ( 100 ), 39 ( 55 ). a mixture of trans , trans - farnesal and cis , trans - farnesal was prepared by dissolving trans , trans - farnesol ( sigma ) in pentane and stirring with mno 2 . farnesal was oxidised to a mixture of cis , trans - and trans , trans - farnesoic acid with silver oxide ( caliezi and schinz , 1947 ). microsomal pellets that contain (+)- germacrene a hydroxylase activity and an enzyme suspension were prepared from deep frozen chicory cubes as described in example 1 . the enzyme suspension was divided in 1 ml aliquots and incubated with 50 μm (+)- valencene in the presence of a 1 mm nadph - regenerating system that consisted of 1 mm nadph , 5 mm glucose - 6 - phosphate , and 1 . 2 iu glucose - 6 - phosphate cehydrogenase . the initial concentration of substrate in each assay was 45 μm and all experiments were done in duplicate . to the blank assays no nadph regenerating system was added , so that cytochrome p450 enzymes ( including the (+)- germacrene a hydroxylase ) were not active . after 60 minutes the incubations were stopped by storing them at − 20 ° c . in a freezer . the enzyme assays were analysed as described in example 1 . incubation of (+)- valencene with a microsomal pellet from chicory roots and nadph yielded ( fig1 ) only a trace of the expected valencene - 12 - ol ([+]- 2 -[ 2r ]- 2 -[ 1 , 2 , 3 , 4 , 6 , 7 , 8 , 8a - octahydro - 8α , 8aβ - dimethyl - 2α - napthalenyl ]- 2 - propen - 1 - ol ) that was identified by comparison of its mass spectrum with a mass spectrum kindly provided by dr . r . näf ( firmenich s a , geneva , switzerland ). eims ( 70 ev ) m / z 220 [ m ] + ( 22 ), 189 ( 52 ), 187 ( 41 ), 161 ( 80 ), 145 ( 54 ), 131 ( 49 ), 21 ( 41 ), 119 ( 58 ), 117 ( 39 ), 107 ( 55 ), 105 ( 84 ), 95 ( 44 ), 93 ( 73 ), 91 ( 100 ), 81 ( 47 ), 79 ( 84 ), 77 ( 51 ), 67 ( 39 ), 55 ( 48 ), 41 ( 63 ). the major product was nootkatone ( table vii ), whereas in most experiments the corresponding β - nootkatol was detected in amounts smaller than the valencene - 12 - ol ( the chromatogram depicted in fig1 is an exception !). it was assumed that this β - nootkatol is an intermediate in the formation of nootkatone . for this reason incubations of 100 μm β - nootkatol were performed at ph 10 with nad ( p ) + and a 150 , 000 g supernatant of chicory roots , such as described for the conversion of (−)- elema - 1 , 3 , 11 ( 13 )- 12 - ol and germacra - 1 ( 10 ), 4 , 11 ( 13 )- trien - 12 - ol by nadp + - dependent dehydrogenases by de kraker et al . ( 2001 ). during incubation the added β - nootkatol was converted for more than 60 % into nootkatone in the presence of either 1 mm nadp + or 1 mm nad + . in the absence of these cofactors conversion still amounted to 25 %, whereas the boiled enzyme extract gave no conversion of β - nootkatol . at ph 7 . 5 , enzyme activity was slightlylower , whereas a 150 , 000 g pellet , as expected , yielded 3 - fold less dehydrogenase activity than the corresponding supernatant . incubations with a mixure of α - and β - nootkatol showed that only β - nootkatol was converted ( fig1 ). to get some more information about the substrate specificity of the dehydrogenase ( s ) present in chicory roots , incubations with the 150 , 000 g supernatant were also performed with 100 μm trans , trans - farnesol as substrate ( fig1 ) in the presence of either nad + or nadp + . trans , trans - farnesol was converted up to 60 % into a mixture of trans , trans - farnesal and cis , trans - farnesal , and small amounts of farnesoic acid were observed as well . various ph - values were tested between 7 . 5 and 11 . 0 with tris , glycine and caps buffers ; the highest conversion of farnesol into farnesal was observed at ph 10 decreasing to 30 % of mnaximum enzyme activity at ph 7 . 