HUE033564T2 - Eljárás (+)-zizaen elõállítására - Google Patents

Description

(12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9188 (2006 01> 15.03.2017 Bulletin 2017/11 (86) International application number: (21) Application number: 10721853.9 PCT/IB2010/052103 (22) Date of filing: 12.05.2010 (87) International publication number: WO 2010/134004 (25.11.2010 Gazette 2010/47)

(54) METHOD FOR PRODUCING (+) -ZIZAENE

VERFAHREN ZUR HERSTELLUNG VON(+) -ZIZAEN PROCEDE DE PRODUCTION DE (+)-ZIZAENE (84) Designated Contracting States: · LIN XIN ET AL: "Genome mining in Streptomyces AL AT BE BG CH CY CZ DE DK EE ES FI FR GB coelicolor: Molecular cloning and GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO characterization of a new sesquiterpene

PL PT RO SE SI SK SM TR synthase" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 128, no. 18, May 2006 (30) Priority: 20.05.2009 PCT/IB2009/052118 (2006-05), pages 6022-6023, XP002591929 ISSN: 0002-7863 (43) Date of publication of application: · CHANDRA PATI L ET AL: "A stereocontrolled 28.03.2012 Bulletin 2012/13 total synthesis of (+/-)-zizaene" TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, (73) Proprietor: Firmenich S.A. AMSTERDAM, NL LNKD- 1211 Geneva 8 (CH) DOI:10.1016/S0040-4020(02)00063-7, vol. 58, no. 9, 25 February 2002 (2002-02-25), pages (72) Inventors: 1773-1778, XP004336421 ISSN: 0040-4020

• SCHALK, Michel · BARKER A J ET AL: "SYNTHETIC

F-74160 Collonges-sous-saleve (FR) PHOTOCHEMISTRY A NEW SYNTHESIS OF

• DEGUERRY, Fabienne RACEMIC ZIZAENE VIA AN INTRA MOLECULAR CH-1211 Geneva 8 (CH) VARIANT OF THE DE MAYO REACTION"

JOURNAL OF THE CHEMICAL SOCIETY PERKIN (74) Representative: Armstrong, lain Cheshire et al TRANSACTIONS I, no. 8,1983, pages 1901-1904,

HGF Limited XP009136188 ISSN: 0300-922X 4th Floor, Merchant Exchange · HONG Y J ET AL: "Consequences of 17-19 Whitworth Street West conformational preorganization in sesquiterpene

Manchester M1 5WG (GB) biosynthesis: Theoretical studies on the formation of the bisabolene, curcumene, (56) References cited: acoradiene, zizaene, cedrene, duprezianene, and

EP-A1- 1 878 792 WO-A1-2009/109597 sesquithuriferol sesquiterpenes" JOURNAL OF WO-A2-2006/134523 THE AMERICAN CHEMICAL SOCIETY 20090617 AMERICAN CHEMICAL SOCIETY USA LNKD-D0l:10.1021/JA9005332, vol. 131, no. 23,17 June 2009 (2009-06-17), pages 7999-8015, XP002591931 ISSN: 0002-7863

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

Description Technical field [0001] The present invention provides a method of producing (+)-zizaene, said method comprising contacting at least one polypeptide with farnesyl pyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce (+)-zizaene, a compound which can be used as precursorfordiverse compounds useful in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing (+)-zizaene is also an object of the present invention.

Prior art [0002] Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Over 300 sesquiterpene hydrocarbons and 3000 sesquiterpenoids have been identified and many new structures are identified each year. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as source of terpenes. Terpene molecules are often used as such, but in some cases chemical reactions are used to transform the terpenes into other high value molecules.

[0003] Biosynthetic production of terpenes involves enzymes called terpene synthases. There is virtually an infinity of sesquiterpene synthases present in the plant kingdom, all using the same substrate (farnesyl pyrophosphate, FPP) but having different product profiles. Genes and cDNAs encoding sesquiterpene synthases have been cloned and the corresponding recombinant enzymes characterized. The biosynthesis of terpenes in plants and other organisms has been extensively studied and is not further detailed in here.

[0004] Generally, the price and availability of plant natural extracts are dependent on the abundance, oil yield and geographical origin of the plants. In addition, the availability and quality of natural extracts is very much dependent on climate and other local conditions leading to variability from year to year, rendering the use of such ingredients in high quality perfumery very difficult or even impossible some years.

[0005] Vetiver oil is one of these natural extracts. It is a relatively expensive perfuming ingredient, which consists of a complex mixture of sesquiterpene alcohols, aldehydes and acids having a complex olfactory profile. The individual constituents of vetiver oil could also be useful as perfuming ingredients but their purification from the oil is not feasible at large scale.

[0006] A plant-independent method for producing the vetiver oil constituents would therefore be very desirable but a cost-effective chemical synthesis of such compounds is so far not available.

[0007] (+)-Zizaene is a naturally occurring sesquiterpene molecule. It can be used as precursorforvarious compounds which are useful in the field of perfumery and flavoring, in particular for constituents of vetiver oil like khusimol, zizaen-12-aland khuzenic acid. A biochemical pathway leading to the synthesis of (+)-zizaene would therefore be of great interest.

[0008] Analysis of the composition of vetiver oil showed that zizaene derivatives are major constituents of this oil and contribute to the vetiver odor. See for example Weyerstahl et al (2000), Flav. Fragr. J., 15, 395-412. Nevertheless, this document does not provide or even suggest an amino acid or nucleotide sequence leading to the production of (+)-zizaene.

[0009] The biosynthesis of vetiver oil in vetiver roots has been investigated in Del Giudice et al (2008), The microbial community ofVetiver root and its involvement into essential oil biogenesis, Environ. Microbiol., 10(10), 2824-2841. This publication describes the production of sesquiterpenes by microorganisms isolated from vetiver roots and supports the idea of the microorganisms implicated in the biosynthesis of vetiver sesquiterpenes. Our results are in opposition to the suggestions made in this article because we show that zizaene is produced by a sesquiterpene synthase expressed by the vetiver roots themselves and not by the microbial community.

[0010] A sesquiterpene synthase capable of synthesizing a precursor of the vetiver oil constituents, in particular (+)-zizaene, has never been disclosed in the prior art.

[0011] The percentage of identity between the known sesquiterpene synthases and the polypeptide of the invention is very low. The closest protein sequence to the (+)-zizaene synthase of the invention is a putative terpene synthase from Zea mays (NCBI access No ACG24265) which shares 56% amino acid sequence identity with the (+)-zizaene synthase of the invention. The products obtained with this putative terpene synthase have not been identified. The closest fully characterized synthase is a (£)-befa-caryophyllene synthase from Zea mays (NCBI access No ABY79212), which is only 51% identical to the synthase of the invention.

[0012] In addition to the difference between the sequences themselves, it also has to be pointed out that the structure and the properties of the products synthesized by the above-mentioned enzyme are very different from those of (+)-zi-zaene. In particular (E)-beta-caryophyllene is not suitable as a precursor for the production ofvetiveroil constituents.

[0013] Despite extensive studies of terpene cyclization, the isolation and characterization of the terpene synthases is still difficult, particularly in plants, due to their low abundance, their often transient expression patterns, and the complexity of purifying them from the mixtures of resins and phenolic compounds in tissues where they are expressed.

[0014] It is an objective of the present invention to provide methods for making (+)-zizaene in an economic way, as indicated above. Accordingly, the present invention has the objective to produce (+)-zizaene while having little waste, a more energy and resource efficient process and while reducing dependency on fossil fuels. It is a further objective to provide enzymes capable of synthesizing (+)-zizaene, which is useful as precursor for perfumery and/or aroma ingredients.

Abbreviations used [0015] bp base pair kb kilo base BSA bovine serum albumin

cDNA complementary DNA CTAB cethyltrimethylammonium bromide DMAPP dimethylallyl diphosphate DNA deoxyribonucleic acid dATP deoxy adenosine triphosphate dNTP deoxy nucleotide triphosphate DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FPP farnesyl pyrophosphate GC gaseous chromatograph idi isopentenyl diphosphate isomerase IPP isopentenyl diphosphate IPTG isopropyl-D-thiogalacto-pyranoside LB lysogeny broth MOPSO 3-(N-morpholino)-2-hydroxypropanesulfonic acid MS mass spectrometer mvaK1 mevalonate kinase mvaK2 mevalonate diphosphate kinase PCR polymerase chain reaction PVP polyvinylpyrrolidone RMCE recombinase-mediated cassette exchange 3’-/5’-RACE 3’ and 5’ rapid amplification of cDNA ends RNA ribonucleic acid mRNA messenger ribonucleic acid

TE tris and EDTA YNB yeast nitrogen base

Description of the invention [0016] The present invention provides a method to biosynthetically produce (+)-zizaene in an economic, reliable and reproducible way.

[0017] A "sesquiterpene synthase" or a "polypeptide having a sesquiterpene synthase activity" is intended here as a polypeptide capable of catalyzing the synthesis of a sesquiterpene molecule or of a mixture of sesquiterpene molecules from the acyclic terpene precursor FPP.

[0018] As a "(+)-zizaene synthase" or as a "polypeptide having a (+)-zizaene synthase activity", we mean here a polypeptide capable of catalyzing the synthesis of (+)-zizaene starting from FPP. (+)-Zizaene may be the only product or may be part of a mixture of sesquiterpenes.

[0019] The ability of a polypeptide to catalyze the synthesis of a particular sesquiterpene (for example (+)-zizaene) can be simply confirmed by performing the enzyme assay as detailed in Example 3.

[0020] According to the present invention, polypeptides are also meant to include truncated polypeptides provided that they keep their sesquiterpene synthase activity as defined in any of the above embodiments and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO:1.

[0021] As intended herein below, "a nucleotide sequence obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof" encompasses any sequence that has been obtained by changing the sequence of SEQ ID NO:2, of SEQ ID NO:11 or of the complement of one of these two sequences using any method known in the art, for example by introducing any type of mutations such as deletion, insertion or substitution mutations. Examples of such methods are cited in the part of the description relative to the variant polypeptides and the methods to prepare them.

[0022] The percentage of identity between two peptidic or nucleotidic sequences is a function of the number of amino acids or nucleotide residues that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity.

[0023] Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the worldwide web. Preferably, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) at http://www.nc-bi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of peptidic or nucleotidic sequences and to calculate the percentage of sequence identity.

[0024] One object of the present invention is therefore a method for producing (+)-zizaene comprising a) contacting FPP with at least one polypeptide having a (+)-zizaene synthase activity and comprising an amino acid sequence at least 70% identical to SEQ ID NO:1; b) optionally, isolating the (+)-zizaene produced in step a).

[0025] According to a preferred embodiment, the method is a method for producing (+)-zizaene as a major product. According to an even more preferred embodiment, (+)-zizaene represents at least 50%, preferably at least 60%, preferably at least 80%, preferably at least 90% of the product produced by the method of the invention.

[0026] The method can be carried out in vitro as well as in vivo, as will be explained in details further on.

[0027] The polypeptide to be contacted with FPP in vitro can be obtained by extraction from any organism expressing it, using standard protein or enzyme extraction technologies. If the host organism is an unicellular organism or cell releasing the polypeptide of the invention into the culture medium, the polypeptide may simply be collected from the culture medium, for example by centrifugation, optionally followed by washing steps and re-suspension in suitable buffer solutions. If the organism or cell accumulates the polypeptide within its cells, the polypeptide may be obtained by disruption or lysis of the cells and further extraction of the polypeptide from the cell lysate.

