BRPI0906468B1 - METHOD TO PRODUCE SCLAREOL - Google Patents

Abstract

METHOD FOR PRODUCING SCLAREOL. The present invention provides a method of producing sclareol, which method comprises contacting a particular polypeptide having a sclareol synthesizing activity with labdenediol diphosphate (LPP). In particular, said method can be carried out in vitro or in vivo to produce sclareol, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of the polypeptide used in the method. A nucleic acid derived from Salvia sclarea and encoding the polypeptide of the invention, an expression vector containing said nucleic acid, as well as an organism or a non-human host cell transformed to host the same nucleic acid, are also parts of this invention.

Description

technical field

(01) The present invention provides a method of producing sclareol, which method comprises contacting at least one polypeptide with geranyl geranyl pyrophosphate (GGPP). In particular, said method can be carried out in vitro or in vivo to produce sclareol, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequences of a polypeptide used in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are part of the present invention. A non-human host organism or a transformed cell to be used in the sclareol production method is also an object of the present invention.

state of the art

(02) Terpenes are found in many organisms (microorganisms, animals and plants). These compounds are made up of five carbon units, called isoprene units, and are classified by the amount of these units present in their structure. Thus, monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms, respectively. Diterpenes, for example, are widely found in the plant kingdom and over 2500 diterpene structures have been described (Connolly and Hill, Dictionary of Terpenoids, 1991, Chapman & Hall, London). Terpene molecules have been important for hundreds of years for their flavoring and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as a source of terpenes. Terpene molecules are often used as such, but in some cases chemical reactions are used to transform terpenes into other high-value molecules.

(03) The biosynthetic production of terpenes involves enzymes called terpene synthases. The synthesis of a diterpene can be carried out by a single enzyme or by two or more enzymes. When two enzymes are involved, the first catalyzes the synthesis of a diterpene diphosphate ester, which, in turn, is converted to the final product by the second enzyme.

(04) Two types of cyclization mechanisms occur in nature and are related to two types of diterpene synthases, which can be classified into class I and class II diterpene synthases (Wendt and Schulz, 1998, Structure. 6(2) :127-33). For some diterpenes, the cyclization mechanism is initiated by the ionization of the disphosphate ester function of GGPP, followed by the resulting carbocation reaction with an internal double bond. The diterpene synthases that catalyze this type of cyclization are class I diterpene synthases. The second mode of cyclization in diterpene biosynthesis, catalyzed by class II diterpene synthases, is initiated by protonation of the terminal double bond of GGPP and leads, after internal rearrangement and proton elimination, to a cyclic diterpene diphosphate intermediate.

(05) Genes and cDNAs encoding diterpene synthases of each of the two classes were cloned and the recombinant enzymes characterized. The availability of genes encoding different types of diterpene synthases provides information about the primary structures of the enzymes. Some amino acid motifs are conserved in diterpene synthases and are related to cyclization-dependent protonation or ionization. A DDxxD motif (where x represents any amino acid) is found in several class I diterpene synthases. Said motif is probably involved in the binding and ionization of the diphosphate moiety. In class II synthases, a conserved DxDD motif (where x represents any amino acid) is found, in which the second aspartate residue is involved as a proton donor.

(06) Sclareol is a naturally occurring diterpene molecule, extensively used as a raw material for the synthesis of fragrance molecules with ambergris notes. These syntheses were developed to provide an alternative to ambergris, a waxy substance secreted by the intestines of the sperm whale. Ambergris is highly prized for its pleasant odor and was historically used as a perfume component. Due to its high price and high demand for ambergris, and particularly due to the protection of whale species, the chemical synthesis of ambergris constituents and molecules with an ambergris character has been developed. Among these molecules, Ambrox® (trademark of Firmenich SA, Switzerland) is the most widely appreciated substitute for Ambergris. The most widely used starting material for the synthesis of Ambrox® is the diterpene diol sclareol.

(07) Generally, the price and availability of natural plant extracts are dependent on the abundance, oil yield and geographic origin of the plants. Furthermore, the availability and quality of natural extracts are much more dependent on climate and other local conditions, which lead to year-to-year variability, making the use of these components in high-quality perfumery very difficult or even impossible some years ago. Therefore, it would be an advantage to provide a source of sclareol that is less subject to fluctuations in availability and quality. Chemical synthesis would seem to be an obvious option for preparing sclareol. However, given its highly complex structure, an economical synthetic process for the preparation of sclareol is still difficult. A biochemical pathway leading to sclareol synthesis would therefore be of great interest.

(08) The biosynthesis of terpenes in plants and other organisms has been extensively studied and is not very detailed at present, but reference is made to Dewick, Nat. Product Rep., 2002, 19, 181-222, which reviews the prior art of terpene biosynthetic pathways.

(09) Several diterpene synthases have already been identified. In particular, terpene synthases, which have a certain percentage of sequence identification with the sequences of the present invention, were also found in sequence databases. However, the percentage of identification between the known diterpene synthases and the polypeptides of the invention is very low.

(010) The closest synthases to the LPP (Labdenediol diphosphate) synthases of the invention are two copalyl diphosphate synthases (one from Solanum lycopersicum (BAA84918) and one from Cucurbita maxima (AAD04293 and AAD04292)), a putative copalyl diphosphate synthase from Scoparia dulcis (BAD91286) and a hypothetical protein from Vitis vinifera. The sequences of these proteins share only 41% identification with the LPP synthases of the invention and none of these sequences are described as being useful in the production of sclareol, and as having LPP synthase activity.

(011) The synthases closest to the sclareol synthase of the invention are a terpenoid cyclase of undefined function (NCBI accession number AAS98912) having 36% identification with the polypeptide of the invention, an ent-kaurene synthase from Cucumis sativus (number accession BAB19275) having 32% identification with the polypeptide of the invention, an ent-casadiene synthase from Oryza sativa (accession number ABH10734 and published in Xu, Wilderman, Morrone Xu, Roy, Margis-Pinheiro, Upadhyaya, Coates and Peters , Functional characterization of the rice caurene synthase-like genetic family, Fitoquímica 68(3), 2007, 312-326) having 32% identification with the polypeptide of the invention and an ent-kaurene synthase from Oryza sativa (accession number AAQ72559 and published in Margis-Pinheiro, Zhou, Zhu, Dennis and Upadhyaya, Isolation and characterization of DS-tagged rice (Oryza sativa L.) GA-responsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway, Plant Cell Rep., 23(12), 2005, 819-833) having 32% identification with the sclareol synthase provided in the present invention. Furthermore, none of these sequences are described as being useful in the production of sclareol and, in particular, as being able to catalyze the transformation of LPP into sclareol.

(012) In addition to the difference between the sequences themselves, it also has to be pointed out that the structure and properties of the products synthesized by the above-mentioned enzymes are very different from those of sclareol and LPP. The properties of copalyl diphosphate are very different from those of labdenediol diphosphate (LPP). In particular, unlike LPP, copalyl diphosphate is not used as an intermediate product of sclareol biosynthesis. Ent-kaurene and ent-cassadiene are also very different from sclareol. Ent-kaurene is a tricyclic diterpene, which does not contain any alcohol functional groups, unlike sclareol, which is a bicyclic diol. Furthermore, ent-kaurene, which is a precursor of a plant growth-regulating hormone, is out of use in the field of perfumery and flavoring, considering that sclareol is of high interest in these technical fields, as explained above.

(013) A prior art document specifically relates to a sclareol synthase (Banthorpe, Brown and Morris, Partial purification of farnesyl pyrophosphate: Drimenol cyclase and geranyl geranyl pyrophosphate: Sclareol cyclase, using cell culture as a material source, Phytochemistry 31, 1992, 3391-3395). In this reference, a partially purified protein from Nicotiana glutinosa is identified as a sclareol synthase, but no indication is given, regarding the amino acid sequence of this protein, the nucleotide sequence of the nucleic acid that encodes it, or the use of this protein in a method for in vitro or in vivo sclareol biosynthesis. Furthermore, this document does not teach or even suggest that two proteins (a class I and a class II diterpene synthase) are involved in catalyzing the transformation of GGPP into sclareol. Rather, it teaches that a single, partially purified protein is responsible for sclareol synthesis.

(014) WO 2008/007031 discloses a protein having a syn-copalyl-8-ol diphosphate synthesis activity, the nucleotide sequence encoding said protein, as well as a vector and a transgenic non-human organism comprising said nucleic acid. This syn-copalyl-8-ol diphosphate synthase, however, is very different from the polypeptide of the invention, because the protein disclosed there has an amino acid sequence only 44% identical to the present LPP-synthase, used in the methods of the present invention in the polypeptide melting point of the invention.

(015) It is an object of the present invention to provide methods for creating sclareol in an economical manner as indicated above. Accordingly, the present invention aims to produce sclareol while having little residue, a more energy and resource efficient process and at the same time reducing dependence on fossil fuels. It is an additional objective to provide enzymes capable of synthesizing sclareol, which is useful as perfumery and/or aroma components.

(016) No prior art document discloses a process for the biosynthetic production of sclareol from the acyclic diterpene precursor GGPP. Abbreviations Used bp base pair kb kilo base BSA bovine serum albumin DMAPP dimethylallyl diphosphate DNA deoxyribonucleic acid cDNA complementary DNA dT deoxy thymine dNTP deoxy nucleotide triphosphate DTT dithiothreitol FPP farnesyl pyrophosphate GC gas chromatography GGPP Geranyl geranyl pyrophosphate: idi isopentenyl diphosphate isomerase IPP isopentenyl diphosphate IPTG isopropyl-D-thiogalactopyranoside LB lysogen broth LPP labdenediol diphosphate MOPSO sulfonic acid 3-(N-morpholino)-2-hydroxypropane MS mass spectrometer mvaK1 mevalonate kinase mvaK2 mevalonate diphosphate kinase PCR polymerase chain reaction 3'-/5'- RACE rapid amplification of 3' and 5' ends of cDNA RMCE recombinase-mediated cassette exchange RT-PCR polymerase chain reaction via reverse transcription ribonucleic acid RNA messenger ribonucleic acid mRNA RuBisCO ribulose-1,5-bisphosphate carboxylase SDS-PAGE polyacrylamide gel electrophoresis - SDS SsLPPs Salvia sclarea labdenediol diphosphate synthase

Description of the invention

(017) The present invention provides a method to biosynthetically produce sclareol in an economical, reliable and reproducible manner.

