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The invention provides the use of enzymes derived from an organism, in particularly from a plant, that contains sesquiterpene lactones, in biocatalysis. The regio- and stereoselective oxidation of organic compounds is still a largely unresolved challenge to organic chemistry (Faber, 2000). We investigated whether the oxidising enzymes of a sesquiterpene containing organism such as an Asteraceae species, are capable of converting for example sesquiterpene olefins to commercially interesting products. We found useful enzymes with a surprisingly broad application. Asteraceae, but also plant species from other families, that contain sesquiterpene lactones also contain the enzymes required for their biosyntheis, and thus can be used as a preferred source for these enzymes. In a preferred embodiment, the invention provides an enzyme that is useful for nootkatone synthesis. Nootkatone is a compound mainly founds in grapefruits and other citrus fruits. Surprisingly, however, we provide here evidence that nootkatone synthesis can also occur with an enzyme that is not derived from a citrus fruit, but from another sesquiterpene containing species instead. Considering the large availability of non-citrus derived enzymes, for example from the large amounts of chicory taproots that remain after the growing of the chicory vegetable or inuline chicory, or from other Asteraceae species, such as lettuces, sunflower, artichoke, radicchio, etc, etc, that can easily and abundantly be grown, we have provided here useful enzymes for for example the flavor, aroma, pharmaceutical or biocide industry. In addition to the Asteraceae, the liverworts (Hepaticae) are another good example of a cheap and widely available source for these oxidising enzymes and several families of the liverworts have been reported to contain sesquiterpene lactones. Sesquiterpene lactones isolated from higher plants contain, essentially without exception, α-methylene-γ-lactone groups in which H-7 is α-oriented. The liverworts produce sesquiterpene lactones of the enantiomeric series. Nevertheless, the oxidising enzymes from these organisms can carry out similar reactions as the enzymes from higher plants and are a valuable addition to the higher-plant enzymes. In particular, the invention provides a method for converting a substrate and generating a stereo- and regioselective conversion product comprising subjecting said substrate to enzymes derived from a plant species producing sesquiterpene lactones, especially wherein said conversion product comprises an alcohol, aldehyde/ketone or carboxylic acid: Also, the conversion products can be further modified using commercial enzymes such as alcohol dehydrogenases or microbial bioconversion systems. Considering that useful Asteraceae and liverwort species can easily be grown, the invention provides an inexpensive resource of enzymes or other biocatalysts (cells, explants, tissue, hairy roots or cell cultures) for bioconversion. Particularly useful crops comprise Cichorium spp, lettuces such as Lactuca spp., Helianthus spp. etc. but are not limited to these. The method as provided herein allows the conversion of a terpene in a useful stereo- and regioselective conversion product, for example linear or branched terpene alcohols, aldehydes/ketones or carboxylic acids, especially where said substrate comprises a sesquiterpene. In particular, a method is provided wherein said enzyme comprises a cytochrome P450 monooxygenase, such as (+)-germacrene A hydroxylase or a sesquiterpenolide C2 hydroxylase These enzymes catalyse the hydroxylation of (+)-germacrene A and a sesquiterpene lactone intermediate in chicory sesquiterpene lactone biosynthesis. Although the eniymes belong to a biochemical pathway, they do not have a high substrate specificity. Hydroxylation of a sesquiterpene occurs when the compound contains, like germacrene A, an isopropenyl substituent or, like germacrene B, an isopropylidene substituent or like valencene an allylic C2-position. The conversion carried out by these hydroxylases occurs with high regio- and stereoselectivity which is a big advantage compared with microbial hydroxylations which usually occur with very low specificity. In the detailed description, examples are given for the conversion of valencene to the commercially important flavor/fragrance compound nootkaton. The substrate, (+)-valencene, is converted to several products; two of them are valencene alcohol (2.61%) and nootkatone (24.4%). Nootkatone is used as a constituent of soft drinks and perfumes. Also, a method is provided wherein the substrate amorpha-4,11-dien-12-ol, is converted to the antimalarial artemisinin or a precursor thereof. Other substrates that were hydroxylated include germacrene A, as one might expect, the substrate with the highest rate of conversion and β-selinene. Alternative substrates that are converted to their corresponding alcohols are: alloisolongifolene (at 1.49% compared to β-selinene). Amorphadiene is converted to two products, at 