[0028] The polypeptide having a (+)-zizaene synthase activity, either in an isolated form or together with other proteins, for example in a crude protein extract obtained from cultured cells or microorganisms, may then be suspended in a buffer solution at optimal pH. If adequate, salts, BSA and other kinds of enzymatic co-factors, may be added in order to optimize enzyme activity. Appropriate conditions are described in more details in the Examples further on.

[0029] The precursor FPP may then be added to the suspension or solution, which is then incubated at optimal temperature, for example between 15 and 400C, preferably between 25 and 35°C, more preferably at 300C. After incubation, the (+)-zizaene produced may be isolated from the incubated solution by standard isolation procedures, such as solvent extraction and distillation, optionally after removal of polypeptides from the solution.

[0030] According to another preferred embodiment, the method of any of the above-described embodiments is carried out in vivo. In this case, step a) comprises cultivating a non-human host organism or cell capable of producing FPP and transformed to express at least one polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:1 and having a (+)-zizaene synthase activity, under conditions conducive to the production of (+)-zizaene.

[0031] According to a more preferred embodiment, the method further comprises, prior to step a), transforming a non human organism or cell capable of producing FPP with at least one nucleic acid encoding a polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:1 and having a (+)-zizaene synthase activity, so that said organism expresses said polypeptide.

[0032] These embodiments of the invention are particularly advantageous since it is possible to carry out the method in vivo without previously isolating the polypeptide. The reaction occurs directly within the organism or cell transformed to express said polypeptide.

[0033] According to a particular embodiment of the invention, the at least one nucleic acid encoding the (+)-zizaene synthase comprises a nucleotide sequence at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, SEQ ID NO: 11 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:2, SEQ ID NO: 11 or the complement thereof. In an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:2, SEQ ID NO: 11 or the complement thereof.

[0034] According to a more preferred embodiment the at least one nucleic acid used in any of the above embodiments comprises a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. According to an even more preferred embodiment, said at least one nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof.

[0035] According to another embodiment, the at least one nucleic acid is isolated from Vetiveria zizanoides.

[0036] The organism or cell is meant to "express" a polypeptide, provided that the organism or cell is transformed to harbor a nucleic acid encoding said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the host organism or cell. The term "express" encompasses "heterologously express" and "over-express", the latter referring to levels of mRNA, polypeptide and/or enzyme activity over and above what is measured in a non-transformed organism or cell. A more detailed description of suitable methods to transform a non-human host organism or cell will be described later on in the part of the specification that is dedicated to such transformed non-human host organisms or cells as specific objects of the present invention and in the examples.

[0037] A particular organism or cell is meant to be "capable of producing FPP" when it produces FPP naturally or when it does not produce FPP naturally but is transformed to produce FPP, either prior to the transformation with a nucleic acid as described herein or together with said nucleic acid. Organisms or cells transformed to produce a higher amount of FPP than the naturally occurring organism or cell are also encompassed by the "organisms or cells capable of producing FPP". Methods to transform organisms, for example microorganisms, so that they produce FPP are already known in the art. Such methods can for example be found in the literature, for example in the following publications Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., and Keasling, J.D. Nat Biotechnol., 2003, 21(7), 796-802 (transformation of E. coli)\ Wu, S., Schalk, M., Clark, A., Miles, R.B., Coates, R., and Chappell, J., Nat Biotechnol., 2006, 24(11), 1441-1447 (transformation of plants); Takahashi, S., Yeo, Y„ Greenhagen, B. T., McMullin, T., Song, L., Maurina-Brunker, J., Rosson, R., Noel, J., Chappell, J, Biotechnology and Bioengineering, 2007, 97(1), 170-181 (transformation of yeast)..

[0038] To carry out the invention in vivo, the host organism or cell is cultivated under conditions conducive to the production of (+)-zizaene. Accordingly, if the host is a transgenic plant, optimal growth conditions are provided, such as optimal light, water and nutrient conditions, for example. If the host is a unicellular organism, conditions conducive to the production of (+)-zizaene may comprise addition of suitable cofactors to the culture medium of the host. In addition, a culture medium may be selected, so as to maximize (+)-zizaene synthesis. Optimal culture conditions are described in a more detailed manner in the following Examples.

[0039] Non-human host organisms suitable to carry out the method of the invention in vivo may be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human host organism used to carry out the invention in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used. Particularly useful plants are those that naturally produce high amounts of terpenes. In a more preferred embodiment, the plant is selected from thefamily of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant is selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha. Preferably, the plant belongs to the species of Nicotiana tabacum.

[0040] In a more preferred embodiment the non-human host organism used to carry out the method of the invention in vivo is a microorganism. Any microorganism can be used but according to an even more preferred embodiment said microorganism is a bacteria oryeast. Most preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

[0041] Some of these organisms do not produce FPP naturally. To be suitable to carry out the method of the invention, these organisms have to be transformed to produce said precursor. They can be so transformed either before the modification with the nucleic acid described according to any of the above embodiments or simultaneously, as explained above.

[0042] Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of the invention in vivo. Suitable eukaryotic cells may be any non-human cell, but are preferably plant or fungal cells.

[0043] According to a preferred embodiment, the at least one polypeptide having a (+)-zizaene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino acid sequence at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:1. According to a more preferred embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO:1. In an even more preferred embodiment, said polypeptide consists of SEQ ID NO:1.

[0044] According to another preferred embodiment, the at least one polypeptide having a (+)-zizaene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino acid sequence that is a variant of SEQ ID NO:1 obtained by genetic engineering. In other terms, said polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. According to a more preferred embodiment, the at least one polypeptide having a (+)-zizaene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments consists of an amino acid sequence that is a variant of SEQ ID NO:1 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof.

[0045] As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they keep their activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO:1.

[0046] Examples of variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or form proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- orC- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of the invention. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention. For example, as detailed in Example 4 below, SEQ ID NO:11 is a variant of SEQ ID NO:2, obtained by artificial mutation of SEQ ID NO:2, leading to a nucleotide sequence which is optimized for expression in E. coli and which encodes the same (+)-zizaene synthase as SEQ ID NO:2 (i.e. SEQ ID NO:1). The sequences SEQ ID NO:2 and SEQ ID NO: 11 are 76% identical.

[0047] Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of the invention. In particular such a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Accordingly, the present invention encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides. Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway, can also be advantageously be used in the methods of the invention.

[0048] According to another embodiment, the at least one polypeptide having a (+)-zizaene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments is isolated from Vetiveria zizanoides.

[0049] An important tool to carry out the method of the invention is the polypeptide itself. A polypeptide having a (+)-zizaene synthase activity and comprising an amino acid sequence at least 70% identical to SEQ ID NO:1 is therefore another object of the present invention.

[0050] According to a preferred embodiment, the polypeptide is capable of producing (+)-zizaene as a major product. According to an even more preferred embodiment, it is capable of producing a mixture of sesquiterpenes wherein (+)-zizaene represents at least 60%, preferably at least 80%, preferably at least 90% of the sesquiterpenes produced.

[0051] According to a more preferred embodiment, the polypeptide has a (+)-zizaene synthase activity.

[0052] According to a preferred embodiment, the polypeptide comprises an amino acid sequence at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:1. According to a more preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO:1. According to an even more preferred embodiment, the polypeptide consists of SEQ ID NO:1.

[0053] According to another preferred embodiment, the polypeptide comprises an amino acid sequence that is a variant of SEQ ID NO:1 obtained by genetic engineering. In other terms, said polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. According to a more preferred embodiment, the polypeptide having a (+)-zizaene synthase activity consists of an amino acid sequence that is a variant of SEQ ID NO:1 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof.

[0054] According to another embodiment, the polypeptide is isolated form Vetiveria zizanoides.

[0055] As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they keep their activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO:1.

[0056] Examples of variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or form proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- orC- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of the invention. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention. For example, as detailed in Example 4 below, SEQ ID NO:11 is a variant of SEQ ID NO:2, obtained by artificial mutation of SEQ ID NO:2, leading to a nucleotide sequence which is optimized for expression in E. coli and which encodes the same (+)-zizaene synthase as SEQ ID NO:2 (i.e. SEQ ID NO:1). The sequences SEQ ID NO:2 and SEQ ID NO:11 are 76% identical.

[0057] Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends are also encompassed by the polypeptides of the invention. In particular such a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be a signal peptide, for example. Accordingly, the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides. Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway, are also encompassed by the polypeptides of the invention.

[0058] As mentioned above, the nucleic acid encoding the polypeptide of the invention is a useful tool to modify nonhuman host organisms or cells intended to be used when the method is carried out in vivo.

[0059] A nucleic acid encoding a polypeptide according to any of the above-described embodiments is therefore also an object of the present invention.

[0060] According to a preferred embodiment, the nucleic acid comprises a nucleotide sequence at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, SEQ ID NO: 11 or the complement thereof. According to a more preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. According to an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:2, SEQ ID NO:11 or the complement thereof.

[0061] According to another embodiment, the nucleic acid is isolated from Vetiveria zizanoides.

[0062] The nucleic acid of the invention can be defined as including deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form (DNA and/or RNA). The terms "nucleotide sequence" should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid. Nucleic acids of the invention also encompass certain isolated nucleotide sequences including those that are substantially free from contaminating endogenous material. The nucleic acid of the invention may be truncated, provided that it encodes a polypeptide encompassed by the present invention, as described above.

[0063] According to a more preferred embodiment, the at least one nucleic acid according to any of the above embodiments comprises a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. Preferably said nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, SEQ ID NO:11 or the complement thereof.

[0064] The nucleic acids comprising a sequence obtained by mutation of SEQ ID NO:2, SEQ ID NO:11 or the complement thereof are encompassed by the invention, provided that the sequences they comprise share at least the defined percentage of identity with the corresponding fragments of SEQ ID NO:2, SEQ ID NO:11 or the complement thereof and provided that they encode a polypeptide having a (+)-zizaene synthase activity, as defined in any of the above embodiments. Mutations maybe any kind of mutations of these nucleic acids, such as point mutations, deletion mutations, insertion mutations and/or frame shift mutations. A variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by a preferred codon. Due to the degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, multiple DNA sequences can code for the same polypeptide, all these DNA sequences being encompassed by the invention.

[0065] Another important tool for transforming host organisms or cells suitable to carry out the method of the invention in vivo is an expression vector comprising a nucleic acid according to any embodiment of the invention. Such a vector is therefore also an object of the present invention.

[0066] An "expression vector" as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of the invention operably linked to at least one regulatory sequence, which controls initiation and/or termination of the transcription and/or translation, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the nucleic acid of the invention.

[0067] The expression vectors of the present invention may be used in the methods for preparing a genetically transformed host organism and/or cell, in host organisms and/or cells harboring the nucleic acids of the invention and in the methods for producing or making polypeptides having a (+)-zizaene synthase activity, as disclosed further below.

[0068] Recombinant non-human host organisms and cells transformed to harbor at least one nucleic acid of the invention so that it heterologously expresses or over-expresses at least one polypeptide of the invention are also very useful tools to carry out the method of the invention. Such non-human host organisms and cells are therefore another object of the present invention.

[0069] A nucleic acid according to any of the above-described embodiments can be used to transform the non-human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides.

[0070] Non-human host organisms of the invention may be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human host organism is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus is suitable to be transformed according to the present invention. Particularly useful plants are those that naturally produce high amounts of terpenes. In a more preferred embodiment, the plant is selected from the family of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant is selected from the genera Nicotiana, Solatium, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa), Gossypium (cotton), Artemisia, Sylvia and Mentha. Preferably, the plant belongs to the species of Nicotiana tabacum.