(018) As proposed in this application, sclareol, GGPP, LPP and all other compounds mentioned in this application are defined by the manner of their formulas, as shown in figure 1.

(019) A "diterpene synthase" or "a polypeptide having a diterpene synthesis activity" is proposed for the purpose of the present application as a polypeptide capable of catalyzing the synthesis of a terpene molecule from the acyclic terpene precursor GGPP or from a diterpene diphosphate ester, such as LPP, or capable of catalyzing the synthesis of a diterpene diphosphate ester from the acyclic diterpene precursor GGPP.

(020) As an "LPP synthase" or as a "polypeptide having an LPP synthesis activity", we refer here to a polypeptide capable of catalyzing the synthesis of LPP starting from GGPP.

(021) As a "sclareol synthase" or as a "polypeptide having a sclareol synthesis activity", we refer here to a polypeptide capable of catalyzing the synthesis of sclareol starting from LPP.

(022) As a “polypeptide capable of catalyzing the transformation of GGPP into sclareol”, we refer here to any polypeptide that is capable of catalyzing said transformation and, in particular, polypeptides that catalyze a two-step mechanism, during which LPP is synthesized as an intermediate product.

(023) The ability of a polypeptide to catalyze the synthesis of a particular diterpene (e.g. sclareol) and/or a particular diterpene diphosphate ester (e.g. LPP) can simply be confirmed by performing the enzymatic assay as detailed in Example 3.

(024) According to the present invention, polypeptides must also include truncated polypeptides, considering that they maintain their diterpene synthesis activity, as defined in any of the above configurations and that they share, at least, the defined percentage identification with the corresponding fragment of SEQ ID NO:1 or 2 or of SEQ ID NO:3. Particularly useful truncated polypeptides are those with a deletion at the N-terminus of the plastid targeting signal.

(025) The percentage of identification between two peptide or nucleotide sequences is a function of the amount of amino acid or nucleic acid residues that are identical in the two sequences, when an alignment of these two sequences was generated. Identical residues are defined as residues that are the same in the two sequences at a given position in the alignment. The sequence identification percentage, as used herein, is calculated from the favorable alignment by taking the amount of identical residues between the two sequences, dividing it by the total amount of residues in the shorter sequence and multiplying by 100. The alignment favorable is the alignment in which the identification percentage is the highest possible. Gaps can be introduced into one or both sequences at one or more positions in the alignment to obtain favorable alignment. These gaps are then taken into account as non-identical residuals for the calculation of the sequence identification percentage.

(026) Alignment for purposes of determining percentage identification of the amino acid or nucleic acid sequence can be achieved in a number of ways, using computer programs and, for example, publicly available computer programs on the world wide web. Preferably, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set for default parameters, available from the National Center for Biotechnology Information (NCBI) at http://www. ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain a favorable alignment of peptide or nucleotide sequences and calculate the percentage of sequence identification.

(027) An object of the present invention is therefore a method of producing sclareol comprising contacting GGPP with at least one polypeptide having an LPP synthesis activity and comprising an amino acid sequence at least 50% identical to that of SEQ ID NO: 1 or 2; contacting the intermediate product produced in step a) with at least one polypeptide having a sclareol synthesizing activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO:3; and optionally, isolating the sclareol produced in step b).

(028) According to a preferred configuration, steps a) and b) of the sclareol production method are carried out simultaneously by contacting the GGPP with said at least one polypeptide having an LPP synthesis activity and with said, at least one, polypeptide having a conjoint sclareol synthesizing activity.

(029) For the purpose of the present application, saying that steps a) and b) are carried out simultaneously, we mean that only one action is necessary for the person, who wants to carry out the invention, to achieve the result of both steps, i.e. that is, contacting the GGPP with at least two polypeptides or with at least one fusion polypeptide, as described below. However, sclareol production will still be carried out in a two-step mechanism, as illustrated by figure 2. LPP is first synthesized in situ from GGPP by the LPP synthase or the fusion polypeptide part, having the sequence of LPP- synthase in the presence of sclareol synthase. The LPP thus produced is, therefore, contacted directly with the sclareol synthase or with the part of the fusion polypeptide, having the sequence of the sclareol synthase, the enzyme that catalyzes the transformation of this precursor into sclareol as soon as it is produced.

(030) According to a preferred configuration, steps a) and b) of the sclareol production method are carried out simultaneously by contacting the GGPP with at least one fusion polypeptide capable of catalyzing the transformation of GGPP into sclareol and comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and an amino acid sequence at least 50% identical to SEQ ID NO:3.

(031) According to a preferred embodiment, the fusion polypeptide capable of catalyzing the transformation of GGPP into sclareol comprises the sequence of a polypeptide having an LPP synthesis activity and an amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and the sequence of a polypeptide, having sclareol synthesizing activity and an amino acid sequence at least 50% identical to SEQ ID NO:3.

(032) The method can be performed in vitro as well as in vivo, as it will be explained in detail later.

(033) The polypeptide that will come into contact with LPP in vitro can be obtained by extraction from any organism that expresses it, using standard enzyme or protein extraction technologies. If the host organism is a unicellular organism or cell that releases the polypeptide of the invention into the culture medium, the polypeptide can simply be collected from the culture medium, for example, by centrifugation, optionally followed by washing steps and resuspension in suitable buffer solutions. . If the organism or cell accumulates the polypeptide in its cells, the polypeptide can be obtained by disrupting or lysing the cells and further extracting the polypeptide from the cell lysate.

(034) The polypeptide, which has a sclareol-synthesizing activity and/or the polypeptide, which has an LPP-synthesizing activity, either in an isolated form or together with other proteins, e.g. in a crude protein extract, obtained from cultured cells or microorganisms, can then be suspended in a buffer solution at a favorable pH. If appropriate, salts, BSA, DTT and other types of enzyme cofactors can be added in order to optimize enzyme activity. Suitable conditions are described in more detail in the Examples later.

(035) The GGPP or LPP precursor can then be added to the suspension or solution, which is then incubated at a favorable temperature, for example between 15 and 40°C, preferably between 25 and 35°C, more preferably at 30°C. After incubation, the LPP or sclareol produced can be isolated from the incubated solution by standard isolation procedures such as solvent extraction and distillation, optionally after removing the polypeptides from the solution.

(036) According to another preferred embodiment, the sclareol production method is carried out in vivo. In such a case, steps a) and b) of the method described above are carried out simultaneously and comprise the culturing of a non-human host cell or organism capable of producing GGPP and transformed to express at least one polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and having an LPP synthesis activity, and at least a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:3, and having a sclareol synthesizing activity under conditions that lead to the production of sclareol.

(037) According to a more preferred configuration, the method further comprises, before step a) and b), the transformation of a non-human organism or cell, capable of producing GGPP with at least one nucleic acid, which encodes a polypeptide, comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and having an LPP synthesizing activity, and having at least one nucleic acid, encoding a polypeptide, comprising a sequence of amino acid at least 50% identical to SEQ ID NO:3 and having a sclareol synthesis activity, such that said organism expresses said polypeptides.

(038) These configurations of the invention are particularly advantageous since it is possible to carry out the method in vivo without previously isolating the polypeptide. The reaction takes place directly within the organism or cell transformed to express said polypeptide.

(039) The non-human host cell or organism can be transformed with both nucleic acids at the same time or separately. When the non-human host cell or organism is transformed with both nucleic acids at the same time, these nucleic acids can be incorporated into a single vector or into different vectors.

(040) According to a particular embodiment of the invention, at least the nucleic acid encoding the LPP synthase comprises a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60 %, preferably at least 65%, preferably 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:4, 5 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:4, 5 or the complement thereof. According to an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:4, 5 or the complement thereof.

(041) According to a particular embodiment of the invention, at least the nucleic acid encoding sclareol synthase comprises a nucleotide sequence, at least 50%, preferably at least 55%, preferably at least 60 %, preferably at least 65%, preferably 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:6 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:6 or the complement thereof. In an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:6 or the complement thereof.

(042) In another particular embodiment of the invention, the organism or non-human host cell can also be transformed with at least one nucleic acid, which encodes a fusion polypeptide, capable of catalyzing the transformation of GGPP into sclareol and comprising a amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and an amino acid sequence at least 50% identical to SEQ ID NO:3.

(043) According to a preferred embodiment, the nucleic acid encoding a fusion polypeptide comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 65% 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:4, 5 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:4, 5 or the complement thereof.

(044) According to another preferred embodiment, the nucleic acid encoding a fusion polypeptide comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 65% 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:6 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:6 or the complement thereof.

(045) According to an even more preferred embodiment, the nucleic acid encoding a fusion polypeptide consists of SEQ ID NO: 4, 5 or the complement thereof and a nucleotide sequence at least 50%, preferably in the at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 75% 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:6 or the complement thereof. Alternatively, the nucleic acid encoding the fusion polypeptide consists of SEQ ID NO:6 or the complement thereof and a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60 %, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, more preferably at least 98% and even more preferably at least 98% identical to SEQ ID NO:4, 5 or the complement thereof. Alternatively, the nucleic acid encoding the fusion polypeptide consists of a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to SEQ ID NO:4, 5 or the complement thereof and a nucleotide sequence, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at at least 95%, more preferably at least 98%, and even more preferably at least 98% identical to SEQ ID NO:6 or the complement thereof. In a more preferred embodiment, the nucleic acid encoding the fusion polypeptide consists of SEQ ID NO:4, 5 or the complement thereof and SEQ ID NO:6 or the complement thereof.