74.0 and 18.7%; the first is amorpha-4,11-dien-12-ol the precursor of artemisinin and the identity of the latter is unknown. The relative rate of conversions of the other substrates were found to be the following: (−)-α-trans-bergamotene (13.5%), (−)-β-elemene (56.2%), germacrene A (110%, probably even higher), germacrene B (3.13% and 8.60%, cis-trans isomers), (+)-γ-gurjunene (54.9%), (+)-ledene (7.20%), and neointermedeol (6.92%). It is particularly useful when said enzymes are derived from an extract of said Asteraceae species, extracts being easily made according to a method known in the art. It is also contemplated herein to use free latex from an Asteraceae as enzyme preparation. In the detailed description, examples are given with enzymes derived from chicory roots. Because the roots of chicory are extremely bitter, they are regarded as a waste product of chicory cultivation. About 100,000 tons of chicory roots are produced annually in The Netherlands, but because of their bitter taste, it is not even possible to use them as cattle feed. Therefore they provide a vast and cheap source for the enzymes described in the invention. For an industrial process, the invention provides a method to make enzyme extracts that contain the P450 enzymes. These can be prepared by homogenising chicory roots in an extraction buffer according to Example 1 containing PVPP. The resulting slurry is then filtered and centrifuged. Low- (20,000×g) followed by high-speed (150,000×g) centrifugation will result in highly-enriched microsomal pellets, but also supernatants obtained after very low speed (for example 2,000×g) centrifugation are active, making it easier to adapt the procedure for industrial processes. The pellets or supernatants can be resuspended in a suitable assay buffer to which the appropriate substrates, and NADPH or an NADPH-regenerating system can be added or the enzymes may be immobilised according to Example 5 to increase their efficiency and lifetime. Cofactor recycling will further increase the economic feasability. After incubation at a suitable temperature, the alcohol products can be easily extracted and separated from the substrate using preparative-scale column chromatography. The suitable substrates can be any sesquiterpene hydrocarbon that could yield an interesting product after hydroxylation at positions equivalent to the positions described in the examples, for example valencene, amorphadiene, (−)-α-trans-bergamotene, γ-gurjunene, ledene, germacrene A, germacrene B, but not limited to those. The corresponding alcohols formed enzymatically from these substrates could be further oxidised to the corresponding aldehydes/ketones or acids—if these are of more value—using a chicory alcohol and aldehyde dehydrogenase or commercially available dehydrogenases. However, another possible method is provided wherein said enzymes are present in a tissue- or cell culture derived from said Asteraceae species, for example a hairy root culture. Hairy root and cell cultures of chicory can be 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, amorphadiene, (−)-α-trans-bergamotene, γ-gurjunene, ledene, germacrene A, germacrene B, but not limited to those, preferably at 200–1000 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. It is also possible to make use of a recombinant enzyme, for example derived from a transgenic plant or micro-organism provided with a nucleic acid encoding an enzyme according to the invention. According to the invention, cytochrome P450 cDNAs can be obtained using random sequencing of a cDNA library of chicory taproots, the organ where the activity of these enzymes is highest. Also a PCR approach is feasible where the sequence homology of cytochrome P450 enzymes is used to design degenerate primers that can be used to generate PCR fragments. These fragments are used to screen a cDNA library to obtain full-length cDNAs. These cDNAs can be expressed in E. coli, yeast or any other micro-organism adapted to large-scale industrial fermentation and suitable for the expression of P450s. In such a fermentation procedure, the transgenic micro-organisms will be fed the desired sesquiterpene hydrocarbon substrates for example valencene, amorphadiene, (−)-α-trans-bergamotene, γ-gurjunene, ledene, germacrene A, germacrene B, but not limited to those. The produced alcohol products can be extracted continuously or in batch. These micro-organisms could also be equipped with the synthase of the desired sesquiterpene hydrocarbon substrate, such as for example the valencene synthase, which would further reduce the costs of production, and an alcohol and/or aldehyde dehydrogenase in order to produce ketones/aldehydes, such as in the case of nootkatone production, or acids—when required.
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- customthe invention provides an inexpensive resource of enzymes or other biocatalysts (cells, explants, tissue, hairy roots or cell cultures) for bioconversion
- customThe method as provided herein
- customIn particular, a method is provided wherein