[0071] In a more preferred embodiment the non-human host organism is a microorganism. Any microorganism is suitable for the present invention, but according to an even more preferred embodiment said microorganism is a bacteria or yeast. Most preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

[0072] Isolated higher eukaryotic cells can also be transformed, instead of complete organisms. As higher eukaryotic cells, we mean here any non-human eukaryotic cell except yeast cells. Preferred higher eukaryotic cells are plant cells or fungal cells.

[0073] The term "transformed" refers to the fact that the host was subjected to genetic engineering to comprise one, two or more copies of each of the nucleic acids required in any of the above-described embodiment. Preferably the term "transformed" relates to hosts heterologously expressing the polypeptides encoded by the nucleic acid with which they are transformed, as well as over-expressing said polypeptides. Accordingly, in an embodiment, the present invention provides a transformed organism, in which the polypeptides are expressed in higher quantity than in the same organism not so transformed.

[0074] There are several methods known in the art for the creation of transgenic host organisms or cells such as plants, fungi, prokaryotes, or cultures of higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, plant and mammalian cellular hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Elsevier, New York and Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press. Cloning and expression vectors for higher plants and/or plant cells in particular are available to the skilled person. See for example Schardl et al. Gene 61: 1-11, 1987.

[0075] Methods for transforming host organisms or cells to harbor transgenic nucleic acids are familiar to the skilled person. For the creation of transgenic plants, for example, current methods include: electroporation of plant protoplasts, liposome-mediated transformation, agrobacterium-mediated transformation, polyethylene-glycol-mediated transformation, particle bombardment, microinjection of plant cells, and transformation using viruses.

[0076] In one embodiment, transformed DNA is integrated into a chromosome of a non-human host organism and/or cell such that a stable recombinant system results. Any chromosomal integration method known in the art may be used in the practice of the invention, including but not limited to recombinase-mediated cassette exchange (RMCE), viral site-specific chromosomal insertion, adenovirus and pronuclear injection.

[0077] In order to carry out the method for producing (+)-zizaene in vitro, as exposed herein above, it is very advantageous to provide a method of making at least one polypeptide having a (+)-zizaene synthase activity as described in any embodiment of the invention. Therefore, the invention provides a method for producing at least one polypeptide according to any embodiment of the invention comprising a) culturing a non-human host organism or cell according to any embodiment of the invention; b) isolating the polypeptide from the non-human host organism or cell cultured in step a).

[0078] According to a preferred embodiment, said method further comprises, prior to step a), transforming a nonhuman host organism or cell with at least one nucleic acid according to any embodiment of the invention, so that said organism expresses the polypeptide encoded by said nucleic acid.

[0079] A nucleic acid according to any of the above-described embodiments can be used.

[0080] Transforming and culturing of the non-human host organism or cell can be carried out as described above for the method of producing (+)-zizaene in vivo. Step b) may be performed using any technique well known in the art to isolate a particular polypeptide from an organism or cell.

[0081] A "polypeptide variant" as referred to herein means a polypeptide having a (+)-zizaene synthase activity and being substantially homologous to the polypeptide according to any of the above embodiments, but having an amino acid sequence different from that encoded by any of the nucleic acid sequences of the invention because of one or more deletions, insertions or substitutions.

[0082] Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. See Zubay, Biochemistry, 1983, Addison-Wesley Pub. Co. The effects of such substitutions can be calculated using substitution score matrices such a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, J. Mol. Biol., 1991,219, 555-565. Other such conservative substitutions, for example substitutions of entire regions having similar hydrophobicity characteristics, are well known.

[0083] Naturally occurring peptide variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides encoded by the sequences of the invention.

[0084] Variants of the polypeptides of the invention may be used to attain for example desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution, increased affinity for the substrate, improved specificity for the production of one or more desired compounds, increased velocity of the enzyme reaction, higher activity or stability in a specific environment (pH, temperature, solvent, etc), or improved expression level in a desired expression system. A variant or site directed mutant maybe made by any method known in the art. Variants and derivatives of native polypeptides can be obtained by isolating naturally-occurring variants, or the nucleotide sequence of variants, of other or same plant lines or species, or by artificially programming mutations of nucleotide sequences coding for the polypeptides of the invention. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods.

[0085] Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used to enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Accordingly, the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo-or polypeptides and/or those which are linked to signal peptides. Fusion polypeptides encompassed by the invention also comprise fusion polypeptides resulting from a fusion of other functional proteins, such as other proteins from the terpene biosynthesis pathway.

[0086] Therefore, in an embodiment, the present invention provides a method for preparing a variant polypeptide having a (+)-zizaene synthase activity, as described in any of the above embodiments, and comprising the steps of: (a) selecting a nucleic acid according to any of the embodiments exposed above; (b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid; (c) transforming host cells or unicellular organisms with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence; (d) screening the polypeptide for at least one modified property; and, (e) optionally, if the polypeptide has no desired variant (+)-zizaene synthase activity, repeating the process steps (a) to (d) until a polypeptide with a desired variant (+)-zizaene synthase activity is obtained; (f) optionally, if a polypeptide having a desired variant (+)-zizaene synthase activity was identified instep (d), isolating the corresponding mutant nucleic acid obtained in step (c).

[0087] According to a preferred embodiment, the variant polypeptide prepared is capable of producing (+)-zizaene as a major product. According to an even more preferred embodiment, it is capable of producing a mixture of sesquiterpenes wherein (+)-zizaene represents at least 60%, preferably at least 80%, preferably at least 90% of the sesquiterpenes produced.

[0088] In step (b), a large number of mutant nucleic acid sequences may be created, for example by random mutagenesis, site-specific mutagenesis, or DNA shuffling. The detailed procedures of gene shuffling are found in Stemmer, DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA., 1994, 91(22): 10747-1075. In short, DNA shuffling refers to a process of random recombination of known sequences in vitro, involving at least two nucleic acids selected for recombination. For example mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion.

[0089] Accordingly, the polypeptide comprising SEQ ID NO:1 may be recombined with any other sesquiterpene synthase encoding nucleic acids, for example isolated from an organism other than Veriveria zizanoides. Thus, mutant nucleic acids may be obtained and separated, which may be used for transforming a host cell according to standard procedures, for example such as disclosed in the present examples.

[0090] In step (d), the polypeptide obtained in step (c) is screened for at least one modified property, for example a desired modified enzymatic activity. Examples of desired enzymatic activities, for which an expressed polypeptide may be screened, include enhanced or reduced enzymatic activity, as measured by KM or Vmax value, modified regio-chemistry or stereochemistry and altered substrate utilization or product distribution. The screening of enzymatic activity can be performed according to procedures familiar to the skilled person and those disclosed in the present examples.

[0091] Step (e) provides for repetition of process steps (a)-(d), which may preferably be performed in parallel. Accordingly, by creating a significant number of mutant nucleic acids, many host cells may be transformed with different mutant nucleic acids at the same time, allowing for the subsequent screening of an elevated number of polypeptides. The chances of obtaining a desired variant polypeptide may thus be increased at the discretion of the skilled person.

[0092] All the publications mentioned in this application are to disclose and describe the methods and/or materials in connection with which the publications are cited.

Description of the drawings [0093]

Figure 1: GC-MS analysis of sesquiterpenes produced by the Vetiveria zizanoides (+)-zizaene synthase (VzZS). A, total ion chromatogram. B, mass spectrum of the major peak (1). C, mass spectrum of an authentic (+)-zizaene standard. Peak 1 was identified as the (+)-zizaene. Peaks 2, 3 and 4 were identified as prezizaene, a-funebrene and β-funebrene respectively. The peaks marked with S were unidentified sesquiterpene compounds.

Figure 2: Structure of the major sesquiterpene compounds produced by the Vetiveria zizanoides (+)-zizaene synthase (VzZS).

Figure 3: Product profile obtained by in-vitro (A) and in-vivo (B) production of (+)-zizaene. The major peak in each analysis is (+)-zizaene.

Specific embodiments of the invention or Examples [0094] The invention will now be described in further detail by way of the following Examples.

Example 1 RNA extraction and cDNA library construction [0095] Vetiver (Vetiveria zizanoides) plants were obtained from a plant nursery (The Austral Plants Company, Les Avirons, The Reunion Island, France). The plants were cultivated in pots in a green house at the Lullier Agronomy research Station (Switzerland) and were propagated vegetatively by dividing six months to one-year-old clumps. For harvesting of the roots, the plants were removed from the pots and rinsed with tap water.

[0096] For preparation of the cDNA library, roots from several plants were combined: young plants (4 to 6 months after propagation), old plants with a well-developed dense root system (1 to 2 years after propagation) and young plants dried at room temperature for 24 to 36 hours after removing them from the pots. The roots were cut off from the aerial part of the plants and frozen in liquid nitrogen. They were first roughly chopped in liquid nitrogen using a Waring Blendor (Waring Laboratory, Torrington, USA) and then ground to a fine powder using a mortar and pestle. Total RNA was extracted following the procedure described in Kolosova et al (Kolosova, Miller, Ralph, Ellis, Douglas, Ritland and Bohlmann, Isolation of high-quality RNA from gym nosperm and angiosperm trees. J. Biotechniques, 36(5), 821-4,2004) with the following modifications. A volume of 20 ml of extraction buffer was used for 2 grams of ground tissue and the extraction buffer was supplemented with 2% (w/v) of PVP (polyvinylpyrrolidone, Sigma-Aldrich). For the CTAB (cethyl-trimethylammonium bromide, Sigma-Aldrich) extraction, the nucleic acid pellet was resuspended in 2 ml TE buffer (10 mM Tris-HCI, pH 8, 1 mM EDTA) and the extraction was performed with 2 ml of 5M NaCI and 1 ml 10% CTAB. For the isopropanol precipitation, the nucleic acid pellet was dissolved in 500 μΙ TE. The final RNA pellet was resuspended in 50 μΙ water.

[0097] A double stranded cDNA library was prepared using the SMART™ PCR cDNA Synthesis Kit (Clontech Laboratories, Mountain View, CA) according to the manufacturer’s instructions and using Superscript™ II RNAse H- reverse transcriptase (Invitrogen, Carlsbad, CA) for the reverse transcription step. An amount of 1 μg of vetiver underground tissue total RNA was used as template for the cDNA synthesis and 15 cycles were performed for the amplification procedure. The library was loaded on a 1 % agarose gel and the fragments of sizes ranging from 1.3 to 3 Kb were eluted. For the sequencing 270 ng of this cDNA library was used.

Example 2

cDNA library sequencing and amplification of a sesquiterpene synthase cDNA

[0098] The technology of massive parallel sequencing of small DNA fragments developed by lllumina (San Diego, California) was used to sequence the whole cDNA library. The preparation of the DNA for sequencing, the sequencing and the assembling of the reads were performed by Fasteris SA (Plan-les-Ouates, Switzerland). The cDNA library was treated following the Genomic Sample Prep Kit (lllumina) and sequenced on the Genome Analyzer system (lllumina). A total 4.2 million of 35 bp reads were obtained (of which 3.6 million were unique sequences). These reads were assembled using EDENA 2.1.1, a software finding overlaps between the reads and assembling de novo contigs (Hernandez D., Frangois P., Farinelli L., Csteras M., and Schrenzel J., De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer. Genome Res. 18(5), 802-809, 2008). After eliminating contigs shorter than 100 bases, 4324 unique contigs were obtained with a maximum length of 1882 bp. Another assembling was performed using the Velvet 1.0 program (Zerbino and Birney (2008), Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18(5), 821-829), providing 9264 unique contigs of length between 100 and 2006 bases.