(046) According to another preferred embodiment, the nucleic acid encoding the fusion polypeptide comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, most preferably at least 95% identical and even more preferably at least 98% identical to SEQ ID NO:87. In an even more preferred embodiment, the nucleic acid encoding the fusion polypeptide comprises the nucleotide sequence SEQ ID NO:87. In a more preferred embodiment, the nucleic acid encoding the fusion polypeptide consists of SEQ ID NO:87.

(047) The non-human organism or cell can advantageously be further transformed with at least one gene encoding a polypeptide involved in the metabolism of GGPP production, such as, for example, enzymes of the MEP pathway, the MVA pathway and /or prenyl transferases. Transformation of a non-human organism or cell, capable of producing GGPP with an LPP synthase and a sclareol synthase, or with a fusion polypeptide, as described in any of the embodiments of the invention, is sufficient for the production of sclareol . However, further transformation with at least one enzyme involved in the production of GGPP and/or a GGPP precursor, i.e., isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), has the advantage of increasing the amount of precursor available for conversion to sclareol.

(048) The organism or cell must “express” a polypeptide, considering that the organism or cell is transformed to host a nucleic acid that encodes said polypeptide, this nucleic acid is transcribed into mRNA and the polypeptide is found in the organism or host cell. The term "express" encompasses "heterologously express" and "overexpress", 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 for transforming a non-human organism or cell will be described later in the part of the specification, which is devoted to such transformed non-human host organisms or cells as specific objects of the present invention and in the Examples.

(049) A particular organism or cell must be “capable of producing GGPP” when it produces GGPP naturally or when it does not naturally produce GGPP but is transformed to produce GGPP, or prior to transformation with a nucleic acid as described in this or together with said nucleic acid. Organisms or cells transformed to produce a greater amount of GGPP than the naturally occurring organism or cell are also encompassed by "organisms or cells capable of producing GGPP". Methods for transforming organisms, for example microorganisms, so that they produce GGPP are known in the art. Such methods can, for example, be found in Huang, Roessner, Croteau and Scott, Engineering Escherichia coli for the synthesis of taxadiene, an important intermediate in the biosynthesis of taxol, Bioorg Med Chem., 9(9), 2001,2237- 2242.

(050) According to a preferred embodiment, the organism accumulates GGPP naturally or is transformed to accumulate such a precursor.

(051) To carry out the invention in vivo, the organism or the host cell is cultivated under conditions that lead to the production of sclareol. Accordingly, if the host is a transgenic plant, favorable growth conditions are provided, such as favorable light, water and nutrient conditions, for example. If the host is a unicellular organism, conditions leading to sclareol production may include the addition of appropriate cofactors to the host's culture medium. In addition, a culture medium can be selected in order to maximize sclareol synthesis. Favorable culture conditions are described in more detail in the following Examples.

(052) Non-human host organisms suitable for carrying out the method of the invention in vivo can 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, prokaryote or 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 the family of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant is selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Brassica (snuff), Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha. Preferably, the plant belongs to the species of Nicotiana tabacum.

(053) In a more preferred embodiment, the non-human host organism used to carry out the invention in vivo is a microorganism. Any microorganism can be used, but according to an even more preferred embodiment, said microorganism is a bacterium or a fungus. Preferably, said fungus is a yeast. More preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

(054) Several of these organisms do not produce GGPP naturally. To be suitable for carrying out the method of the invention, these organisms have to be transformed to produce said precursor. They can be transformed prior to modification with the described nucleic acid according to any of the above configurations or simultaneously as explained above.

(055) Isolated larger eukaryotic cells can also be used, instead of whole organisms, as hosts to carry out the method of the invention in vivo. Suitable eukaryotic cells can be any non-human cell, but are preferably plant cells.

(056) According to a preferred embodiment, at least one polypeptide having an LPP synthesis activity, used in any of the above-described configurations or encoded by nucleic acid, used in any of the above-described configurations comprises an amino acid sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at 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 or 2. According to a more preferred embodiment, the said polypeptide comprises amino acid sequence SEQ ID NO:1 or 2. In an even more preferred embodiment, said polypeptide comprises SEQ ID NO:1 or 2.

(057) According to another preferred embodiment, at least one polypeptide having a sclareol synthesizing activity, used in any of the above-described configurations or encoded by nucleic acid, used in any of the above-described configurations comprises an amino acid sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% identical to SEQ ID NO:3. According to a more preferred embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO:3. In an even more preferred embodiment, said polypeptide consists of SEQ ID NO:3.

(058) According to a further preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, comprises an amino acid sequence of at least 55%, preferably, at least 60%, preferably at least 65%, preferably 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 or 2. According to a more preferred embodiment, said fusion polypeptide comprises the amino acid sequence SEQ ID NO:1 or 2.

(059) According to another preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, comprises an amino acid sequence at least 55%, preferably, in the at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 85% 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:3. According to a more preferred embodiment, said fusion polypeptide comprises the amino acid sequence SEQ ID NO:3.

(060) According to a still more preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, consists of SEQ ID NO: 1 or 2 and a sequence amino acid at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:3. Alternatively, it consists of SEQ ID NO:3 and an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at 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 or 2. Alternatively, the fusion polypeptide consists of an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 further more preferably at least 98% identical to SEQ ID NO:1 or 2 and of an amino acid sequence at least 55%, preferably at least 60%, preferably at least 65%, most preferably 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 at least 98% identical to SEQ ID NO:3. In a more preferred embodiment, the fusion polypeptide consists of SEQ ID NO:1 or 2 and SEQ ID NO:3.

(061) According to another particularly preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations comprises an amino acid sequence at least 50%, preferably, at least 55% preferably at least 60% preferably at least 65% preferably 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:88. In a more preferred embodiment, the fusion polypeptide comprises the amino acid sequence SEQ ID NO:88. According to an even more preferred embodiment, the fusion polypeptide consists of SEQ ID NO:88.

(062) According to a further specific embodiment of the invention, the polypeptide having an LPP synthesis activity, as proposed in any embodiment of the method of the invention, is a polypeptide comprising an amino acid sequence at least 50% identical to any of SEQ ID NO:36 or 39, which are truncated forms of SEQ ID NO:1. Preferably, said polypeptide comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 any one of SEQ ID NO: 36 to 39. According to a more preferred embodiment, said polypeptide comprises any one of SEQ ID NO:36 to 39. According to an even more preferred embodiment, said polypeptide comprises any one of SEQ ID NO:36 to 39.

(063) According to another specific embodiment of the invention, the polypeptide having a sclareol synthesis activity, as proposed in any embodiment of the method of the invention, is a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:74, which is a truncated form of SEQ ID NO:3. Preferably, said polypeptide comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:74. According to a more preferred embodiment, said polypeptide comprises SEQ ID NO:74. According to an even more preferred embodiment, said polypeptide consists of SEQ ID NO:74.

(064) According to another particularly preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, comprises an amino acid sequence at least 50%, preferably, at least 55% preferably at least 60% preferably at least 65% preferably 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 any one of SEQ ID NOs:36 to 39. In an even more preferred embodiment, this comprises any one of SEQ ID NOs: 36 to 39.

(065) According to another particularly preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations comprises an amino acid sequence at least 50%, preferably, at least 55% preferably at least 60% preferably at least 65% preferably 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:74. In an even more preferred embodiment, this comprises SEQ ID NO:74.

(066) In an even more preferred embodiment, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, consists of any one of SEQ ID NO:36 to 39 and a amino acid sequence, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:74. Alternatively, the fusion polypeptide, used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations, consists of SEQ ID NO:74 and an amino acid sequence, at least 50%, preferably, in the at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 75% 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to any one of SEQ ID NOs: 36 to 39. Alternatively, the fusion polypeptide consists of an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 any one of SEQ IDs NO:36 to 39 and of an amino acid sequence, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:77. In a more preferred embodiment, the fusion polypeptide used in any of the above-described configurations or encoded by the nucleic acid of any of the above-described configurations consists of any one of SEQ ID NO:36 to 39 and SEQ ID NO:74 .

(067) According to an additional embodiment, the nucleic acid encoding an LPP synthase, used in any of the configurations described above, comprises a nucleotide sequence at least 50% identical to any one of SEQ ID NO:28 to 31, which are truncated forms of SEQ ID NO:4 or the complement thereof. Preferably, said nucleic acid comprises a nucleotide sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 most preferably at least 98% identical to any one of SEQ ID NO: 28 to 31 or their complement. According to a more preferred embodiment, said nucleic acid comprises any one of SEQ ID NOs: 28 to 31 or the complement thereof. In an even more preferred embodiment, the nucleic acid consists of any one of SEQ ID NOs: 28 to 31 or the complement thereof.

(068) According to another specific embodiment of the invention, the nucleic acid encoding a sclareol synthase, used in any of the configurations described above, comprises a nucleotide sequence at least 50% identical to SEQ ID NO:73, which is a truncated form of SEQ ID NO:6 or the complement thereof. Preferably, said nucleic acid comprises a nucleotide sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 most preferably at least 98% identical to SEQ ID NO:73 or complement of it. According to a more preferred embodiment, said nucleic acid comprises SEQ ID NO:73 or the complement thereof. In an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:73 or the complement thereof.

(069) According to another specific embodiment, the nucleic acid encoding a fusion polypeptide as used in any of the above-described embodiments comprises a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 any one of SEQ ID NOs: 28 to 31 or the complement thereof. In an even more preferred embodiment, this comprises the nucleotide sequence of any one of SEQ ID NOs: 28 to 31 or the complement thereof.

(070) According to another specific embodiment, the nucleic acid encoding a fusion polypeptide as used in any of the above-described embodiments comprises a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:73 or the complement thereof. In an even more preferred embodiment, this comprises the nucleotide sequence of SEQ ID NO:73 or the complement thereof.