0 . a chicory enzyme extract efficiently catalysed the conversion of (+)- valencene via β - nootkatol into nootkatone ( fig1 and 18 ). possibly these reactions are catalysed by the same enzymes that are involved in the biosynthesis of leucodin from (+)- costunolide ( fig2 ; example 2 ). on basis of its structural resemblance with leucodin , the same might have occurred to (+)- ledene ( see example 2 , table i ), but this compound was only hydroxylated and not converted into a ketone . formation of nootkatone catalysed by enzymes of chicory proceeds via β - nootkatol that is subsequently oxidised to nootkatone by nad ( p ) + - dependent dehydrogenase ( s ). these dehydrogenases are operationally soluble enzymes , but apparently partially end up in the microsomal pellet , just as the germacrene alcohol dehydrogenase ( s ). the dehydrogenase ( s ) involved in formation of nootkatone have a strong preference for β - nootkatol over α - nootkatol and this may indicate the involvement of a specific enzyme that perhaps in planta is responsible for the oxidation of the alcohol precursor of leucodin . in generalthe dehydrogenase ( s ) present in a 150 , 000 g supernatant of chicory do not seem to act with a high substrate specificity . in addition to the conversion of germacrene alcohol and elemene alcohollaldehyde ( example 1 ) they are also capable of converting trans , trans - farnesol into farnesal an farnesoic acid . nad ( p ) + - dependent oxidation of farnesol to farnesal , including the observed isomerisation of farnesal , has also been reported for other crude plant extracts , ( chayet et al ., 1973 ; overton and roberts , 1974 ). whether in uivo the isolated dehydrogenase activities are exclusively involved in sesquiterpene lactone biosynthesis or have ( also ) a different function is uncertain . nootkatone is a much sought - after aromatic substance with a distinctive flavor of grapefruit that is widely used in the flavor and fragrance and food industry . nootkatone was also shown to be an important constituent of anti - ulcer medication ( yamahara et al ., 1990 ). for this reason the possibilities for conversion of the less valuable (+)- valencene has intensively been studied , but the yields obtained by either chemical methods or microbiological methods were not satisfying or too laborious ( dhavlikar and albroscheit , 1973 ; könst et al . 1975 ; lamare and furstoss , 1990 ). remarkably , there is not yet any direct proof that in grapefruit , the source of valencene and nootkatone , a biochemical pathway exists for the conversion of (+)- valencene via β - nootkatol into nootkatone ( del rio et al ., 1992 ). generally , the production of new materials for flavors and fragrances has been a powerful driving force in the research on the . hydroxylation of terpenes by micro - organisms . although in some cases successful these microbiological conversions often yield a broad spectrum of products , including epoxides and diols ( lamare and furstoss , 1990 ; drauz and waldmann , 1995 ; faber , 2000 ) and the oxidations often occur at double bonds . this is in strong contrast to the highly specific oxidation of valencene to nootkatoie as catalysed by the chicory enzyme extract . carboxylic acids are important flavours and fragrances . for instance , fatty acids of various chain lengths are very characteristic for the flavour of cheese ( west , 1996 ), whereas higher fatty acids contribute to the taste and smell of peanuts ( hashim et al ., 1993 ). branched chain fatty acids are important flavour notes in mutton and sheep cheese ( heinsman , 2000 ; heinsman et al ., submitted ). the chicory dehydrogenases responsible for the conversion of germacrene alcohol to germacrene acid have a rather low substrate specificity . therefore we have investigated whether these dehydrogenases are capable of oxidising other kinds of terpene alcohols and aldehydes as well as linear and branched aliphatic alcohols and aldehydes . a chicory enzyme preparation or extract containing the dehydrogenases is made according to de kraker et al . ( 2001a ). incubation with an alcohol or aldehyde , for instance amorpha - 4 , 11 - diene - 12 - ol or the corresponding aldehyde , in the presence of nadp + at ph 10 leads to the formation of the corresponding carboxylic acid in high yield . in case of amorpha - 4 , 11 - diene - 12 - ol , a mixture of artemisinic and dihydroartemisinic acid are produced . apparently , the dehydrogenases are not only capable of oxidising amorphadiene to artemisinic acid but also the reductase responsible for the reduction of the double bond in the isopropenyl group in the pathway after costunolide ( example 2 ) is capable of reducing artemisinic acid to dihydroartemisinic acid . as the conversion of dihydroartemisinic acid to artemisinin is supposed to proceed non - enzymatically , chicory may be a suitable production organism for artemisinin , or a source of genes that can be used to produce artemisinin in other organisms . in the same way , octanol or octanal and 4 - methyloctanol or 4 - methyloctanal are oxidised to the corresponding octanoic acid and 4 - methyloctanoic acid , respectively . since 4methyloctanoic acid is chiral , we checked the stereochemical composition of the product , which appears to contain the r - enantiomer in preference . large scale bioconversion of sesquiterpenes using enzymes isolated from chicory roots fresh roots of cultivated chicory are processed as described in example 1 , and the 150 , 000 g pellets are used for the following experiment . two grams of pellets are suspended in 50 ml of demineralised water containing 25 mm tris ph 7 . 5 , 10 % glycerol , 1 mm ascorbic acid and 2 mm dtt . to this is added 10 u of an nadph - regenerating enzyme like glucose 6 - phosphate dehydrogenase , hydrogenase , or , preferably , formate dehydrogenase ( seelbach et al ., 1996 ) and 100 g of a solid support like deae - sepharose , polyacrylamide or , preferably , accurelo beads . the mixture is stirred overnight at 4 ° c . after which the immobilised enzymes are filtered , washed with deminerallsed water containing 25 mm tris ph 7 . 5 , 10 % glycerol and 1 mm ascorbic acid , and stored at 4 ° c . until usage . alternatively , the mixture of chicory enzymes and cofactor - recycling enzyme is brought into contact with a hydrophobic membrane ( flat membrane or hollow fibre unit ) for one night at 4 ° c ., after which the membrane is washed and the membrane ( containing the immobilised enzymes ) is stored at 4 ° c . until usage . the inumobilised enzyme is placed in a bioreactor and suspended in 500 ml of demineralised water containing 25 mm tris ph 7 . 5 , 10 % glycerol , and 1 mm ascorbic acid , at room temperature . to this mixture , nadph is added up to 0 . 3 mm , together with a suitable amount of cosubstrate , for cofactor recycling . typically , 1 . 5 mm of glucose 6 - phosphate is added as a cosubstrate when using glucose 6 - phosphate dehydrogenase for the recycling of nadph , whereas 100 mm of formic acid / sodium formate ph 7 . 5 is added when formate dehydrogenase is the enzyme of choice . then , the sesquiterpene substrate ( 100 mm in ethanol ) is pumped into the bioreactor at a speed of 0 . 1 ml / min , under continuous gentle stirring . at the same time , oxygen is pumped into the bioreactor using a thin - walled silicon tube which allows bubble - free aeration ( rissom et al ., 1997 ). this system is chosen since it is known that gas - liquid interfaces like air bubbles ) frequently cause inactivation of monooxygenases . pumping is stopped when 50 ml of substrate solution ( 5 mmol , about 1 g of sesquiterpene ) has been added , and the reaction mixture is stirred at room temperature until the concentration of product does no longer increase ( as monitored by off - line gc ). at that time , the immobilised enzyme is ifitered off and the product is obtained by extraction of the aqueous medium with pentane , followed by chromatography over silica ( using petroleum ether / ethyl acetate mixtures as the solvents ). in this way , 0 . 5 g of pure hydroxylated sesquiterpene can be obtained , but higher yields are possible , depending on the substrate used . also nootkatone can be obtained in this way in yields around 50 % or higher , using valencene as the substrate . the immobilised enzymes are washed with buffer and can be reused many times . enzymatic reactions at temperatures higher than ambient are possible but lead to a decreased lifetime of the enzyme preparation . in case of hydrogenase , more advantageous results are obtained when this enzyme is immobilised separately from the chicory enzymes , and a two - compartment bioreactor is used . the two compartments are separated by a highly porous membrane which is permeable for the nadp ( h ) cofactor used . one compartment contains immobilised hydrogenase and a thin - walled silicon tube , through which hydrogen gas is pumped . the other compartment contain n the immobilised chicory enzymes and a thin - walled silicon tube , through which oxygen gas is pumped . in this way , both enzymes have access to their substrates and