[0099] All the contigs generated were compared against a protein sequences database (containing a selection of 7000 plant protein sequences) using the Blastx algorithm (Altschul etal, J. Mol. Biol. 215,403-410,1990). The contigs showing significantsequence homology with plantsesquiterpene synthases were retained and the homology was further confirmed by performing, for each selected contig, a blast search against the NCBI non-redundant protein sequences (NCBI; http://www.ncbi.nlm.nih.gov). In this way, 15 contigs were confirmed as being fragments of sesquiterpene encoding cDNA.

[0100] One of the selected contigs (VzCtg306, SEQ ID NO:3) was of 1090 bp length and sequence comparisons with full-length terpene synthases showed that the 3’end and the 5’end were missing. Two forward primers (ctg306-3R1 (SEQ ID NO:4) and ctg306-3R2 (SEQ ID NO:5)) and two reverse primers (ctg306-5R1 (SEQ ID NO:6) and ctg306-5R2, (SEQ ID NO:7)) were designed from this sequence and used for the Rapid Amplification of cDNA Ends (RACE). The SMART™ RACE cDNA Amplification Kit (Clontech Laboratories, Mountain View, CA) was used with the PrimeScript reverse transcriptase (TaKaRa Bio, Shiga, Japan). Thus, a SMART™ 5’RACE-Ready cDNA and a SMART™ 3’RACE-Ready cDNA pool were prepared each from 1.2 μg vetiver root total RNA. For the 5’RACE, a first round PCR was performed with the UPM primers (Clontech Laboratories) and the ctg306-5R1 primer (SEQ ID NO:6) followed by a second round PCR with the NUP primer (Clontech Laboratories) and the ctg306-5R2 primer (SEQ ID NO:7). For the 3’RACE, a first round PCR was performed with the UPM primers (Clontech Laboratories) and the ctg306-3R1 (SEQ ID NO:4) primer followed by a second round PCR with the NUP primer (Clontech Laboratories) and the ctg306-3R2 primer (SEQ ID NO:5). The amplifications were performed in the conditions detailed in the manufacturer manual (Clontech).

[0101] The combination of the 5’ and 3’RACE allowed the reconstitution of a 1925 bp cDNA (VzZS, SEQ ID NO:8) containing an open reading frame of 1668 bp (SEQ ID NO:2) encoding fora protein of 555 amino acids length (SEQ ID NO:1).

Example 3

Heterologous expression and enzyme characterization [0102] The full-length VzZS open reading frame (VzZS-ORF, SEQ ID NO:2) was amplified from the SMART™ 5’RACE-Ready cDNA pool using the primer ctg306-start (SEQ ID NO:9) and ctg306-stop (SEQ ID NO:10). The amplification of this cDNA for the expression constructs were performed using the Pfu DNA polymerase (Promega, Madison, Wl, USA), in a final volume of 50 μΙ containing 5 μΙ of Pfu DNA polymerase 10X buffer, 200 μΜ each dNTP, 0.4 μΜ each forward and reverse primer, 2.9 units Pfu DNA polymerase and 2.5 μΙ of the cDNA (prepared as described above). The thermal cycling conditions were as follows: 1.5 min at95°C; 30 cycles of 45 sec at 95°C, 30 sec at54°C and 4 min at 72°C; and 10 min at 72°C.

[0103] The PCR products were inserted into the pET101/D-TOPO vector using the Champion pET101 Directional TOPO Expression Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Several clones were selected and the plasmid inserts sequenced to confirm that the sequence was identical to the sequence obtained by RACE.

[0104] The plasmid pET101-VzZS was used to transform BI21(DE3) E. Coli cells (Novagen, Madison, Wl). Single colonies of transformed cells were used to inoculate 5 ml LB medium. After 5 to 6 hours incubation at 37°C, the cultures were transferred to a 20°C incubator and left 1 hour for equilibration. Expression of the protein was then induced by the addition of 1 mM IPTG and the culture was incubated over-night at 20°C. The next day, the cells were collected by centrifugation, resuspended in 0.1 volume of 50 mM MOPSO pH 7, 10% glycerol and lyzed by sonication. The extracts were cleared by centrifugation (30 min at 20,000 g) and the supernatants containing the soluble proteins were used for further experiments.

[0105] The crude E coli protein extracts containing the recombinant protein was used for the characterization of the enzymatic activities. Farnesyl-diphosphate (FPP) was synthesized as described by Keller, R.K., and Thompson, R., J. Chromatogr. 645(1), 161-167, 1993. The assays were performed in 1 to 4 mL of 50 mM MOPSO pH 7, 10% glycerol, 1 mM DTT, 10 mM MgCI2 in the presence of 10 to 100 μΜ of substrate and 0.1 to 0.5 mg of crude protein. The tubes were incubated 12 to 24 hours at 30°C and extracted twice with one volume of pentane. After concentration under a nitrogen flow, the extracts were analysed by GC and GC-MS and compared to extracts from assay with control proteins. The GC analysis was performed on an Agilent 6890 Series GC system equipped with a flame ionization detector using a 0.25 mm inner diameter by 30 m SPB-1 capillary column (Supelco, Bellefonte, PA). The carrier gas was He at a constant flow of 1 mL/min. The initial oven temperature was 80°C (1 min hold) followed by a gradient of 10°C/min to 300°C. The GC-MS analysis was performed in the same conditions and the spectra were recorded on an Agilent 5975 mass detector.

[0106] In these conditions, the recombinant protein encoded by the VzZS cDNA produced one major sesquiterpene representing 75% of the sesquiterpene mixture produced. This major product was identified as being (+)-zizaene by matching of the mass spectrum and retention index with authentic standards and published data (Joulain, D., and Konig, W.A., The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998). The enzyme produces also 6.9% of prezizaene, 2.8% of a-funebrene, 2.7% of β-funebrene and at least 3 other sesquiterpenes at proportions between 0.85 and 8.7% (Figure 2). Thus, the VzZS cDNA isolated from Vetlverla zlzanoldes encoded for a (+)-zizaene synthase (SEQ ID NO:1) producing the hydrocarbon precursor of the most abundant sesquiterpenes in vetiver roots (Khusimol, zizaen-12-al, khuzenic acid). The enzyme also produced as secondary products some of the precursors of minor constituents of vetiver roots.

Example 4

Use of the recombinant VzZS protein for in-vivo production of (+)-zizaene in bacteria [0107] For optimal expression of the VzZS in E coli, the DNA sequence of the ORF of the VzCtg306 cDNA was redesigned to take into account the host codon usage and other parameters influencing the stability of the mRNA and its translation. The optimized sequence(VzZS-opt, SEQ ID NO:11) was designed and synthesized with the Nde\ and Kpn\ restriction sites and the 3’ and 5’ends (DNA 2.0, Menlo Park, CA, USA) and subcloned into the pETDuet-1 plasmid (Novagene, Madison, Wl) providing the plasmid pETDuet-VzZS-opt.

[0108] To evaluate the in-vivo production of (+)-zizaene, E. coli cells were transformed with the pETDuet-VzZS-opt plasmid and the production of sesquiterpenes from the endogenous FPP pool was evaluated. To increase the productivity of the cells, an FPP synthase and the genes encoding for a partial mevalonate pathway were also expressed in the same cells. These later genes were organized in a single operon and encoded for a mevalonate kinase (mvaK1), a phosphomevalonate kinase (mvaK2), a mevalonate diphosphate decarboxylase (MvaD) and an isopentenyl diphosphate isomerase (idi) and converted exogenous mevalonate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the two substrates of the FPP synthase.

[0109] The yeast FPP synthase gene was amplified from S. cerevisiae genomic DNA using the primers FPPy_Ncol (SEQ ID NO:12) and FPPy-Eco (SEQ ID NO:13). The genomic DNA was isolated from S. cerevisiae using the Qiagen RNA/DNA Maxi Kit (Qiagen AG, Basel, Switzerland). The PCR was performed with the Pfu DNA polymerase (Promega AG, Dubendorf, Switzerland) in a final volume of 50 μΙ containing 0.4 μΙ of each primer, 200 μΜ dNTPs, 0.5 μΙ DNA polymerase 5 μΙ S. cerevisiae genomic DNA. The PCR cycling condition were as follows: 90 sec at 95°C; 28 cycles of 45 sec at 95°C, 30 sec at 54°C and 4 min at 72°C; 10 min at 72°C. The amplified DNA was ligated as Ndel-EcoRI fragment in the first multi cloning site(MCS1)ofthe pACYCDuet-1 plasmid (Novagen, Madison, Wl) providing the plasmid pACYCDuet-FPPs harbouring the FPPs gene under the control of a T7 promoter.

[0110] An operon containing the genes encoding for mvaK1, mvaK2, MvaD and idi was amplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334, LGC Standards, Molsheim, France) with the primers MVA-up1-start (SEQ ID NO:14) and MVA-up2-stop (SEQ ID NO:15). The PCR was performed using the PfuUltra™ II Fusion HS DNA polymerase (Stratagene, Agilent Technologies Inc., Santa Clara, CA, USA). The composition of the PCR mix was according to the manufacturer instructions. The thermal cycling condition were 2 min at 95°C; 30 cycles of 20 sec at 95°C, 20 sec at 58°C and 90 sec at 72°C; and 3 min at 72°C. The 3.8 Kb fragment was purified on an agarose gel and ligated using the In-Fusion™ Dry-Down PCR Cloning Kit (Clontech Laboratories) into the second MCS of the pACYCDuet-FPPs plasmid digested with Nde\ and Xho\ providing the plasmid pACYCDuet-4506. The sequences of the two inserts were fully sequenced to exclude any mutation.

[0111] BL21 Star™(DE3) E. coli cells (Invitrogen, Carlsbad, CA) were transformed with the plasmid pETDuet-VzZS- opt or co-transformed with the same plasmid and with the plasmid pACYCDuet-4506. Transformed cells were selected on carbenicillin (50 μς/ιτιΙ) and chloramphenicol (34 μς/ιτιΙ) LB-agarose plates. Single colonies were used to inoculate 5 mL liquid LB medium supplemented with the same antibiotics. The culture was incubated overnight at 37°C. The next day 2 mL of TB medium supplemented with the same antibiotics were inoculated with 0.2 mL of the overnight culture. After 6 hours incubation at 37°C, the culture was cooled down to 28°C and 1 mM IPTG, 2 mg/mL mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5N NaOH at a concentration of 1 g/mL and incubating the solution for 30 min at 37°C) and 0.2 ml decane were added to each tube. The cultures were incubated for 48 hours at 28°C. The cultures were then extracted twice with 2 volumes of ethyl- acetate, the organic phase was concentrated to 500 μί and analyzed by GC-MS as described above in Example 3. With the cells producing the (+)-zizaene synthase, the FPP synthase and the four mevalonate pathway enzymes, a productivity of 0.1 mg/mL was obtained and the product profile was identical to the profiles observed with the in-vitro assays (Figure 3).

[0112] This example shows that an E. coli cell transformed with a (+)-zizaene synthase, as defined in the present invention, is capable of producing (+)-zizaene. The other enzymes with which the E. coli cell is transformed are not essential for the production of (+)-zizaene. Indeed (+)-zizaene is also produced when an E. coli cell is transformed with the (+)-zizaene synthase only, but in lower amounts. The other enzymes with which the E. coli cell is transformed are added for the only purpose of increasing the amount of precursor available to the (+)-zizaene synthase.