(071) In an even more preferred embodiment, the nucleic acid encoding a fusion polypeptide as used in any of the above-described embodiments consists of any one of SEQ ID NOs: 28 to 31 or the complement thereof and a sequence of nucleotide at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:73 or complement of it. Alternatively, the nucleic acid encoding a fusion polypeptide, as used in any of the above-described configurations, consists of any one of SEQ ID NO:73 at 50% or the complement thereof and a nucleotide sequence of at least 55 %, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% and even more preferably at least 98% identical to any one of SEQ ID NOs: 28 to 31 or the complement thereof. Alternatively, the nucleic acid encoding the fusion polypeptide consists of a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, even more preferably at least 95%, and more preferably at least 98% identical to any one of SEQ ID NOs: 28 to 31 or the complement thereof and a nucleotide sequence, at least 50%, preferably at least 55%, preferably at least 55% 60%, preferably at least 65%, preferably 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:73 or the complement thereof. In a more preferred embodiment, the nucleic acid encoding the fusion polypeptide, as used in any of the above-described embodiments, consists of any one of SEQ ID NO:28 to 31 or the complement thereof and SEQ ID NO:73 or its complement.

(072) According to another preferred embodiment, the polypeptide or nucleic acid used in the method of any of the above embodiments is derived from Salvia sclarea.

(073) An important tool for carrying out the method of the invention is the fusion polypeptide itself. A fusion polypeptide, capable of catalyzing the transformation of GGPP to sclareol and comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 2 and an amino acid sequence at least 50% identical to SEQ ID NO:3 is therefore another object of the present invention.

(074) According to a preferred embodiment, the fusion polypeptide comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 or 2. In a more preferred embodiment, the fusion polypeptide comprises the amino acid sequence SEQ ID NO:1 or 2.

(075) According to another preferred embodiment, the fusion polypeptide comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:3. According to a more preferred embodiment, said fusion polypeptide comprises the amino acid sequence SEQ ID NO:3.

(076) In an even more preferred embodiment, the fusion polypeptide consists of SEQ ID NO:1 or 2 and an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60 %, preferably at least 65%, preferably 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:3. Alternatively, the fusion polypeptide consists of SEQ ID NO:3 and an amino acid sequence, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 further more preferably at least 98% identical to SEQ ID NO:1 or 2. In a more preferred embodiment, the fusion polypeptide consists of SEQ ID NO:1 or 2 and SEQ ID NO:3.

(077) According to a particular embodiment of the invention, the fusion polypeptide comprises the sequence of a truncated form of SEQ ID NO:1, such as SEQ ID NO:36 to 39 and/or the sequence of a truncated form of SEQ ID NO:3, as SEQ ID NO:74.

(078) Therefore, according to a particular embodiment of the invention, the fusion polypeptide comprises an amino acid sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 90% 95% and even more preferably at least 98% identical to any one of SEQ ID NOs:36 to 39. In an even more preferred embodiment, it comprises any one of SEQ ID NOs:36 to 39.

(079) According to another specific embodiment, the fusion polypeptide comprises an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 further more preferably at least 98% identical to SEQ ID NO:74. In an even more preferred embodiment, this comprises SEQ ID NO:74.

(080) In an even more preferred embodiment, the fusion polypeptide consists of any one of SEQ ID NO: 36 to 39 and an amino acid sequence of at least 50%, preferably at least 55%, preferably at least 50% at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 85% 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:74. Alternatively, the fusion polypeptide consists of SEQ ID NO:74 and an amino acid sequence, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 further more preferably at least 98% identical to any one of SEQ ID NOs:36 to 39. In a more preferred embodiment, the fusion polypeptide consists of any one of SEQ ID NOs:36 to 39 and SEQ ID NO:74 .

(081) According to another particularly preferred embodiment, the fusion polypeptide of the invention comprises an amino acid sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, even more preferably at least 90% 95% and more preferably at least 98% identical to SEQ ID NO:88. In a more preferred embodiment, the fusion polypeptide comprises the amino acid sequence SEQ ID NO:88. In an even more preferred embodiment, the fusion polypeptide consists of SEQ ID NO:88.

(082) As used herein, the polypeptide is proposed as a polypeptide or peptide fragment that includes the amino acid sequences identified herein, as well as truncated polypeptides or variants, provided that they retain their activity as defined above and that they share , at a minimum, the defined percentage of identification with the corresponding fragment of SEQ ID NO:1, 2 or 3.

(083) Examples of variant polypeptides are naturally occurring proteins that result from alternative mRNA combination events or form proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences at the N- or C-terminus upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from 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 later, are also covered by the invention.

(084) As mentioned above, the nucleic acid encoding the fusion polypeptide of the invention is a useful tool for modifying non-human host organisms or cells, proposed to be used when the method is carried out in vivo.

(085) A nucleic acid encoding a fusion polypeptide, according to any of the configurations described above, therefore, is also an object of the present invention.

(086) According to a preferred embodiment, the nucleic acid comprises a nucleotide sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:4, 5 or the complement thereof. In a more preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:4, 5 or the complement thereof.

(087) According to another preferred embodiment, the nucleic acid comprises a nucleotide sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:6 or the complement thereof. According to a more preferred embodiment, said fusion polypeptide comprises the amino acid sequence SEQ ID NO:6 or the complement thereof.

(088) In an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:4, 5 or the complement thereof and a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at at least 95% and even more preferably at least 98% identical to SEQ ID NO:6 or the complement thereof. Alternatively, the nucleic acid consists of SEQ ID NO:6 or the complement thereof and a nucleotide sequence of at least 55%, at least 60%, preferably at least 65%, preferably at least 70 %, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, more preferably at least, and further more preferably at least 98% identical to SEQ ID NO:4, 5 or the complement thereof. Alternatively, the nucleic acid consists of a nucleotide sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:4 , 5 or the complement thereof and a nucleotide sequence, at least 55%, preferably at least 60%, preferably at least 65%, preferably 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: 6 or its complement. In a more preferred embodiment, the nucleic acid consists of SEQ ID NO:4, 5 or the complement thereof and SEQ ID NO:6 or the complement thereof.

(089) Particularly useful nucleic acids are those encoding fusion polypeptides, comprising truncated forms of SEQ ID NO:1 and/or 3. Therefore, nucleic acid comprising a truncated form of SEQ ID NO:4, such as SEQ ID NO :28 to 31 or the complement thereof and/or comprising a truncated form of SEQ ID NO:6, such as SEQ ID NO:73, or the complement thereof are particularly useful embodiments of the invention.

(090) Therefore, according to another specific configuration, the nucleic acid comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65% preferably 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 any one of SEQ ID NOs: 28 to 31 or the complement thereof. In an even more preferred embodiment, this comprises the nucleotide sequence of any one of SEQ ID NOs: 28 to 31 or the complement thereof.

(091) According to another specific embodiment, the nucleic acid comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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:73 or the complement thereof. In an even more preferred embodiment, this comprises the nucleotide sequence of SEQ ID NO:73 or the complement thereof.

(092) In an even more preferred embodiment, the nucleic acid consists of any one of SEQ ID NO: 28 to 31 or the complement thereof and a nucleotide sequence of at least 50%, preferably at least 55% preferably at least 60% preferably at least 65% preferably 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:73 or the complement thereof. Alternatively, the nucleic acid consists of SEQ ID NO:73 or the complement thereof and a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 90% 95%, and even more preferably at least 98%, identical to any one of SEQ ID NOs: 28 to 31 or the complement thereof. In a more preferred embodiment, the nucleic acid consists of any one of SEQ ID NO:28 to 31 or the complement thereof and SEQ ID NO:73 or the complement thereof.

(093) According to another particularly preferred embodiment, the nucleic acid comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 further more preferably at least 98% identical to SEQ ID NO:87. In a more preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:87. According to an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:87.

(094) The nucleic acid of the invention can be defined as including deoxyribonucleotide or ribonucleotide polymers in single or double stranded form (DNA and/or RNA). The term "nucleotide sequence" is also to be understood to include 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 include certain isolated nucleotide sequences, including those that are substantially free of containing endogenous material. The nucleic acid of the invention may be truncated, provided it encodes a polypeptide encompassed by the present invention, as described above.

(095) Nucleic acids, which comprise a sequence obtained by mutating SEQ ID NO:4, 5 or the complement thereof and/or a sequence obtained by mutating SEQ ID NO:6 or the complement thereof, are also covered by the invention, provided that the sequences they comprise share at least the defined percentage of identification with the corresponding fragments of SEQ ID NO: 4, 5 or 6 or their complement, and provided that they encode a fusion polypeptide, capable of catalyzing the transformation of GGPP to sclareol as defined above. Mutations can be any type of mutations of these nucleic acids, such as point mutations, deletion mutations, insertion mutations and/or frameshift mutations. A variant nucleic acid can 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 the amino acids are encoded by a preferred codon. Due to the degeneracy of the genetic code, where more than one codon can encode the same amino acid, multiple DNA sequences can encode the same polypeptide, all of these DNA sequences being covered by the invention.

(096) Another important tool for transforming organisms or host cells suitable for carrying out the method of the invention in vivo is an expression vector, comprising a nucleic acid, according to any embodiment of the invention. This vector is therefore also an object of the present invention.

(097) An "expression vector" as used herein includes any linear or circular recombinant vector, including but not limited to viral, bacteriophage, and plasmid vectors. The expert is able to select 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 transcription, translation, initiation and termination, such as a promoter, operator, or transcriptional enhancer sequence. or a ribosomal mRNA binding site, and optionally including, at a minimum, a selectable marker. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the nucleic acid of the invention.

(098) The expression vectors of the present invention can be used in the method for preparing a genetically transformed organism and/or host cell, in organisms and/or host cells that harbor the nucleic acids of the invention and in the methods of producing or creating polypeptides having a sclareol synthesizing activity, as disclosed further below.