cofactors whereas the hydrogenase does not suffer from inactivation by oxygen . alternatively , enzymes that are co - immobilised on solid beads can be used in a fed - batch bioreactor where the outlet is equipped with a hydrophobic membrane such that the product is able to pass whereas the cofactors and the enzymes remain inside the reactor . for this purpose , a membrane reactor in which both enzyme preparations are immobilised on the membrane is most advantageous . using this system , the enzymes can be used continuously since they are stable for a prolonged period , allowing the facile preparation of several hundred grams of hydroxylated sesquiterpenes or nootkaton . application of chicory and other asteraceae cell and hairy root cultures for the bioconversion of ( sesqui ) terpenes cultures of de - differentiated cells or callus , hairy roots , shoots and other tissues have been used extensively for the bioconversion of a wide range of substrates . for example cell suspension cultures of grape were able to convert citral to nerol , geraniol and geranylacetate . also asteraceae species are suitable for this approach . hairy root and cell cultures of chicory are obtained using standard protocols ( hairy root cultures : song et al ., 1995 ; cell cultures : dubois et al ., 1988 ). the cultures are supplied with sesquiterpenes such as valencene , α - gurjunene , amorpha - 4 , 11 - diene and α - trans - bergamotene at 200 mg / l . after one week of growth in the presence of the sesquiterpenes , the reaction products are extracted from the culture medium and the hairy roots / cells using pentane / ether . the reaction products are characterised using gc - ms . incubations with valencene yield an efficient conversion to nootkatone . incubations with α - gurjunene , amorphadiene and α - trans - bergamotene yield an efficient conversion to the corresponding alcohols with a high regioselectivity : hydroxylation occurs only in the isopropenyl group . isolation and characterisation of valencene synthase from grapefruit , pomelo , orange and chamaecyparis nootkatensis recently we have demonstrated that the sesquiterpenoid backbone of the sesquiterpene lactones in chicory is formed by a (+)- germacrene a synthase which cyclizes fpp to (+)- germacrene a ( de kraker et al ., 1998 ; bouwmeester et al ., 1999b ). these socalled terpene synthases catalyse the first committed step in all terpenoid biosynthetic pathways . the terpene synthases are a large group of enzymes that all convert the ubiquitous substrates geranyl diphosphate ( to the monoterpenes ), farnesyl diphosphate ( to the sesquiterpenes ), geranylgeranyl diphosphate ( to the diterpenes ) or squalene epoxide ( to the triterpenes ) ( bohlmann et al , 1998 ). the sesquiterpenes exhibit an exceptional large structural diversity , and over 7000 different compounds have been described . like the other terpene synthases ( leading to mono -, di - and triterpenes ), sesquiterpene synthases exhibit a fair degree of sequence similarity , which allows them — in most cases — to be recognized as a sesquiterpene synthase , and enables the design of degenerate primers to be used in pcr to generate fragments that can be used to screen libraries or can be extended using race - pcr to obtain the full - length cdnas ( bohlmann et al . 1998 ). particularly in specialised tissues or enriched libraries also random sequencing may be used to obtain these ( sesqui ) terpene synthases ( bohlmann et al ., 1998 ). the valencene synthase gene is isolated from grapefruit , orange , pomelo and chamaecyparis nootkatensis using pcr with degenerate primers based on the sequence homology existing between sesquiterpene synthases ( see for example bouwmeester et al ., 1999b ) or using the sequence information of the putative sesquiterpene synthase isolated from grapefruit , as published in genbank ( af411120 ). total rna is isolated from grapefruit , orange and pomelo albedo and c . nootkatensis using the purescript rna isolation kit ( biozym ). dnase i ( deoxyribonuclease i , rnase free ) is used to remove dna from the rna isolate . the dnase i is removed with a phenol / chloroform extraction after which the rna is precipitated ( ethanol precipitation with naac ). poly ( a )+ rna is extracted from 20 μg of total rna using 2 μg poly - d ( t ) 25v oligonucleotides coupled to 1 mg paramagnetic beads ( dynal a . s .). the poly ( a )+ rna is resuspended in 20 μl h 2 o . the reverse transcription reaction is carried out in a 50 μl reaction containing 10 μl poly ( a )+ mixture , 0 . 