Example 5

Use of the recombinant VzZS protein for in-vivo production of (+)-zizaene in yeast [0113] For in-vivo production of sesquiterpenes in yeast cells, a Saccharomyces cerevisiae strain (YNP5) in which the ERG9 gene (coding for the squalene synthase, the enzyme converting FPP to squalene) has been down-regulated by replacing the native ERG9 promoter with the MET3 promoter, thus providing a strain with reduced ergosterol biosynthesis and higher FPP pool available for sesquiterpene synthases (Asadollahi, M.A., Maury, J., Moller., K, Nielsen, K.F., Schalk, M., Clark, A., and Nielsen, J., Biotechnology and Bioengineering 99(3), 666-677, 2008).

[0114] The VzZS cDNA was amplified from the pETDuet-VzZS-opt plasmid with the primers Ctg306_start_opt (SEQ ID NO:16) and Ctg306_stop_opt (SEQ ID NO:17). The PCR was performed with the Pfu DNA Polymerase (Promega) using the following thermal cycling conditions: 90 sec at 94°C; 35 cycles of 30 sec at 94°C, 30 sec at 55°C, 4 min at 72°C; and 10 min at 72°C. The amplified cDNA was purified and, in order to add 3’ A overhangs, was incubated 15 min at 72°C in the presence of 0.2 mM dATP and 1U HotStart Taq DNA polymerase in the appropriate buffer (Qiagen). The cDNA was ligated into pYES2.1/V5-His-TOPO® plasmid using the pYES2.1 TOPO® TA Expression Kit (Invitrogen, Carlsbad, CA). The plasmids were selected for correct sequence and orientation of the insert and were used to transform the YNP5 yeast cells using the S.c. EasyComp™ Transformation Kit (Invitrogen, Carlsbad, CA).

[0115] One single colony of transformed yeast strains were used to inoculate 20 ml of YNB medium (5 g/L (NH4)2S04; 3 g/L KH2P04; 0.5 g/L MgS04.7 H20; 1 mL/L trace metal solution) supplemented with 2% glucose. The culture was incubated for 24 hours at 28°C. The cells were recovered by centrifugation and resuspended in 20 mL of YNB medium supplemented with 2% galactose. After on 1 hour culture, methionine at 0.5 mM final concentration and 2 mL decane were added to the culture. After 24 hours incubation at 28°C, the cultures were extracted with ethyl acetate and analyzed by GC-MS as described in Example 3. The total quantity of sesquiterpenes produced by the yeast cells in these conditions was estimated at 25 mg/L.

SEQUENCE LISTING

[0116]

<110> Firmenich SA <120> METHOD FOR PRODUCING (+)-ZIZAENE <130> 7640 PCT zizaene synthase <160> 17 <170> Patentln version 3.5 <210> 1 <211> 555

<212> PRT <213> Vetiveria zizanoides <400> 1

Met Ala Thr Thr Ala Ala Phe Cys Leu Thr Thr Thr Pro lie Gly Glu 15 10 15

Pro Val Cys Arg Arg Gin Tyr Leu Pro Thr Val Trp Gly Ser Phe Phe 20 25 30

Leu Thr Tyr Gin Pro Cys Thr Pro Glu Glu Val Gin Ser Met Glu Glu 35 40 45

Arg Ala Leu Ala Lys Lys Thr Glu Val Gly Arg Met Leu Gin Glu Val 50 55 60

Ala Ala Ser Ser Asn Leu Ala Arg Lys Leu Gly Leu Val Asp Glu Leu 65 70 75 80

Glu Arg Leu Gly Val Asp Tyr His Tyr Lys Thr Glu lie Asn Asp Leu 85 90 95

Leu Gly Ala lie Tyr Asn Gly Lys Asp Asp Asp Asn Gly Gly Ser Asp 100 105 110

Asp Asp Leu Tyr lie Thr Ser Leu Lys Phe Tyr Leu Leu Arg Lys His 115 120 125

Gly Tyr Ala Leu Ser Ser Asp Val Phe Leu Lys Phe Arg Asp Glu Gin 130 135 140

Gly Asn lie Ser Ser Asp Asp Val Lys Cys Leu lie Met Leu Tyr Asp 145 150 155 160

Ala Ser His Leu Arg lie His Glu Glu Lys lie Leu Asp Asn lie Asn 165 170 175

Ser Phe Thr Lys Ser Cys Leu Gin Ser Val Leu Glu Thr Asn Leu Glu 180 185 190

Pro Ala Leu Gin Glu Glu Val Arg Cys Thr Leu Glu Thr Pro Arg Phe 195 200 205

Arg Arg Val Glu Arg lie Glu Ala Lys Arg Phe lie Ser Ala Tyr Glu 210 215 220

Lys Asn lie Ala Arg Asp Asp Ala Leu Leu Glu Phe Ala Arg Leu Asp 225 230 235 240

Tyr Asn lie Val Gin lie Leu Tyr Cys Lys Glu Leu Lys Glu Leu Thr 245 250 255

Val Trp Trp Lys Glu Phe His Ser Arg Thr Asn Leu Thr Phe Ala Arg 260 265 270

Asp Arg lie Val Glu Met Tyr Phe Trp Val Met Ala lie lie Tyr Glu 275 280 285

Pro Cys Tyr Ser Tyr Ser Arg lie Trp Val Thr Lys Met Phe Leu Ser 290 295 300

Val Ala Leu Leu Asp Asp lie Tyr Asp Asn Tyr Thr Ser Thr Glu Glu 305 310 315 320

Ser Asn lie Phe Thr Thr Ala Met Glu Arg Trp Asp Val Lys Ala Thr 325 330 335

Glu Gin Leu Pro Ala Asn Met Arg Thr Phe Tyr Asp Tyr Leu lie Cys 340 345 350

Thr Thr Asp Glu Val Val Glu Glu Leu Lys Leu Gin Asn Asn Lys Asn 355 360 365

Ala Glu Leu Val Lys Lys Val Leu lie Asp Ala Ala Lys Cys Tyr His 370 375 380

Ser Glu Val Lys Trp Arg Asp Asp His Tyr Val Pro Asn Asp Val Gly 385 390 395 400

Glu His Leu Gin Leu Ser Met Arg Ser lie Ala Ala Met His Ser lie 405 410 415

Asn Phe Val Phe lie Ser Leu Gly Ala Val Cys Thr Arg Glu Ala Val 420 425 430

Glu Cys Ala Phe Thr Tyr Pro Lys lie lie Arg Gly lie Cys Val His 435 440 445

Ala Arg lie Ser Asn Asp lie Ala Ser His Glu Arg Glu Gin Ala Ser 450 455 460

Glu His Met Ala Ser Thr Leu Gin Thr Cys Met Lys Gin Tyr Gly lie 465 470 475 480

Thr Val Glu Glu Ala Ala Glu Lys Leu Arg Val lie Asn Glu Glu Ser 485 490 495

Trp Met Asp lie Val Glu Glu Cys Leu Tyr Lys Asp Gin Tyr Pro Leu 500 505 510

Ala Leu Ser Glu Arg Val Val Ala Phe Ala Gin Ser lie Cys Phe Met 515 520 525

Tyr Asn Gly Val Asp Lys Tyr Thr lie Pro Ser Lys Leu Lys Asp Ser 530 535 540

Leu Asp Ser Leu Tyr Val Asn Leu lie Pro Val 545 550 555 <210>2 <211 > 1668

<212> DNA <213> Vetiveria zizanoides <400>2 atggcgacga ctgccgcctt ctgcctcacc accactccga tcggcgagcc agtctgtcgc 60 cggcagtacc tcccaaccgt ctggggcagc ttcttcctca cctaccagcc atgcacgccg 120 gaagaggtcc agtccatgga ggagagggct ctggccaaga agacggaggt ggggcgcatg 180 ttgcaggagg tcgccgcctc cagtaacctc gcacggaagc tgggccttgt cgatgagcta 240 gagcggctcg gggtggacta tcactacaag acggagatca acgacttgct gggtgccatt 300 tataatggca aggacgacga taatggaggt tctgatgacg acctctatat cacatcgctt 360 aagttctatc tgctcaggaa gcatgggtac gctttatctt cagatgtgtt tctgaagttc 420 agagatgagc aaggaaatat ttcaagtgat gatgtgaaat gcctgatcat gttgtatgat 480 gcctcacatt tgaggattca tgaggagaaa attcttgaca acatcaacag tttcaccaag 540 agctgcctcc aatcagtttt agaaacaaat ttggaaccgg ctctccaaga ggaggtgcgg 600 tgcacattgg agacacctcg attcagaagg gttgagagaa tcgaagcgaa acgctttatc 660 tcagcgtacg aaaagaacat agcacgagat gacgccctac tagagtttgc aaggctggac 720 tacaatatcg tgcaaattct ctactgcaag gagctgaaag aacttacagt atggtggaag 780 gagttccatt cacggacaaa tctgacattt gcacgagata gaattgtgga gatgtatttc 840 tgggtcatgg caattattta cgagccttgt tactcgtatt cacggatatg ggttacaaaa 900 atgtttctat ccgtggcatt gttggatgac atctatgaca attatacgag cacagaggag 960 agcaatatct ttactacggc catggaaagg tgggatgtga aggccaccga acaactgcca 1020 gcaaacatga ggacattcta cgattactta atttgtacaa cagatgaggt cgtagaagaa 1080 ttgaaacttc agaataataa gaatgctgaa ttagtcaaga aagtgctgat tgacgccgct 1140 aaatgctacc attcggaggt caaatggcgt gatgaccact acgtccctaa tgatgttgga 1200 gagcacctgc agctttcaat gcgaagcatt gcagctatgc actccatcaa ctttgtcttc 1260 atttcactgg gagctgtgtg tactagggag gcggttgagt gtgctttcac ttatccaaaa 1320 attattagag gtatatgtgt tcacgcacgt attagtaacg atatcgcgtc acatgagcga 1380 gaacaagctt cggagcatat ggcatcaacg ttgcaaactt gcatgaagca gtatgggatt 1440 acagtagagg aagctgctga aaagctcaga gtaataaacg aggagtcatg gatggacatc 1500 gttgaggaat gcctttataa ggaccagtat cccctggcgc tttcggagag ggtggtggct 1560 tttgcacaat caatatgttt catgtacaat ggtgtagata aatacaccat accatcaaaa 1620 ctcaaggaca gtctagactc attgtacgtc aatttgattc cagtttga 1668