(099) Recombinant non-human host cells and organisms, transformed to host at least a nucleic acid encoding a polypeptide having an LPP synthesizing activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO :1 or 2 and at least one nucleic acid encoding a polypeptide having a sclareol synthesizing activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO:3 such that it expresses heterologously or super - express said polypeptides are also very useful tools to carry out the method of the invention. These non-human host organisms and cells are therefore another object of the present invention.

(0100) According to a preferred embodiment, said polypeptide having an LPP synthesis activity comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably 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 or 2. According to a more preferred embodiment, said polypeptide comprises SEQ ID NO:1 or 2. According to an even more preferred embodiment, said polypeptide consists of SEQ ID NO:1 or 2.

(0101) According to another preferred embodiment, said polypeptide having a sclareol synthesis activity comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably, 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:3. According to a more preferred embodiment, said polypeptide comprises SEQ ID NO:3. According to an even more preferred embodiment, said polypeptide consists of SEQ ID NO:3.

(0102) According to a further preferred embodiment, the nucleic acid encoding a polypeptide having an LPP synthesis activity comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably 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:4, 5 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises SEQ ID NO:4, 5 or the complement thereof. According to an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:4, 5 or the complement thereof.

(0103) According to a further preferred embodiment, the nucleic acid encoding a polypeptide having a sclareol synthesizing activity comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably 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:6 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises SEQ ID NO:6 or the complement thereof. According to an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:6 or the complement thereof.

(0104) According to another preferred embodiment, said polypeptide having an LPP synthesis activity comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably, 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 any one of SEQ ID NOs:36 to 39. According to a more preferred embodiment, said polypeptide comprises any one of SEQ ID NOs:36 to 39. According to an even more preferred embodiment , said polypeptide consisting of any one of SEQ ID NO:36 to 39.

(0105) According to another preferred embodiment, said polypeptide having a sclareol synthesis activity comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably, 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:73. According to a more preferred embodiment, said polypeptide comprises SEQ ID NO:73. According to an even more preferred embodiment, said polypeptide consists of SEQ ID NO:73.

(0106) According to a further preferred embodiment, said nucleic acid encoding a polypeptide having an LPP synthesis activity comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 90% 95% and even more preferably at least 98% identical to any one of SEQ ID NOs: 28 to 31 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises any one of SEQ ID NOs: 28 to 31 or the complement thereof. According to an even more preferred embodiment, said nucleic acid consists of any one of SEQ ID NOs: 28 to 31 or the complement thereof.

(0107) According to a further preferred embodiment, the nucleic acid encoding a polypeptide having a sclareol synthesizing activity comprises a nucleotide sequence of at least 55%, preferably at least 60%, preferably at least 60% 65%, preferably 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:73 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises SEQ ID NO:73 or the complement thereof. According to an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:73 or the complement thereof.

(0108) According to another preferred embodiment, non-human host organisms and cells are transformed to host at least one nucleic acid encoding a fusion polypeptide, as described in any of the above embodiments of the invention, in a manner that it heterologously expresses or overexpresses said fusion polypeptide.

(0109) The non-human organism or cell can advantageously be further transformed with at least one gene encoding a polypeptide involved in the metabolism of GGPP production, such as, for example, enzymes of the MEP pathway, the MVA pathway and /or prenyl transferases. Transformation of a non-human organism or cell, capable of producing GGPP with an LPP synthase and a sclareol synthase, or with a fusion polypeptide, as described in any of the embodiments of the invention, is sufficient for the production of sclareol . However, further transformation with at least one enzyme involved in the production of GGPP and/or a GGPP precursor, i.e., isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), has the advantage of increasing the amount of precursor available for conversion to sclareol.

(0110) Non-human host organisms of the invention can be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human host organism of the invention is a plant, prokaryote or 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, Solanum, Sorghum, Arabidopsis, Brassica (snuff), Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha. Preferably, the plant belongs to the species of Nicotiana tabacum.

(0111) 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 bacterium or a fungus. Preferably, said fungus is yeast. More preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

(0112) Isolated larger euraryotic cells can also be transformed, rather than whole organisms. By larger eukaryotic cells, we mean here any non-human eukaryotic cell except yeast cells. The preferred larger eukaryotic cells are plant cells. The. The term "transformed" refers to the fact that the host has been genetically engineered to comprise one, two or more copies of each of the required nucleic acids in any of the configurations described above. Preferably, the term "transformed" relates to hosts that heterologously express the polypeptides encoded by the nucleic acid with which they are transformed, as well as over-express said polypeptides. Accordingly, in one embodiment, the present invention provides a transformed organism in which the polypeptides are expressed in greater amounts than in the same non-transformed organism. B. There are several methods known in the art for creating transgenic host organisms or cells, such as plants, fungi, prokaryotes, or cell cultures of larger eukaryotic organisms. Suitable 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 Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press. Cloning and expression vectors for plants and/or larger plant cells, in particular, are available to the skilled person. See, for example, Schardl et al. Gene 61: 1-11, 1987. c. Methods of transforming host organisms or cells to host 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, agrobacteria-mediated transformation, polyethylene glycol-mediated transformation, particle bombardment, microinjection of plant cells, and transformation using viruses. d. In one embodiment, transformed DNA is integrated into a chromosome of a non-human host cell and/or organism such that a stable recombinant system results. Any method of chromosomal integration known in the art may be used in the practice of the invention, including but not limited to recombinase-mediated cassette exchange (RMCE), site-specific viral chromosome insertion, adenovirus, and pronuclear injection.

(0113) In order to carry out the method of producing sclareol in vitro, as set out above, it is very advantageous to provide a method of creating at least one polypeptide having a diterpene synthesis activity, as described in any of the configurations of the invention. Therefore, the invention provides a method for producing at least one fusion polypeptide capable of catalyzing the transformation of GGPP into sclareol comprising

(0114) the cultivation of a non-human host organism or cell transformed with the expression vector of the invention, so that it hosts a nucleic acid, according to the invention and expresses or overexpresses a polypeptide encoded by said acid Nucleic and able to catalyze the transformation of GGPP into sclareol;

(0115) isolation of the polypeptide capable of catalyzing the transformation of GGPP into sclareol from the host organism or cell, non-human, cultured in step a).

(0116) According to a preferred configuration, said method further comprises, before step a), the transformation of a non-human host organism or cell with at least one expression vector of the invention, so that it hosts at least one nucleic acid according to the invention and express or overexpress the polypeptide encoded by said nucleic acid.

(0117)Transformation and cultivation of the non-human host organism or cell can be performed as described above for the in vivo sclareol production method. Step b) can be carried out using any technique well known in the art for isolating a particular polypeptide from an organism or a cell.

(0118) A "polypeptide variant", as referred to herein, means a fusion polypeptide, capable of catalyzing the transformation of GGPP to sclareol and being substantially homologous to the polypeptide, according to any of the above embodiments, but having a sequence of amino acid different from that encoded by any of the nucleic acid sequences of the invention due to one or more deletions, insertions or substitutions.

(0119) Variants may encompass conservatively substituted sequences, meaning that a given amino acid residue is replaced with a residue having similar physicochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, 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 Gln and Asn. See Zubay, Biochemistry, Addison-Wesley Pub. Co., (1983). The effects of these substitutions can be calculated using substitution score matrices such as PAM-120, PAM-200 and PAM-250, as discussed in Altschul, (J. Mol. Biol. 219:555-65, 1991). Other conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.

(0120) Naturally occurring peptide variants are also encompassed by the invention. Examples of such variants are proteins that result from alternative mRNA splicing events or proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences at the N- or C-terminus 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. Variants of the polypeptides of the invention may be used to obtain, for example, a desired elevated or reduced enzymatic activity, modified regiochemistry or stereochemistry or altered substrate utilization or product distribution, a high affinity for the substrate, an improved specificity for the production of one or more desired compounds, an increased activity different enzymatic enzyme, an increase in enzyme reaction rate, increased activity or stability in a specific environment (pH, temperature, solvent, etc.), or an improved expression level in a desired expression system. A variant or site targeted mutant can be created by any method known in the art. Variants and derivatives of native polypeptides can be obtained by isolating naturally occurring variants or nucleotide sequence variants, from other plant strains or species or the same, or by artificially programming mutations of nucleotide sequences encoding the fusion polypeptides of the invention. Alterations to the native amino acid sequence can be performed by any of a number of conventional methods.

(0121)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 increase the expression of the polypeptides, be useful in purifying the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences can 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.

(0122) Therefore, in one embodiment, the present invention provides a method for preparing a variant fusion polypeptide capable of catalyzing the transformation of GGPP into sclareol and comprising the steps of: (a) selecting a nucleic acid according to any of the settings set out 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) evaluating the polypeptide for at least one modified property; and, (e) optionally, if the polypeptide does not have the desired variant sclareol synthesis activity, repeating steps (a) to (d) of the process until a polypeptide having a desired variant sclareol synthesis activity is obtained; (f) optionally, if a polypeptide having a desired variant sclareol synthesis activity has been identified in step d), isolating the corresponding mutant nucleic acid obtained in step (c).

(0123) In step (b), a large amount of mutant nucleic acid sequences can be created, for example, by random mutagenesis, site-specific mutagenesis or DNA shuffling. Detailed genetic reassortment procedures are found in Stemmer, Reassembly of DNA by Random Fragmentation and Reassembly: In Vitro Recombination for Molecular Evolution. Proc Natl Acad Sci USA., 1994, 91(22): 107471075. In summary, 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 sites by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow ligation with fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution or deletion. Alternatively, site-specific, oligonucleotide-directed mutagenesis procedures can be employed to provide an altered gene where predetermined codons can be altered by substitution, deletion or insertion.

(0124) Accordingly, the fusion polypeptide comprising SEQ ID NO:4 or 5 and SEQ ID NO:6 may be recombined with any other diterpene synthase encoding nucleic acids, for example isolated from an organism other than Salvia clarify. In this way, mutant nucleic acids can be obtained and separated, which can be used to transform a host cell, according to standard procedures, for example, as disclosed in the present Examples.