3 μg oligo ( dt ) 25 v , 1 mm each datp , dttp , dctp and dgtp , 50 mm tris - hcl ph 8 . 3 , 80 mm kcl , 10 mm mgcl 2 and catalyzed with 12 u amv reverse transcriptase ( pharmacia ). after an incubation for 2 h at 42 ° c . the reaction is stopped and the cdna purified with the wizard pcr preps dna purification system ( promega ). the cdna is resuspended in 50 μl h 2 o . based on comparison of sequences of terpenoid synthases , two degenerated primers were designed for two conserved regions : sense primer : 5 ′- gay gar aay ggi aar tty aar ga - 3 ′; anti - sense primer : 5 ′- cc rta igc rtc raa igt rtc rtc - 3 ′ ( wallaart et al ., 2001 ) ( primers from eurogentec , seraing , belgium ) ( bouwmeester et al ., 1999b ; wallaart et al ., 2001 ). pcr is performed in a total volume of 50 82 l containing 0 . 5 μm of each of the two primers , 0 . 2 mm dntp , 1 u super taq polymerase / 1 × pcr buffer ( ht biotechnology ltd , cambridge , england ) and 10 μl cdna . the reaction mixture is incubated in a . thermocycler ( robocycler , stratagene ) with 1 min denaturation at 94 ° c ., 1 . 5 min annealing at 42 ° c . and 1 min elongation at 72 ° c . during 40 cycles . agarose gel electrophoresis revealed a single specificpcr product of 550 bp that blast comparison shows to have homology with sesquiterpene synthases . the pcr product is purified using the wizard pcr preps dna purification system ( promega ) and subeloned using the pgemt system . e . coli jm101 is transformed with this construct . the full length cdna is obtained using race - pcr . for functional expression , the cdna clone is subdloned in frame into the expression vector pet 11d ( stratagene ). the construct and pet 11d without an insert ( as negative control ) are transformed to e . coli bl 21 ( de3 ) ( stratagene ), and grown overnight on lb agar plates supplemented with ampicillin at 37 ° c . cultures of 50 ml lb medium supplemented with ampicillin ( 100 μg / ml ) and 0 . 25 mm isopropyl - 1 - thio - β - d - galactopyranoside ( iptg ) are occulated with these over night cultures to a 600 = 0 . 5 and grown for 3 h at 27 ° c . the cells are harvested by centrifugation during 8 minutes at 2000 g and resuspended in 1 . 2 ml buffer containing 15 mm mopso ( ph 7 . 0 ), 10 % ( v / v ) glycerol , 10 mm mgcl2 , 1 mm sodium ascorbate and 2 mm dtt ( buffer a ). the resuspended cells are sonicated on ice during 4 min ( 5 sec on , 30 sec oft ), centrifuged for 5 minutes at 4 ° c . ( 14 . 000 rpm ) and the supernatant used for assays . for determination of product identity , 20 μm [ 3 h ]- fdp is added to 0 . 5 ml of the enzyme preparations diluted 1 : 1 with buffer a containing 0 . 1 % tween - 20 . after the addition of a 1 - ml redistilled pentane overlay , the tubes are carefully mixed and incubated for 1 h at 30 ° c . following the assay , the tubes are mixed , the organic layer is removed and passed over a short column of aluminum oxide overlaid with anhydrous mgso 4 . the assay is re - extracted with 1 ml of pentane : diethyl ether ( 80 : 20 ), which is also passed over the aluminum oxide column , and the column washed with 1 . 5 ml of pentane : diethyl ether ( 80 : 20 ). the extract is analysed using radio - glc and gc - ms ( bouwmeester et al ., 1999a , b ). radio - glc analysis shows that the cdna formed a functionally active protein catalysing the formation of one radiolabelled sesquiterpene from [ 3 h ]- fdp . the negative control ( vector without insert ) produced no radioactivity . the samples are also analysed by gc - ms using a hp 5890 series ii gas chromatograph equipped with an hp5 - ms column ( 30 m × 0 . 25 mm i . d ., 0 . 25 μm df ) and hp 5972a mass selective detector ( hewlett - packard ). the oven is programmed at an initial temperature of 70 ° c . for 1 min , with a ramp of 5 ° c . min − 1 to 210 ° c . and final time of 5 min . the injection port ( splitless mode ), interface and ms source temperatures are 150 , 290 and 180 ° c ., respectively , and the he inlet pressure is controlled by electronic pressure control to achieve a constant column flow of 1 . 