<210>3 <211 > 1090 <212> DNA <213> Vetiveria zizanoides <400 3 tgtgaaatgc ctgatcatgt tgtatgatgc ctcacatttg aggattcatg aggagaaaat 60 tcttgacaac atcaacagtt tcaccaagag ctgcctccaa tcagttttag aaacaaattt 120 ggaaccggct ctccaagagg aggtgcggtg cacattggag acacctcgat tcagaagggt 180 tgagagaatc gaagcgaaac gctttatctc agcgtacgaa aagaacatag cacgagatga 240 cgccctacta gagtttgcaa ggctggacta caatatcgtg caaattctct actgcaagga 300 gctgaaagaa cttacagtat ggtggaagga gttccattca cggacaaatc tgacatttgc 360 acgagataga attgtggaga tgtatttctg ggtcatggca attatttacg agccttgtta 420 ctcgtattca cggatatggg ttacaaaaat gtttctatcc gtggcattgt tggatgacat 480 ctatgacaat tatacgagca cagaggagag caatatcttt actacggcca tggaaaggtg 540 ggatgtgaag gccaccgaac aactgccagc aaacatgagg acattctacg attacttaat 600 ttgtacaaca gatgaggtcg tagaagaatt gaaacttcag aataataaga atgctgaatt 660 agtcaagaaa gtgctgattg acgccgctaa atgctaccat tcggaggtca aatggcgtga 720 tgaccactac gtccctaatg atgttggaga gcacctgcag ctttcaatgc gaagcattgc 780 agctatgcac tccatcaact ttgtcttcat ttcactggga gctgtgtgta ctagggaggc 840 ggttgagtgt gctttcactt atccaaaaat tattagaggt atatgtgttc acgcacgtat 900 tagtaacgat atcgcgtcac atgagcgaga acaagcttcg gagcatatgg catcaacgtt 960 gcaaacttgc atgaagcagt atgggattac agtagaggaa gctgctgaaa agctcagagt 1020 aataaacgag gagtcatgga tggacatcgt tgaggaatgc ctttataagg accagtatcc 1080 cctggcgctt 1090

<210>4 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 catagcacga gatgacgccc tactagagt 29

<210> 5 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400 5 gcaaggctgg actacaatat cgtgca 26

<210>6 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 cggtggcctt cacatcccac ctttccat 28

<210> 7 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 aagatattgc tctcctctgt gctcgtat 28

<210> 8 <211> 1925 <212> DNA <213> Vetiveria zizanoides <400> 8 gaagcaaagc catctgccgt gctatcactc tagcaaatta tactgagtgg ataaacttaa 60 taccacacca gacgttttgc attcatggcg acgactgccg ccttctgcct caccaccact 120 ccgatcggcg agccagtctg tcgccggcag tacctcccaa ccgtctgggg cagcttcttc 180 ctcacctacc agccatgcac gccggaagag gtccagtcca tggaggagag ggctctggcc 240 aagaagacgg aggtggggcg catgttgcag gaggtcgccg cctccagtaa cctcgcacgg 300 aagctgggcc ttgtcgatga gctagagcgg ctcggggtgg actatcacta caagacggag 360 atcaacgact tgctgggtgc catttataat ggcaaggacg acgataatgg aggttctgat 420 gacgacctct atatcacatc gcttaagttc tatctgctca ggaagcatgg gtacgcttta 480 tcttcagatg tgtttctgaa gttcagagat gagcaaggaa atatttcaag tgatgatgtg 540 aaatgcctga tcatgttgta tgatgcctca catttgagga ttcatgagga gaaaattctt 600 gacaacatca acagtttcac caagagctgc ctccaatcag ttttagaaac aaatttggaa 660 ccggctctcc aagaggaggt gcggtgcaca ttggagacac ctcgattcag aagggttgag 720 agaatcgaag cgaaacgctt tatctcagcg tacgaaaaga acatagcacg agatgacgcc 780 ctactagagt ttgcaaggct ggactacaat atcgtgcaaa ttctctactg caaggagctg 840 aaagaactta cagtatggtg gaaggagttc cattcacgga caaatctgac atttgcacga 900 gatagaattg tggagatgta tttctgggtc atggcaatta tttacgagcc ttgttactcg 960 tattcacgga tatgggttac aaaaatgttt ctatccgtgg cattgttgga tgacatctat 1020 gacaattata cgagcacaga ggagagcaat atctttacta cggccatgga aaggtgggat 1080 gtgaaggcca ccgaacaact gccagcaaac atgaggacat tctacgatta cttaatttgt 1140 acaacagatg aggtcgtaga agaattgaaa cttcagaata ataagaatgc tgaattagtc 1200 aagaaagtgc tgattgacgc cgctaaatgc taccattcgg aggtcaaatg gcgtgatgac 1260 cactacgtcc ctaatgatgt tggagagcac ctgcagcttt caatgcgaag cattgcagct 1320 atgcactcca tcaactttgt cttcatttca ctgggagctg tgtgtactag ggaggcggtt 1380 gagtgtgctt tcacttatcc aaaaattatt agaggtatat gtgttcacgc acgtattagt 1440 aacgatatcg cgtcacatga gcgagaacaa gcttcggagc atatggcatc aacgttgcaa 1500 acttgcatga agcagtatgg gattacagta gaggaagctg ctgaaaagct cagagtaata 1560 aacgaggagt catggatgga catcgttgag gaatgccttt ataaggacca gtatcccctg 1620 gcgctttcgg agagggtggt ggcttttgca caatcaatat gtttcatgta caatggtgta 1680 gataaataca ccataccatc aaaactcaag gacagtctag actcattgta cgtcaatttg 1740 attccagttt gacgacatcg catcaagtat taattctagg cttaatataa tgccagtaaa 1800 catcatatgt aagggatatt tactttcgtg aatccaaata atttgagggg tcctgtgttc 1860 ctcttaccaa ggatatgtca tcaagttgaa aaatatagcc agcaaaaaaa aaaaaaaaaa 1920 aaaaa 1925

<210>9 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 caccatggcg acgactgccg ccttct 26

<210> 10 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 cgatgtcgtc aaactggaat ca 22

<210> 11 <211> 1671 <212> DNA <213> Artificial sequence <220> <223> Redesigned sequence for E. coli obtained from SEQ ID NO:8 <400> 11 atggctacta cggctgcttt ttgtctgact actactccta ttggtgagcc ggtgtgtcgt 60 cgccaatacc tgccgactgt gtggggtagc ttcttcttga cctaccaacc gtgcaccccg 120 gaagaggtgc aaagcatgga ggagcgtgca ttggccaaaa agaccgaggt tggccgtatg 180 ttgcaagagg tggcggccag cagcaacctg gcccgcaagc tgggtttggt tgacgagctg 240 gagcgtctgg gtgtggacta ccattacaaa accgagatta acgatctgct gggcgcgatc 300 tataatggta aggacgatga caacggcggt agcgacgatg atctgtacat tacgtctctg 360 aaattctatc tgctgcgtaa gcatggttat gcattgagca gcgatgtttt tctgaaattt 420 cgcgacgagc agggtaacat tagctccgac gacgtcaagt gcctgatcat gttgtacgac 480 gcgagccact tgcgtattca tgaggagaaa atcctggata atatcaattc cttcacgaag 540 agctgcctgc aaagcgttct ggaaaccaat ctggaaccgg cgctgcagga agaggttcgc 600 tgtacgctgg agacgccgcg tttccgccgt gtcgaacgta ttgaagcaaa acgcttcatt 660 agcgcgtacg agaagaacat tgcgcgtgac gacgctctgc tggagttcgc gcgcctggac 720 tacaacattg tccagattct gtattgcaaa gagctgaaag aactgacggt gtggtggaag 780 gagttccaca gccgcaccaa tctgacgttt gcacgtgatc gcatcgtgga aatgtacttt 840 tgggtcatgg caattatcta cgagccttgc tactcgtata gccgcatttg ggtcaccaaa 900 atgtttttga gcgtcgcact gctggatgat atttacgaca actatactag cacggaggaa 960 agcaacattt tcaccacggc gatggagcgc tgggacgtga aagcgaccga acaactgccg 1020 gcgaatatgc gtacctttta tgactatctg atctgcacca ccgacgaagt tgttgaagaa 1080 ctgaaactgc agaataacaa gaacgcggaa ctggttaaga aggtgctgat cgacgcggcc 1140 aaatgctatc atagcgaagt taaatggcgc gacgatcact acgttccgaa tgacgttggt 1200 gaacatctgc agctgtcgat gcgttccatt gcggcgatgc acagcattaa tttcgtgttc 1260 atttccctgg gcgccgtgtg tactcgcgag gcagtcgaat gcgcgtttac ttatccgaag 1320 atcattcgcg gtatttgtgt ccatgcccgt atctccaatg acattgcctc ccatgagcgc 1380 gagcaggcat ccgagcacat ggctagcact ctgcaaacct gcatgaagca gtatggcatt 1440 accgttgagg aggcggcaga aaaactgcgt gtgattaacg aggagagctg gatggatatc 1500 gtcgaggagt gcctgtacaa ggatcagtat ccgctggcat tgtctgagcg cgtcgttgca 1560 tttgcccaga gcatttgttt tatgtacaat ggcgtggaca agtacaccat tccgagcaag 1620 ctgaaggata gcctggatag cttgtatgtc aacctgattc cggtttaatg a 1671

<210> 12 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 ctagccatgg cttcagaaaa agaaattagg 30

<210> 13 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 13 ccggaattcc tatttgcttc tcttgtaaac tttgttcaag 40 <210> 14 <211> 42

<212> DNA <213> Artificial sequence <220> <223> Primer <400> 14 aaggagatat acatatgaca aaaaaagttg gtgtcggtca gg 42

<210> 15 <211> 43 <212> DNA <213> Artificial sequence <220 <223> Primer <400> 15 ctttaccaga ctcgagttac gcctttttca tctgatcctt tgc 43

<210 16 <211> 25 <212> DNA <213> Artificial sequence <220 <223> Primer <400 16 atggctacta cggctgcttt ttgtc 25

<210> 17 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 tcattaaacc ggaatcaggt tgacatac 28 Claims 1. A polypeptide having a (+)-zizaene synthase activity and comprising an amino acid sequence at least 70% identical to SEQ ID NO:1. 2. The polypeptide of claim 1, characterized in that it comprises an amino acid sequence at least 80%, preferably at least 90% identical to SEQ ID NO:1. 3. The polypeptide of claim 1, characterized in that it comprises the amino acid sequence SEQ ID NO:1. 4. The polypeptide of claim 1, characterized in that it consists of SEQ ID NO:1. 5. A nucleic acid encoding a polypeptide according to any one of claims 1 to 4, characterized in that it comprises a nucleotide sequence at least 75% identical to SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. 6. The nucleic acid of claim 5, characterized in that it comprises the nucleotide sequence SEQ ID NO:2, SEQ ID NO:11 or the complement thereof. 7. The nucleic acid of claim 5, characterized in that it consists of SEQ ID NO:2, SEQ ID NO: 11 or the complement thereof. 8. An expression vector comprising the nucleic acid of any one of claims 5 to 7. 9. The expression vector of claim 8, in the form of a viral vector, a bacteriophage or a plasmid. 10. The expression vector of claim 8 or 9, including the nucleic acid of the invention operably linked to at least one regulatory sequence, which controls initiation and/or termination of the transcription and/or translation, such as a transcriptional promoter, operator or enhancer or an mRNAribosomal binding site and, optionally, including at least one selection marker. 11. A non-human host organism or cell transformed to harbor at least one nucleic acid according to any one of claims 5 to 7, so that it heterologously expresses or over-expresses at least one polypeptide according to any one of claims 1 to 4. 12. The non-human host organism of claim 11, characterized in that it is a plant, a prokaryote or a fungus. 13. The non-human host organism of claim 11, characterized in that it is a microorganism, preferably a bacteria oryeast. 14. The non-human host organism of claim 13, characterized in that said bacteria is E. coli and said yeast is Saccha-romyces cerevisiae. 15. The non-human host cell of claim 11, characterized in that it is a plant cell or a fungal cell. 16. A method for producing (+)-zizaene comprising a) contacting FPP with at least one polypeptide according to any one of claims 1 to 4; b) optionally, isolating the (+)-zizaene produced in step a). 17. The method of claim 16, characterized in that step a) comprises cultivating a non-human host organism or cell according to any one of claims 11 to 14, under conditions conducive to the production of (+)-zizaene. 18. The method of claim 17, characterized in that it further comprises, prior to step a), transforming a non-human host organism or cell capable of producing FPP with at least one nucleic acid according to any one of claims 5 to 7, so that said organism expresses the polypeptide encoded by said nucleic acid. 19. A method for producing at least one polypeptide according to any of claims 1 to 4 comprising a) culturing a non-human host organism or cell according to any one of claims 11 to 14; b) isolating the polypeptide from the non-human host organism or cell cultured in step a). 20. The method of claim 19, characterized in that it further comprises, prior to step a), transforming a non-human host organism or cell with at least one nucleic acid according to any one of claims 5 to 7, so that said organism expresses the polypeptide encoded by said nucleic acid. 21. A method for preparing a variant polypeptide having a (+)-zizaene synthase activity comprising the steps of: (a) selecting a nucleic acid according to any one of the claims 5 to 7; (b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid; (c) transforming host cells or unicellular organisms with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence; (d) screening the polypeptide for at least one modified property; and, (e) optionally, if the polypeptide has no desired variant (+)-zizaene synthase activity, repeating the process steps (a) to (d) until a polypeptide with a desired variant (+)-zizaene synthase activity is obtained; (f) optionally, if a polypeptide having a desired variant (+)-zizaene synthase activity was identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c).