(0125) In step (d), the polypeptide obtained in step (c) is evaluated with respect to at least one modified property, for example, a desired modified enzymatic activity. Examples of desired enzyme activities for which an expressed polypeptide can be evaluated include increased or reduced enzyme activity as measured by the KM or Vmax value, modified regiochemistry or stereochemistry, and altered substrate utilization or product delivery. The evaluation of enzymatic activity can be carried out according to procedures familiar to the person skilled in the art and those disclosed in the present Examples.

(0126) Step (e) prepares for repeating steps (a)-(d) of the process, which can preferably be carried out in parallel. Accordingly, by creating a significant amount of mutant nucleic acids, many host cells can be transformed with different mutant nucleic acids at the same time, allowing the subsequent evaluation of a high amount of polypeptides. The chances of obtaining a desired variant polypeptide can therefore be high at the discretion of the skilled person.

(0127) All publications referenced in this application are incorporated by reference into the disclosure and describe methods and/or materials in connection with which publications are cited.

Description of drawings

(0128) Figure 1: Structures of the various compounds mentioned in the description.

(0129) Figure 2: Mechanism of sclareol biosynthesis from GGPP. Enzymatic steps 1 and 2 can be catalyzed by two distinct proteins (an LPP synthase and a sclareol synthase) or by a single bifunctional enzyme (fusion polypeptide).

(0130) Figure 3: Deduced alignment of SsLPPs3 (SEQ ID NO:4) and SsLPPs9 (SEQ ID NO:5) from the amino acid sequences SEQ ID NO:1 and 2, two closely related diterpene synthases, which encode cDNAs, isolated for the purpose of the present invention. Identical residues are in white letters and different residues between the two sequences are in black letters.

(0131) Figure 4: SDS-PAGE analysis of crude soluble protein extracts from E. coli cells expressing SsLPPs3 and SsLPPs9 proteins (SEQ ID NO: 1 and 2). Lines 1 and 8: molecular weight standards; lines 2 and 7: control proteins, obtained from cells transformed with the plasmid without insert; lanes 3 and 4: proteins from cells transformed with pETDue-SsLPPs3; lanes 5 and 6: proteins from cells transformed with pETDuet-SsLPPs9. The gel was stained for total protein using Coomassie Blue.

(0132) Figure 5: SDS-PAGE analysis of the affinity of SsLPPs3 (SEQ ID NO:1) of purified recombinant sage diterpene synthase expressed in E. coli. Line M, molecular weight standard; lane 1, crude soluble protein extract from control cells; lane 2, crude soluble protein extract from cells transformed with pET28-SsLPPs3; line 3, through-flow fractions; lines 4 to 7, wash fractions; lanes 8 to 10, elution fractions with 250mM L-histidine. The gel was stained for total protein using Coomassie Blue.

(0133) Figure 6: GC analysis of products obtained after incubation of affinity purified SsLPPs3 (SEQ ID NO:1) with GGPP. (A) Direct solvent extract, (B) Solvent extract of the same sample after treatment with alkaline phosphatase.

(0134) Figure 7: (A) N-terminal sequences of full-length recombinant diterpene synthases and truncated SsLPPs3 (SEQ ID NO:1 and SEQ ID NO:36 to 39). (B) SDS-PAGE analysis of full length and truncated versions of SsLPPs3 diterpene synthases expressed in E. coli. Line M, molecular weight standard; lane 1, crude soluble protein extract from control cells; lane 2, crude soluble protein extract from cells transformed with pET28-SsLPPs3; lane 3, purified histidine-tagged SsLPPs3; row 4 and 5: respectively 1 and 0.5μL of crude soluble protein extract from cells transformed with pETDuet-SsLPPs3; lines 5 to 9, 0.5μL of crude soluble protein extract from cells transformed with pETDuet containing the four sequential deletions. The gel was stained for total protein using Coomassie Blue.

(0135) Figure 8: Alignment of amino acid sequences of class II diterpene synthase-like fragments with the sequence of Oriza sativa stemodeno synthase (Accession No. AAZ76733).

(0136) Figure 9: Alignment of the amino acid sequences of the 1132 constructs (SEQ ID NO:3 and 74) for heterologous expression in E coli.

(0137) Figure 10: GC analysis of the products obtained after incubation of the different 1132 recombinant proteins with LPP. Crude protein extracts from E. coli expressing recombinant SsTps1132 (1132-1-3; SEQ ID NO:3) and 1132-2-5 (SEQ ID NO:74) proteins were incubated with LPP in a final volume of 1mL of 50mM MOPSO, pH 7, supplemented with 15mM MgCl2.

(0138) Figure 11: CG-MS analysis of products generated from LPP by recombinant 1132-2-5 protein (SEQ ID NO:74). (A) Total ion chromatogram of the products obtained from the incubation of LPP with a crude protein extract of E. coli, transformed with pET101-1132-2-5. (B) Mass spectrum of the peak at retention time of 14.3. (C) Mass spectrum of an authentic sclareol standard.

(0139) Figure 12: GC analysis of the products obtained after co-incubation of recombinant proteins 1132 (SEQ ID NO:3 and 74) with the recombinant protein SsLPPs3 (SEQ ID NO:1) in the presence of GGPP.

(0140) Figure 13: Structure of the plasmids used for the transformation of E. coli cells in Examples 10 and 11.

(0141) Embodiments Specific to the Invention or Examples

(0142) The invention will now be described in further detail by way of the following Examples.

Example 1

(0143) Isolation of LPP synthase, which encodes Salvia clarea DNAs by a PCR approach

A. Extraction of plant matter and RNA.

(0144) Developing flower buds of Salvia sclarea (1.5 to 2cm long, 1-2 days old) were collected from the fields of Bassins (Switzerland) and directly frozen in liquid nitrogen. Total RNA was extracted using Invitrogen Concert™ Plant RNA Reagent (Carlsbad, CA) and mRNA was purified by oligodT-cellulose affinity chromatography using the FasTrack® 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA) CA), according to the manufacturer's instructions. A cDNA library was constructed using the Marathon™ cDNA Amplification Kit (Clontech, Mountain View, CA).

B. Polymerase Chain Reactions for amplification of diterpene synthase cDNAs

(0145) Amino acid sequences of class I and II diterpene synthases from different plants were aligned and conserved motifs were selected. Sequences of degenerative oligonucleotides were deduced from these conserved amino acid motifs. The DxDDTAM Motif (x being any amino acid), found in the central part of the amino acid sequences of diterpene synthases and postulated to be involved in internalization with the diphosphate moiety of GGPP in class II diterpene synthases, was used to create the primer inverse DT3F (5'-(SEQ ID NO:7)). Another motif, DVW(I/L)GK(T/S), found in some diterpene synthases, was used to create the reverse primer D/T4R (SEQ ID NO:8)).

(0146) PCR were performed using these primers on all possible combinations of reverse and reverse primers. The PCR mix contained 0.4μM of each primer, 300μM of each dNTPs, 5μL of 10X HotStartTaq® DNA polymerase buffer (Qiagen), 2μL of cDNA dissolved in 100 fold, 0.5μL of HotStartTaq® DNA polymerase in a final volume of 50μL. The cycling conditions were: 35 cycles of 45 sec at 94°C, 45 sec at 50°C and 2 min at 72°C; and 10 min at 72°C. Sizes of PCR products were evaluated on a 1% agarose gel. Strands corresponding to the expected size were excised from the gel, purified using the QIAquick® Gel Extraction Kit (Qiagen), and cloned into the pCR®2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Inserted cDNA fragments were then subjected to DNA sequencing and the sequence was compared against the GenBlank non-redundant protein database (NCBI) using the BLASTX algorithm (Altschul et al, J. Mol. Biol. 215, 403410, 1990). From different PCRs performed, only the combination of primers DT3F (SEQ ID NO:7) and DT4R (SEQ ID NO:8) provided a DNA fragment with the expected size and sequence homology to diterpene synthases. All fragments emitted from this amplification had the exact same sequence. This 354 bp sequence was named FN23 (SEQ ID NO:9).

C. Isolation of full-length cDNA by rapid cDNA end-to-end amplification (RACE).

(0147) Oligonucleotides specific for the FN23 sequence (SEQ ID NO:9) were created. FN23-F1' (SEQ ID NO:10)), FN23-F2 (SEQ ID NO:11)) and FN23-F3 (SEQ ID NO:12)). These primers were used in RT-PCR in combination with oligodT primers, extended with an adapter sequence (SEQ ID NO:13). The composition of the RT-PCR reaction mixture was as follows: 10μl Qiagen OneStep 5X RT-PCR Buffer, 400μM each dNTP, 400nM each primer, 2μl Qiagen OneStep RT-PCR Enzyme Mixture, 1μl RNasin® Ribonuclease (Promega Co., Madison, WI) and 1250ng of total RNA in a final volume of 50ml. The thermocycler conditions were: 30 min at 50°C (reverse transcription); 15 min at 95°C (DNA polymerase activation); 35 cycles of 45 sec at 94°C, 45 sec at 50°C and 90 sec at 72°C; and 10 min at 72°C. A second round of PCR was performed using the RT-PCR products as a template with the adapterP primer (SEQ ID NO:14) in combination with the same or harbored FN23-specific primers. This PCR approach yielded a 1271 bp cDNA fragment (FN30 (SEQ ID NO:15)) having a perfect 192 bp overlap with the FN23 fragment (SEQ ID NO:9) and containing the 3' end, including the codon of interruption and the 3' uncoding sequence of the corresponding cDNA.