0 ml min − 1 . ionization potential is set at 70 ev , and scanning is performed from 30 - 250 amu . the negative control produced no sesquiterpenes , whereas in assays with the expression products valencene is the major product . the identity of the latter is confirmed by analysis of an authentic standard and comparison of the mass spectra with the authentic standard . isolation of the genes encoding sesquiterpene hydroxylases and dehydrogenases from chicory two strategies are used to isolate the sesquiterpene hydroxylases and dehydrogenase from chicory . one is based on the presence of sequence homology between p450 genes on the one hand and dehydrogenases on the other . the other method uses random sequencing of a c . intybus root cdna library which has been shown to be a powerful tool for the isolation of biosynthetic genes ( aharoni , et al ., 2000 ). total rna is isolated from chicorychicons using the purescript rna isolation kit ( biozym ). dnase i ( deoxyribonuclease i , rnase free ) is used to remove dna from the rna isolate . the dnase i is removed with a phenol / chloroform extraction after which the rna is precipitated ( ethanol precipitation with naac ). poly ( a )+ rna is extracted from 20 μg of total rna using 2 μg poly - d ( m125v oligonucleotides coupled to 1 mg paramagnetic beads ( dynal a . s .). the poly ( a )+ rna is resuspended in 20 μl h 2 o . the reverse transcription reaction is carried out in a 50 μl reaction containing 10 μl poly ( a )+ mixture , 0 . 3 μg oligo ( dt ) 25 v , 1 mm each datp , dttp , dctp and dgtp , 50 mm tris - hcl ph 8 . 3 , 80 mm kcl , 10 mm mgcl 2 and catalyzed with 12 u amv reverse transcriptase ( pharmacia ). after an incubation for 2 h at 42 ° c . the reaction is stopped and the cdna purified with the wizard pcr preps dna purification system ( promega ). the cdna is resuspended in 50 μl h 2 o . based on comparison of sequences of cytochrome p450 and dehydrogenase enzymes , degenerate primers are designed for conserved regions such as the er targeting signal , the heme - binding domain , the central region of helix i and the perf - motif . pcr is performed in a total volume of 50 μl containing 0 . 5 μm of each of the two primers , 0 . 2 mm dntp , 1 u super taq polymerase / 1 × pcr buffer ( ht biotechnology ltd , cambridge , england ) and 10 μl cdna . the reaction mixture is incubated in a thermocycler ( robocycler , stratagene ) with 1 min denaturation at 94 ° c ., 1 . 5 min annealing at 42 ° c . and 1 min elongation at 72 ° c . during 40 cycles . agarose gel electrophoresis revealed a single specific pcr product . a cdna library is constructed using the unizap xr custom cdna library service ( stratagene ). for library screening 200 ng of the pcr amplified probes are gel - purified , randomly labelled with [ α - 32 p ] dctp , according to manufacturer &# 39 ; s recommendation ( ready - to - go dna labelling beads (− dctp ), pharmacia ) and used to screen replica filters of 10 4 plaques of the cdna library plated on e . coli xl1 - blue mrf ′ ( stratagene ). the plaque lifting and hybridization are carried out according to standard protocols . positive clones are isolated using a second and third round of hybridization . in vivo excision of the pbluescript phagemid from the uni - zap vector is performed according to manufacturer &# 39 ; s instructions ( stratagene ). in addition , 1500 clones from the cdna library are randomly sequenced and compared to sequences in the databases using blast . promising p450 and dehydrogenase - like sequences were found and are further characterised by heterologous expression . [ heading - 0180 ] expression of the isolated genes in e . coli and yeast . for functional expression , the cdna clones are subcloned into suitable expression vectors and transformed to a suitable expression host . induced cells are harvested by centrifugation during 8 minutes at 2000 g and resuspended in 1 ml assay buffer . the resuspended cells are either sonicated on ice during 4 min ( 5 sec on , 30 sec off ), centrifuged for 5 minutes at 4 ° c . ( 14 . 