Patentansprüche 1. Polypeptid, aufweisend eine (+)-Zizaen-Synthase-Aktivität und umfassend eine Aminosäuresequenz, mindestens 70% identisch mit SEQ ID NO:1. 2. Polypeptid nach Anspruch 1, dadurch gekennzeichnet, dass es eine Aminosäuresequenz, mindestens 80%, vorzugsweise mindestens 90%, identisch mit SEQ ID NO:1, umfasst. 3. Polypeptid nach Anspruch 1, dadurch gekennzeichnet, dass es die Aminosäuresequenz SEQ ID NO:1 umfasst. 4. Polypeptid nach Anspruch 1, dadurch gekennzeichnet, dass es aus SEQ ID NO:1 besteht. 5. Nucleinsäure, codierend ein Polypeptid gemäß einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass sie eine Nucleotidsequenz, mindestens 75% identisch mit SEQ ID NO:2, SEQ ID NO: 11 oder dem Komplement davon, umfasst. 6. Nucleinsäure nach Anspruch 5, dadurch gekennzeichnet, dass sie die Nucleotidsequenz SEQ ID NO:2, SEQ ID NO: 11 oder das Komplement davon umfasst. 7. Nucleinsäure nach Anspruch 5, dadurch gekennzeichnet, dass sie aus SEQ ID NO:2, SEQ ID NO: 11 oder dem Komplement davon besteht. 8. Expressionsvektor, umfassend die Nucleinsäure nach einem der Ansprüche 5 bis 7. 9. Expressionsvektor nach Anspruch 8 in der Form eines viralen Vektors, eines Bakteriophagen oder eines Plasmids. 10. Expressionsvektor nach Anspruch 8 oder 9, einschließend die Nucleinsäure der Erfindung, operabel verknüpft mit mindestens einer regulatorischen Sequenz, welche Initiation und/oderTermination derTranskriptionund/oderTrans-lation kontrolliert, wie beispielsweise einem transkriptioneilen Promotor, Operator oder Enhancer, oder einer ribo-somalen Bindungsstelle der mRNA, und wahlweise einschließend mindestens einen Selektionsmarker. 11. Nicht-humaner Wirtsorganismus oder Zelle, transformiert, um mindestens eine Nucleinsäure gemäß einem der Ansprüche 5 bis 7 zu beherbergen, so dass er oder sie heterolog mindestens ein Polypeptid gemäß einem der Ansprüche 1 bis 4 exprimiert oder überexprimiert. 12. Nicht-humaner Wirtsorganismus nach Anspruch 11, dadurch gekennzeichnet, dass er eine Pflanze, ein Prokaryot oder ein Pilz ist. 13. Nicht-humaner Wirtsorganismus nach Anspruch 11, dadurch gekennzeichnet, dass er ein Mikroorganismus, vorzugsweise eine Bakterie oder Hefe, ist. 14. Nicht-humaner Wirtsorganismus nach Anspruch 13, dadurch gekennzeichnet, dass die Bakterie E. coli ist und die Hefe Saccharomyces cerevisiae ist. 15. Nicht-humane Wirtszelle nach Anspruch 11, dadurch gekennzeichnet, dass sie eine Pflanzenzelle oder eine Pilzzelle ist. 16. Verfahren zum Erzeugen von (+)-Zizaen, umfassend a) Inkontaktbringen von FPP mit mindestens einem Polypeptid gemäß einem der Ansprüche 1 bis 4; b) wahlweise Isolieren des (+)-Zizaens, erzeugt in Schritt a). 17. Verfahren nach Anspruch 16, dadurch gekennzeichnet, dass Schritt a) Kultivieren eines nicht-humanen Wirtsorganismus oder einer Zelle gemäß einem der Ansprüche 11 bis 14 unter Bedingungen, förderlich für die Erzeugung von (+)-Zizaen, umfasst. 18. Verfahren nach Anspruch 17, dadurch gekennzeichnet, dass es weiterhin umfasst, vor Schritt a), einen nichthumanen Wirtsorganismus oder eine Zelle, fähig zum Erzeugen von FPP, mit mindestens einer Nucleinsäure gemäß einem der Ansprüche 5 bis 7 zu transformieren, so dass der Organismus das Polypeptid, codiert durch die Nucleinsäure, exprimiert. 19. Verfahren zum Erzeugen von mindestens einem Polypeptid gemäß einem der Ansprüche 1 bis 4, umfassend a) Kultivieren eines nicht-humanen Wirtsorganismus oder einer Zelle gemäß einem der Ansprüche 11 bis 14; b) Isolieren des Polypeptids aus dem nicht-humanen Wirtsorganismus oder der Zelle, kultiviert in Schritt a). 20. Verfahren nach Anspruch 19, dadurch gekennzeichnet, dass es weiterhin umfasst, vor Schritt a), einen nichthumanen Wirtsorganismus oder eine Zelle mit mindestens einer Nucleinsäure gemäß einem der Ansprüche 5 bis 7 zu transformieren, so dass der Organismus das Polypeptid, codiert durch die Nucleinsäure, exprimiert. 21. Verfahren zum Herstellen eines Varianten Polypeptids, aufweisend eine (+)-Zizaen-Synthase-Aktivität, umfassend die Schritte: (a) Auswählen einer Nucleinsäure gemäß einem der Ansprüche 5 bis 7; (b) Modifizieren der ausgewählten Nucleinsäure, um mindestens eine mutante Nucleinsäure zu erhalten; (c) Transformieren von Wirtszellen oder unizellulären Organismen mitdermutanten Nucleinsäuresequenz, um ein Polypeptid, codiert durch die mutante Nucleinsäuresequenz, zu exprimieren; (d) Screenen des Polypeptids für mindestens eine modifizierte Eigenschaft; und (e) wahlweise, wenn das Polypeptid keine gewünschte variante (+)-Zizaen-Synthase-Aktivität aufweist, Wiederholen der Verfahrensschritte (a) bis (d), bis ein Polypeptid mit einer gewünschten Varianten (+)-Zizaen-

Synthase-Aktivität erhalten wird; (f) wahlweise, wenn ein Polypeptid, aufweisend eine gewünschte variante (+)-Zizaen-Synthase-Aktivität, in

Schritt (d) identifiziert wurde, Isolieren der entsprechenden mutanten Nucleinsäure, erhalten in Schritt (c).

Revendications 1. Polypeptide ayant une activité de (+)-zizaène synthase et comprenant une séquence d’acides aminés au moins 70% identique à la SEQ ID NO:1. 2. Polypeptide selon la revendication 1, caractérisé en ce qu’il comprend une séquence d’acides aminés au moins 80%, de préférence au moins 90%, identique à la SEQ ID NO:1. 3. Polypeptide selon la revendication 1, caractérisé en ce qu’il comprend la séquence d’acides aminés SEQ ID NO:1. 4. Polypeptide selon la revendication 1, caractérisé en ce qu’il est constitué de la SEQ ID NO:1. 5. Acide nucléique codant pour un polypeptide selon l’une quelconque des revendications 1 à 4, caractérisé en ce qu’il comprend une séquence de nucléotides au moins 75% identique à la SEQ ID NO:2, la SEQ ID NO:11 ou le complément de celles-ci. 6. Acide nucléique selon la revendication 5, caractérisé en ce qu’il comprend la séquence de nucléotides SEQ ID NO:2, SEQ ID NO:11 ou le complément de celles-ci. 7. Acide nucléique selon la revendication 5, caractérisé en ce qu’il est constitué de la SEQ ID NO:2, de la SEQ ID NO:11 ou du complément de celles-ci. 8. Vecteur d’expression comprenant l’acide nucléique selon l’une quelconque des revendications 5 à 7. 9. Vecteur d’expression selon la revendication 8, sous la forme d’un vecteur viral, d’un bactériophage ou d’un plasmide. 10. Vecteur d’expression selon la revendication 8 ou 9, incluant l’acide nucléique de l’invention lié de manière fonctionnelle à au moins une séquence régulatrice, qui contrôle l’initiation et/ou la terminaison de la transcription et/ou traduction, telle qu’un promoteur, un opérateur ou un amplificateur de transcription ou un site de fixation ribosomal d’ARNm et, optionnellement, incluant au moins un marqueur de sélection. 11. Organisme ou cellule hôte non humain transformé pour héberger au moins un acide nucléique selon l’une quelconque des revendications 5 à 7, de sorte qu’il exprime ou sur-exprime de façon hétérologue au moins un polypeptide selon l’une quelconque des revendications 1 à 4. 12. Organisme hote non humain selon la revendication 11, caracterise en ce qu’il est une plante, un procaryote ou un champignon. 13. Organisme hote non humain selon la revendication 11, caracterise en ce qu’il est un microorganisme, de preference une bacterie ou une levure. 14. Organisme hote non humain selon la revendication 13, caracterise en ce que ladite bacterie est E. coli et ladite levure est Saccharomyces cerevisiae. 15. Cellule hote non humaine selon la revendication 11, caracterisee en ce qu’elle est une cellule de plante ou une cellule de champignon. 16. Methode pour la production de (+)-zizaene comprenant a) la mise en contact de FPP avec au moins un polypeptide selon I’une quelconque des revendications 1 a 4; b) optionnellement, I’isolement du (+)-zizaene produit dans I’etape a). 17. Methode selon la revendication 16, caracterisee en ce que I’etape a) comprend la culture d’un organisme ou d’une cellule hote non humain selon I’une quelconque des revendications 11 a 14, dans des conditions favorables a la production de (+)-zizaene. 18. Methode selon la revendication 17, caracterisee en ce qu’elle comprend en outre, avant I’etapea), la transformation d’un organisme ou d’une cellule hote non humain capable de produire FPP avec au moins un acide nucleique selon I’une quelconque des revendications 5 a 7, de sorte que ledit organisme exprime le polypeptide code par ledit acide nucleique. 19. Methode pour la production d’au moins un polypeptide selon I’une quelconque des revendications 1 a 4 comprenant: a) la culture d’un organisme ou d’une cellule hote non humain selon I’une quelconque des revendications 11 a 14; b) I’isolement du polypeptide a partir de I’organisme ou de la cellule hote non humain cultive dans I’etape a). 20. Methode selon la revendication 19, caracterisee ence qu’elle comprend en outre, avantl’etapea), la transformation d’un organisme ou d’une cellule hote non humain avec au moins un acide nucleique selon I’une quelconque des revendications 5 a 7, de sorte que ledit organisme exprime le polypeptide code par ledit acide nucleique. 21. Methode pour la preparation d’un polypeptide variant ayant une activite de (+)-zizaene synthase comprenant les etapes de: (a) selection d’un acide nucleique selon I’une quelconque des revendications 5 a 7; (b) modification de I’acide nucleique selectionne pour obtenir au moins un acide nucleique mutant; (c) transformation de cellules hotes ou d’organismes unicellulaires avec la sequence d’acide nucleique mutant pour exprimer un polypeptide code par la sequence d’acide nucleique mutant; (d) le criblage du polypeptide sur au moins une propriete modifiee; et (e) optionnellement, si le polypeptide n’a pas d’activite de (+)-zizaene synthase variante desiree, repetition des etapes de precede (a) a (d) jusqu’a ce qu’un polypeptide avec une activite de (+)-zizaene synthase variante desiree soit obtenu; (f) optionnellement, si un polypeptide ayant une activite de (+)-zizaene synthase variante desiree a ete identifie dans I’etape (d), isolement de I’acide nucleique mutant correspondent obtenu dans I’etape (c).