(0148) For amplification of the 5' end of the cDNA, antisense oligonucleotides, specific for FN23 (SEQ ID NO:9) were created: FN23-R1 (SEQ ID NO:16)), FN23-R2 (SEQ ID NO: 17)), FN23-R3 (SEQ ID NO:18)). These primers were used for 5' RACE using the S. sclarea cDNA library following the Marathon™ cDNA Amplification Kit protocol (Clontech, Mountain View, CA). The thermal cycling conditions were as follows: 1 min at 94°C, 5 cycles of 30 sec at 94°C and 4 min at 72°C, 5 cycles of 30 sec at 94°C and 4 min at 70°C, 20 cycles of 30 sec at 94°C and 4 min at 68°C. This 5' RACE yielded a 1449 bp cDNA fragment (FN40 (SEQ ID NO:19) having a perfect 227 bp overlap with FN23 (SEQ ID NO:9). Comparison with known diterpene synthase sequences revealed that the fragment FN40 (SEQ ID NO:19) contained the translation initiation codon and an 87 bp clear region. Assembly of the three cDNA fragments (FN23, FN30 and FN40 (SEQ ID NO:9, 15 and 19) provided a full length cDNA sequence (SaTpsl) of 2655 bp with an open reading frame (SEQ ID NO:20) encoding 2355 bp for a protein of residues 785 (SEQ ID NO:21), having strong homology to diterpene synthases and, that is, with copalyl diphosphate synthases. The DxDD motif, involved in protonation-initiated cyclization, was present in the amino acid sequence (position 372) and the DDxD motif, involved in ionization-initiated cyclization, was not found. , this protein sequence has the typical characteristics of a class II diterpene synthase, which exclusively catalyzes the protonation-dependent cyclizations of GGPP. The heterologous expression and enzymatic characterization of this protein is detailed in Examples 2-4 below.

Example 2

(0149) Heterologous expression of S. sclarea LLP synthase in E. coli

(0150) pETDuet-1 (Novagen, Madison, WI), engineered for expression under the control of a T7 promoter, was used for expression in E. coli cells. To construct the expression plasmid, the open reading frame of SaTps 1 (SEQ ID NO:20) was PCR amplified from the cDNA library with the reverse and reverse primers SaTps-Nde (SEQ ID NO:22) and SaTps- Kpn (SEQ ID NO:23)), designed to introduce an NdeI site immediately before the start codon and a KpnI site after the stop codon. Since the open reading frame has an NdeI site at position 1614 of the open reading frame, this amplification was performed in two steps by overlap extension PCR (Horton et al, Gene 78, 68, 1989), using the primers SaTps-Nde (SEQ ID NO:22) and SaTps-Kpn (SEQ ID NO:23) in combination with primers Satps-mut1f (SEQ ID NO:24)) and Satps-mut1r (SEQ ID NO:25)), designed to remove the NdeI site without changing the amino acid sequence. The resulting cDNA was first ligated into the PCR2.1-Topo plasmid using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) and the sequences of the inserts were verified prior to subcloning as an NdeI-KpnI fragment into the pETDuet-1 vector.

(0151) Sequence analysis of several clones obtained by amplification of the cDNA library with SaTps1 specific primers showed some variability at several positions of the cDNA sequence. Seven positions have been identified where two different amino acids can be found. A position was found where insertion of a serine residue occurred in some of the clones. These positions are listed in the table below.

(0152) These variations appeared to occur in a random fashion in eleven different sequenced clones, suggesting that at least two closely related isoforms of a diterpene synthase are present in the S. sclarea genome and that the PCR amplification approach led to the mixing of the sequences. Two clones, SsLPPs3 (SEQ ID NO:4) and SsLPPs9 (SEQ ID NO:5), representing sequence variability (Figure 3), were selected for heterologous expression and enzyme characterization experiments.

(0153) Plasmids pETDuet-SsLPPs3 and pETDuet-SsLPPs9 were transferred into E. coli Bl21 (DE3) cells (Novagen, Madison, WI). Single colonies of transformed cells were used to inoculate 5ml of LB medium. After 5 to 6 hours of incubation at 37°C, the cultures were transferred to a 20°C incubator and allowed to equilibrate for 1 hour. Protein expression was then induced by the addition of 1mM IPTG and the culture was incubated overnight at 20°C. The following day, cells were collected by centrifugation, resuspended in 0.1 volume of 50mM MOPSO, pH 7, 10% glycerol and lysed by sonication. Extracts were cleared by centrifugation (30 min at 20,000g) and supernatants containing soluble proteins were used for further experiments.

(0154) Crude protein extracts from pETDuet-SsLPPs3 and pETDuet-SsLPPs9 transformed cells were analyzed by SDS-PAGE and compared with protein extracts obtained from cells transformed with the empty pETDuet plasmid. Recombinant SsLPPs3 and SsLPPs9 proteins (SEQ ID NO:1 and 2) were clearly detected and the apparent molecular weight estimated at 90 KDa, a value in agreement with the calculated molecular weight of 83 KDa (Figure 4).

Example 3

(0155) Purification of S. sclarea LPP synthase and enzymatic activities

(0156) To further characterize the recombinant diterpene synthases, we ensured to purify the enzymes SsLPPs3 and SsLPPs9 (SEQ ID NO:1 and 2).

(0157) Plasmids PCR2.1-Topo containing the cDNA of SsLPPs3 and SsLPPs9 (SEQ ID NO:4 and 5) (Example 2) were digested with NdeI and SacI and the inserts were ligated into plasmid pET28a(+) (Novagen) . The resulting expression plasmids (pET28-SsLPPs3 and pET28-SsLPPs9) contain the cDNAs with a 5'-end modification (SEQ ID NO:26 and 27), created to express the proteins with an N-terminal hexahistidine tag. Purification was performed under native conditions using the ProBondTM Purification System (Invitrogen), following the manufacturer's protocol, except that for the elution, imidazole was substituted for L-histidine to minimize enzyme inhibition. Using this approach, recombinant enzymes SsLPPs3 and SsLPPs9 could be purified to apparent homogeneity (Figure 5). The. Affinity purified enzymes were incubated 12 hours at 30°C with 200μM GGPP and 1mM DTT in MOPSO, pH 7, 10% glycerol. No diterpene products were observed when extracting the pentane incubation and analyzing the extract by GC or GC-MS. Treatment of the same extract by alkaline phosphatase (Sigma, 6 units/ml), followed by pentane extraction and GC analysis, showed the formation of labdenediol (Figure 6) and demonstrated the enzymatic formation of labdenediol-diphosphate (LPP) as a product of GGPP by recombinant diterpene synthase.

(0158) GC analysis was performed on an Agilent 6890 Series GC system (Agilent Technologies), equipped with a flame ionization detector, using a 0.25mm ID per 30mm SPB-1 capillary column (Supelco, Bellefonte, PA). The carrier gas was He at a constant flow rate of 1mL/minute. The initial oven temperature was 100°C (1 minute of suspension), followed by a gradient of 10°C/minute to 300°C.

(0159) GC-MS analysis was performed on a Serial 6890 GC system (Agilent), coupled to a 5975 mass detector (Agilent Technologies). The column was equipped with an internal diameter of 0.25mm per 30m long DB-1MS column (Agilent Technologies). The carrier gas was He at a constant flow rate of 1mL/minute. The initial oven temperature was 80°C, followed by a gradient of 10°C/minute to 280°C. Spectra were recorded at 70eV with an electron multiplier voltage of 2200V. Product identification was confirmed by concordance of retention times and equivalence of the mass spectrum with the spectrum of authentic standards.

Example 4

(0160) N-terminal deletions of LPP synthase from Salvia sclarea a. In plants, diterpene synthases are located in plastids. This compartmentalization is controlled by a processing mechanism that recognizes an N-terminal transit peptide signal. Thus, diterpene synthases are generally expressed as preproteins and are processed in plastids by cleavage of the signal peptide resulting in a protein mature. N-terminal sequence analysis of SsLPPs3 and SsLPPs9 (SEQ ID NO:1 and 2) using the ChloroP method (Emanuelsson et al, Protein Science 8, 978-984, 1999; http://www.cbs.dtu. dk/services/ChloroP/) did not reveal any clear evidence of the presence of a transit peptide. Experiments were therefore performed to assess the effect of N-terminal deletions on enzyme activity. Four truncated cDNAs were created for SsLPPs3 resulting in deletion of 17, 37, 53 and 63 amino acids, respectively, (SEQ ID NO: 28 to 31). Each construct was created by PCR using four different reverse primers each, engineered to stiffen at the position of the desired truncated shape and introducing an NdeI restriction site, followed by an ATG translation initiation codon (SsLPPs3_del1, (SEQ ID NO:32 ); SsLPPs3_del2, (SEQ ID NO:33); SsLPPs3_del3, (SEQ ID NO:34), SsLPPs3_del4, (SEQ ID NO:35)). These primers were used in combination with the SaTps-Kpn primer (SEQ ID NO:23) (Example 2) and the four cDNAs obtained (SEQ ID NO:28 to 31) were ligated into plasmid pETDuet-1. Heterologous expression of the proteins (SEQ ID NO:36 39) was performed in E. coli, as described in Example 2. Figure 7 shows an SDS-PAGE analysis comparing the production level of the heterologous proteins obtained with the different extension constructs full and truncated. An improved expression level was clearly seen especially for the two major deletions. These results are typical for plastid-located terpene synthases and reflect an improved solubility and/or stability of the mature protein compared to the preprotein.

Example 5

(0161) Massively parallel sequencing of a cDNA library from the S. sclarea flower

(0162) We used the massively parallel sequencing technology of small DNA fragments, developed by Illumina (San Diego, California) to obtain sequence information of all transcripts (transcriptome) present in Salvia sclarea flowers. This sequencing technique uses reversible terminator-based sequencing chemistry and the Cluster Station and Genome Sequencer apparatus developed by Solexa and Illumina (www.illumina.com).