000 rpm ) and the supernatant used for assays or used as intact cells . in both systems germacrene a , valencene and nootkatol are used as substrates . ether extracts of the assays are analysed using gc - ms ( see example 7 ). the negative control produced no germacrene a alcohol nootkatol or nootkatone , whereas in assays with the expression products of both genes conversion of germacrene a to germacrene a alcohol , valencene to nootkatol and of nootkatol to nootkatone , respectively , occurred . transformation of chicory with the valencene or amorrhadiene synthase gene to obtain nootkatone or artemisinin producing chicory chicory is transformed as described in pct / ep00 / 02130 ( bouwmeester et al ., 1999b ) with a construct harboring the valencene or amorphadiene synthase . after regeneration , transgenic plants are screened for the activity of the introduced gene by examining sesquiterpene synthase activity in enzyme extracts . hereto , 100 mg of tissue , ground in liq n 2 , is extracted in a 2 - ml eppendorf vial , using a plastic probe to further homogenize the tissue , in 1 . 0 ml of extraction buffer containing containing 50 mm mopso ( ph 6 . 8 ), 20 % ( v / v ) glycerol , 50 mm sodium ascorbate , 50 mm nahso 3 , 1 % pvp - 40 , 10 mm mgcl 2 and 5 mm dtt . after extraction , the samples are centrifuged for 20 min at 20 , 000 g at 4 ° c . 0 . 5 ml of the supernatant are diluted 2 - fold with buffer a ( example 7 ) also containing 6 mm sodium orthovanadate ( an inhibitor of phosphohydrolase activity ). after addition of 20 μm 3 h - fpp and a 1 ml redistilled pentane overlay , the tubes are carefully mixed and incubated for 1 h at 30 ° c . the assays are extracted and analysed using radio - gc and gc - ms as described for example 7 . the wildtype and gus - construct controls all exhibit a similar sesquiterpene synthase activity , which is due to the presence of endogenous germacrene a synthase activity . the transformants exhibit analtered sesquiterpene profile , with varying amounts of valencene and amorphadiene , respectively in addition to germacrene a . for some transformants 90 % of the in vitro produced sesquiterpene is valencene . extracts of the transgenic plants that were analysed using gc - ms showed the presence of valencene , nootkatol and nootkatone on the one hand and artemisinic acid , dihydroartemisinic acid and artemisinin on the other hand , respectively . expression of valencene synthase , sesquiterpene lactone c2 hydroxylase and corresponding dehydrogenase in micro - organisms for the production of nootkatone saccluzromyces cereuisiae and pichia pastoris are transformed using the s . cerevisae easycomp ™ transformation kit ( invitrogen ) according to manufacturer &# 39 ; s instructions with constructs harboring : 1 . the valencene hydroxylase and a general , commercial or chicory dehydrogenase 2 . the valencene synthase and valencene hydroxylase 3 . the valencene synthase , valencene hydroxylase and general , commercial or chicory dehydrogenase transgenic cells are grown in a reactor , and products of the cells are analysed after extraction . transgenic yeast cells harboring constructs 2 and 3 produce nootkatone , showing that the yeast dehydrogenases oxidise the nootkatol produced ( in 2 ). yeast cells harboring construct 1 produce nootkatone upon feeding of valencene . expression of sesquiterpene synthases , and sesquiterpene hydroxylases in micro - organisrns for the production of regio - and stereospecific sesquiterpene alcohols saccharomyces cerevisiae and pichia pastoris are transformed using the s . cereuisae easycomp ™ transformation kit ( invitrogen ) according to manufacturer &# 39 ; s instructions with a construct harboring a sesquiterpene synthase , such as , but not limited to , amorphadiene synthase , (−)- α - trans - bergamotene synthase , alloisolongifolene synthase , γ - gurjunene synthase and a sesquiterpene hydroxylase cdna ( obtained as described in example 8 ). transgenic cells are grown in a reactor , and products of the cells are analysed after extraction . transgenic yeast cells are harboring the constructs produce amorphadien - 12 - ol , ( e )- trans - bergamota - 2 , 12 - dien - 14 - ol ; alloisologifolene alcohol ; and 5 , 11 ( 13 )- guaiadiene - 12 - ol , respectively . ando m , ibayashi k , minami n , nakamura t , isogai k ( 1994 ) studies on the synthesis of sesquiterpene lactones , 16 . the synthesis of 11β , 13 - 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