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description • WEYERSTAHL et al. Flav. Fragr. J„ 2000, vol. 15, · Proc Natl Acad Sci USA., 1994, vol. 91 (22), 395-412 [0008] 10747-1075 [0088] • GIUDICEetal. The microbial community of Vetiver · KOLOSOVA; MILLER; RALPH; ELLIS; root and its involvement into essential oil biogenesis. DOUGLAS; RITLAND ; BOHLMANN. Isolation of

Environ. Microbiol., 2008, vol. 10 (10), 2824-2841 high-quality RNAfrom gymnosperm and angiosperm [0009] trees. J. Biotechniques, 2004, vol. 36 (5), 821-4 • TATIANA etaLFEMSM/crob/o/Leif., 1999, vol. 174, [0096] 247-250 [0023] · HERNANDEZ D. ; FRANQOIS P.; FARINELLI L.; • MARTIN, V.J.; PITERA, D.J.; WITHERS, S.T.; 0STERAS M.; SCHRENZEL J. De novo bacterial NEWMAN, J.D.; KEASLING, J.D. Nat Biotechnol., genome sequencing: Millions of very short reads as- 2003, vol. 21 (7), 796-802 [0037] sembled on a desktop computer. Genome Res, 2008, • WU, S.; SCHALK, M.; CLARK, A.; MILES, R.B.; vol. 18 (5), 802-809 [0098] COATES, R.; CHAPPELL, J. Nat Biotechnol., 2006, · ZERBINO ; BIRNEY. Velvet: algorithms for de novo vol. 24 (11), 1441-1447 [0037] short read assembly using de Bruijn graphs. Genome • TAKAHASHI, S.; YEO, Y.; GREENHAGEN, B. T.; Res, 2008, vol. 18 (5), 821-829 [0098] MCMULLIN, T.; SONG, L.; MAURINA-BRUNKER, · ALTSCHUL et al. J. Mol. Biol., 1990, vol. 215, J.; ROSSON, R.; NOEL, J.; CHAPPELL, J. Bio- 403-410 [0099] technology and Bioengineering, 2007, vol. 97 (1), · KELLER, R.K.; THOMPSON, R. J. Chromatogr., 170-181 [0037] 1993, vol. 645 (1), 161-167 [0105] • POUWELSetal. Cloning Vectors: A Laboratory Man- · JOULAIN, D.; KONIG, W.A. The Atlas of Spectral ual. Elsevier, 1985 [0074] Data of Sesquiterpene Hydrocarbons. EB Verlag, • SAMBROOK et al. Molecular Cloning: A Laboratory 1998 [0106]

Manua. Cold Spring Harbor Laboratory Press, 1989 · ASADOLLAHI, M.A. ; MAURY, J.; M0LLER., K ; [0074] NIELSEN, K.F.; SCHALK, M. ; CLARK, A.; • SCHARDL et al. Gene, 1987, vol. 61, 1-11 [0074] NIELSEN, J. Biotechnology and Bioengineering, • ZUBAY. Biochemistry, 1983 [0082] 2008, vol. 99 (3), 666-677 [0113] • ALTSCHUL. J. Mol. Biol., 1991, vol. 219, 555-565 [0082]

Claims (6)

1. SZABADALMI lOÉNYFDN'lOK I. FÓll|leptig, Iteelynék: van {^»äisiäeiSÜ.üÄ^' aktivltsSg: iiïïî&amp;iy tariafjnsií mtpoosav SzekvÓtelh isméi y legalább "Ö^-baa amatos SEQ ID NO:t-cl. A, .M: I: ίφχ^φΜ :$®φί4 pohgepikl, aszal jellemezve,; hogy ásatemaz mnisiOsav s««kvaudik aitely S legalább Ihfb^xi, elteyiseh legidâbb 9ö%-b&amp;*t ssoaos SE'Q illfNOilte.
2. 3. Μ l, Igénypépt: sxérimi pojjpepifc!;: Misai jellemezve, tegy tamimszza az sröteas. székriritölát SBQ ΙΟ NO.l.
3. 4. Az I. igénypont szerinti poHpeptid. aszal jellemezve., hogy SEQ H) NO: I -bd) áll.
4. 5. Nuklemsav. amely kódolja az. 1 -4. igénypontok bármelyike szerinti po.iipeplide·, azzal jellemezve, 1 # llögy íáííalmaz îégÉÂ 1|%^0:««ó»os SË0 10 NO:2-\ el. SEO ID NOri1~ cl. vagy annak komplementéivel. 6 Az 5. Ígés^ppt szeíisd pä tartalmazza az nuk lentid szekvenciái ShQ ID NO:2, SEQ I I) NO: 1 1 vagy annak kompiemerxtjet ?. Az S, ígénypom szexhífi xmkleiasav. azzal jeiknxiezvc. hogy SBj) ID NO:2-bÓi, SEQ ID NK): 1 -Dől 15 vagy atrmik kompkmemjébői áll
5. 8. Expresszié vektor., amely tanaimazza az 5·?, ígéxtyponmk bármelyike szerinti mtkktexavnt é. A :t. igénypt szerinti: sxpmsszió vektor vimiis vektor, haktorióllg vagy piazmid &amp;κη$$&amp;βκ 1(1, A 8. vagy f: igéaypoi«: szerinti expresszié vektor axsiely imlaimazza s találmány szerinti mikldnsavaí. amely mökédésiieg össze isa kapcsolva legalább egy szabályozási szeteerteiávah amely 20 vezérli a transzkripció es/vagy transteeio indításai és vagy befejezésé!, nnnt transzkripciós pcomoterrel, operátorral vagy enhaneertel vagy mP.NA riboszőmális köiéhdhel és. opcionálisan amely tartalmat legalább egy szelekció marker^· i l. Dem emberi host organizmus vagy sejk amely tipbsztemálva vas, hogy elhelyezzen; ispMbb egy: óUldéiiisavat áz 50, igéxtygóntbk: báinöelyike szerlhg ágy hogy lieíerolbg, iri&amp;tkm expresszál Vágy 25 táiexpmBSzáliiégáiább egy poiipepudekaz 1-4, i#őypiöÍ!!É:Mm«Iyite*eriöte 1,2:. A 1 kigbayponf szettel sem: embéfí host grpmaraas, azzal jdléxnezvs, hegy tevén v, proksrtota vagy gomba. Ill A U. igénypont szerimt nem: er#eri ItoSEotganiztnns,: azzal jelletHezye, hóik miktooxganizxmiB, déswössö bakiÉten vagy élesztő. 3b |4. A O. igénypont ázérhíli x-exs emberi bőst orgmnzntna, azzal jellemezve, hogy a baktérium &amp;< coli és az élesztó Saccharomyces ecrevssiaa. :15, A i I. igényposi nem etnbexi host se-t, azzal jellemezve, bogy növényi aejt vagy gépiin sejt. :10, Eljárás .(·+ j-zízagp; előállítását», amely tartalmazza a> ÉPP koirmkiálav.n legalább egy ixilipepílbdek az: 1-4 igésypoxtiök. htpxstelyäkp: permi; b) ppekmállsan, ki tejcKtzajga: 35 izolálását. :7. A 16. Igénypont szerinti #Ä; azzal jellemezve, hogy lépés aiiariaimäxgä s Ι7··1^· bàmïelyïke ^sce.tissts ecsn eshhes* hősi orgassssxus '-agy stgpienyoszvéss?, silÿniî.-kôrinniôn.yS'l'' b " elősegítik {: ysizaen cibáilMsát. :1.¾. A 17. Igénypont szenttti eljárás. azzal jellemezve. bogy amabba tanalmazz*. lepe*·^·*·^« maj S címben bosl orgam.mius vagy sejt ö&amp;nsz formálását. .‘.mely kepe^ $e>á?»«toS»t«* . vé .put idr.s.r. V ai .1,· ··'· {génypsMstok bármelyike szennt, ágy hogy a? orgmiiztnHs* ezptvsszálj« .b,,Jk..e.hv<>.. álul hódolt pohpept?Jet. '10:, píjátáa legalább egy polippid öjöilMísSára.m 14, ágánpoméfc bármelyiké a:icnm> hs-almazra 10 A) a ! 1-14, Sgósypöéíök bármspké sseíMÍ nem emberi hostetganlissnis vagy sejt^i^vn.t, bj ν$&amp;&amp;ήβά:Μ3φ&amp; á ii« :«S:béfí; Irnat eíganfötsnistél vsgy sbiíiökaniely lepsb« «ί vol?: tenyésztve. 2b. A 19, igénypont saM «près,, srzsl jebsmesv'e, hegy továbbá hníabnams iêges si «mÆ mm ember! "fcosb -orgasiznntäs vagy sejt transzíórnjáfásái legalább egy x;uktea?savval a? >"· »ja.-*yp»*MaK |á bármely? ke szerint. úgy begy az orgsonxnes esprosszáljs a inikleinsav alt a! kockát ponpepho^v
6. 21, Eljárás variáns; pohpeptid előállítását.*. amelynek van (;·) doaen szmiáz akisvimsa, atudy tartalmazza a kővetkező léptékek (8J az 5-7. igénypontok bármelyike szerinti nukleinsav ksváfextásás; (h) a irivüiasztotl tmkldnsav tnodoshását hogy legalább egy mutáns nokkissavm kapjunk; 20 (ej hősi sejtek vagy egységű organismes transxibnnálásá? a mmáss nökleissav szekvenciával , hogy espmssMijós poipepssÄ asmlyét a ;nstá;ss noktóesa.v szekvencia köböl : (d) a pobpeptld seteeiningiét legalább egy rsótiosbnO tulajdonságta; és. (<íj otiesonálisaík ha a txdlöeptidösfc nines árivá hi variáns l'h-jhzizae-h sstpiáz nkbvtiâs«, as ejjázáss lépesek isítséilésbí (ajstéjoOjijgyoiig ígsoj, kapudé pel|pepbáet kívánt variáns gy.i~Ktxsön ssiniaz hstivitassat; 2-5 (fi opcionálisaiig la? poiipeptid, amelynek vas; kivárni variáns· fkjrslaann ssinlár akhxi-ssig nxohöshvss: vídt,lépésben (dg izoláljuk. a lepra ej-lmn lkaptntsmgSbleló: tnuíáns nsktetnssvat. 30