(0163) Technology and equipment were prepared at Fasteris SA (Geneva, Switzerland) and DNA sample preparation and sequencing were performed by Fasteris SA. An aliquot (1μg) of the cDNA library, generated from developing S. sclarea flowers and using the Marathon™ cDNA Amplification Kit (Clontech, Mountain View, CA) (Example 1), was treated using the Sample Preparation Kit Genomics (Illumina). Briefly, the DNA is fragmented by spraying, the ends are repaired to generate tight ends, adapters are ligated to the ends of the DNA fragments, and the adaptor-modified DNA fragments are amplified by PCR. After controlling the quality of the library by gel electrophoresis, the generation of DNA clusters on the flow cell and the sequencing reaction is carried out in the Cluster Station and Genome Sequencer equipment. Using this technology, 1.9 million short sequences (reads) of 35 bases were obtained.

(0164) Edena software (Hernandez et al, Genome res. 15(5), 802-809, 2008) was used to reassemble contiguous sequences. The last five bases were first removed from each read due to possible wrong incorporations due to low fidelity in the last cycles of the sequencing procedure. The software parameters were set to allow a minimum length of 15 bases for overlays with strict identification (100%). Contigs (contiguous sequences) with a length of at least 50 bases were retained. Under these conditions, 2054 contigs from 50 to 1330 bases in length could be reconstituted.

(0165) To assess assembly quality, contigs were screened for sequence identification with the DNA sequence of SsLPPs3, the class II diterpene synthases, first isolated from the S. sclarea cDNA library (SEQ ID NO :4, Example 2). This search was performed using the BLASTn method (Altschul et al, J. Mol Biol. 215, 403-410, 1990). Surprisingly, only 3 contigs of lengths of 81, 73 and 52 bases (contigs 1 to 3, SEQ ID NO:40 to 42) were found and only forty reads were used by the Eland software to generate these contigs. Alignment with the SsLPPs3 reference sequence showed that the 3 contigs covered only 8.7% of the full-length sequence, albeit with a 99% identification.

(0166) Very limited sequence information has been reported in public databases for Salvia sclarea. The only genetic sequence available from the NCBI database (http://www.ncbi.nlm.nih.gov) was the large subunit sequence of ribulose-1,5-bisphosphate carboxylase (RuBisCO) from salvia sclarea (accession no. NCBI Z37450). Searching the contigs for DNA identification with this S. sclarea RuBisCO DNA sequence (BLASTn Search) yielded two contigs of 870 and 547 bases, respectively (contigs 4 and 5, SEQ ID NO:43 and 44) . Alignment of the two contigs with the RuBisCO sequence showed 98% coverage: only 27 bases (between position 858 and 884) out of the 1420 bases were not present in the contigs. In addition to this almost complete coverage, the identification between the reference sequence and the contigs was 99.5%, representing a difference of only 7 nucleotides.

(0167) All reads (unassembled data) were then searched for sequence identification with the sequence of SsLPPs3 (SEQ ID NO:4). Eland software (Illumina) was used to perform this search, allowing a maximum of 2 divergences from the reference sequence. A total of 616 readings were retrieved. Alignment of selected fragments with the reference sequence revealed that the SsLPPs3 sequence was covered to the fullest extent with slightly greater coverage (more reads) towards the 3' end. The same manipulation with the RuBisCO sequence showed that 1650 reads were obtained for this sequence. Coverage of the reference sequence with the reads was much higher for RuBisCO than for SsLPPs3. For SsLPPs3 (SEQ ID NO:4), several small gap regions and regions with sequence ambiguity between reads were found. This incomplete coverage prevents complete reassembly and is certainly the reason for generating only a few very small contigs.

Example 6

(0168) Extraction of class I diterpene synthase-like sequences from sequencing data

(0169) The Blast algorithm (Altschul et al, J. Mol. Biol. 215, 403-410, 1990) was used to search for homology of the deduced amino acid sequences with sequences of class I diterpene synthases.

(0170) A Blastx search against a protein database was first performed on the 2054 contigs. This search yielded only one contig (contig 1610, SEQ ID NO:45), showing sequence homology with class I diterpene synthases. The deduced amino acid sequence of this contig contained the DDxxD motif, characteristic of cyclization initiated by prenyl-ionization. diphosphates.

(0171) A fraction of the raw data, representing approximately 3x105 reads, was then searched for homology to class I diterpene synthases. The reads were searched using the tBlastn algorithm with five class I diterpene synthase amino acid sequences. I selected (NCBI accession numbers AAC39443, BAB19275, BAB12441, AAD34295, AAS98912). This search selected 462 reads, which were then processed using the CAP program (Huang, Genomics 14(1), 18-25, 1992) to identify overlapping sequences. A small portion of the reads could be assembled into short contigs of a maximum length of 111 bases. These contigs, as well as the remaining isolated reads, were used to screen Blastx against a protein database to confirm its identification with class I diterpene synthases. Finally, 5 DNA fragments were retained (SEQ ID NO:46 to 50).

(0172) Amino acid sequences (SEQ ID NO:51 to 55) were deduced from the selected fragments and were aligned with reference diterpene synthase sequences, allowing their relative positioning. Figure 8 shows an alignment of these sequences with the full-length diterpene synthase sequence of Oriza sativa estemodeno synthase (Morrone et al, 2006; NCBI Accession No. AAZ76733) collected as a reference.

Example 7

(0173) PCR amplification of full-length class I diterpene synthase cDNAs

(0174) A set of reverse and reverse oligonucleotides was deduced from DNA sequences related to diterpene synthases selected from the sequencing of the S. sclarea cDNA library (Example 6). These primers were used in combination with cDNA adapter primers in 3'/5' RACE PCR amplifications. Amplifications were performed using the S. sclarea cDNA library, prepared as described above in Example 1, following the Marathon™ cDNA Amplification Kit protocol (Clontech, Mountain View, CA). The thermal cycling conditions were as follows: 1 min at 94°C, 5 cycles of 30 sec at 94°C and 4 min at 72°C, 5 cycles of 30 sec at 94°C and 4 min at 70°C, 20 cycles of 30 sec at 94°C and 4 min at 68°C.

(0175) Using the primer Cont250-Fwd (SEQ ID NO:56), a 547 bp DNA sequence (1130Cont250, SEQ ID NO:66) was obtained. Sequence analysis revealed that it corresponded to the 5' end of a diterpene synthase cDNA and contained 348 bp of coding region. With the primer Cont147_fw1 (SEQ ID NO:57) and Cont147_fw2 (SEQ ID NO:58), we obtained a 1473 bp sequence (1132Cont147, SEQ ID NO:67), containing the 3' end and 1293 bp of the coding region of a diterpene synthase cDNA. The primers Cont147_rev1 (SEQ ID NO:59) and Cont147_rev2 (SEQ ID NO:60) allowed the amplification of a 464 bp DNA fragment (1134Cont147, SEQ ID NO:68). The deduced amino acid showed homology with diterpene synthases, but alignment with other diterpene synthase sequences suggested that 200 to 300 codons were still missing to reach the 5' end. All sequences obtained by this round of amplification differed significantly from sequences from previously isolated SsLPPs (SsLPPs3 and SsLPPs9, SEQ ID NO:4 and 5).

(0176) A 5'RACE approach was used to identify the 5' end of the ORF, corresponding to sequence 1132Cont147 (SEQ ID NO:67). Using primers 1132_race1 (SEQ ID NO:62) and 1132_race2 (SEQ ID NO:63), a 536 bp sequence (1132RACE, SEQ ID NO:69) was obtained, which overlapped by 41 bases with the fragment 1132Cont147 ( SEQ ID NO:67). The N-term of this RACE product was identical to the previously obtained 1134Cont147 sequence (SEQ ID NO:68) and therefore no extension to the 5' end was observed. As previously noted, this sequence had homology to diterpene synthases, but appeared shorter by at least 200 codons than all other published diterpene synthase sequences. 5'RACE experiments were performed in order to try to extend the sequence towards the 5' end of the 1132Cont147 sequence (SEQ ID NO:67) and to identify the true translation initiation codon. Several sets of oligonucleotides were created, but no further sequence information was obtained. This led us to assume that one of the ATG codons in sequence 1134Cont147 (SEQ ID NO:68) was actually the start codon of the corresponding diterpene synthase gene. The nucleotide sequence of this putative diterpene synthase (named SsTps1132, SEQ ID NO:6) was reconstructed from sequences 1132Cont147 (SEQ ID NO:67) and 1132RACE (SEQ ID NO:69). Taking the first ATG, the 1728 bp ORF of SsTps1132 (SEQ ID NO:6) encoded a protein of 575 amino acids (SEQ ID NO:3). This protein contained the ionization-dependent motif (DDFFD) and shared, but relatively low, homology with the published diterpene synthases; the closest sequence being a terpene synthase from Nicotiana tabacum (NCBI Accession No. AAS98912), identification 37%. This protein also shared only 23% identification with SsLPPs isolated from S. sclarea in Examples 1 to 4. SsTps1132 was aligned with selected diterpene synthase sequences. These alignments showed that SsTps1132 (SEQ ID NO: 3) is truncated at the N-terminal end by 150 to 240 amino acids compared to other diterpene synthases.

(0177) The ChloroP method (Emanuelsson et al, Protein Science 8, 978-984, 1999; http://www.cbs.dtu.dk/services/ChloroP/) was used to predict the presence of a transit peptide of chloroplast in SsTps1132 (SEQ ID NO:3). A 51 amino acid chloroplast transit peptide was predicted, supporting a chloroplast localization of this protein.

(0178) Searching all reads for sequences identical to the SsTps1132 DNA sequence (SEQ ID NO:6) yielded 425 reads. The expression level of SsTps1132 (220 reads/Kb) was similar to the expression level of SsLPPs (SEQ ID NO:4 and 5) (260 reads/Kb). With the hypothesis that enzyme catalytic steps in the same metabolic pathway are generally expressed at a similar level, it can be speculated that SsTps1132 is involved in the same metabolic pathway as SsLPPs.

(0179) Contigs generated with the Edena software (Example 5) were searched for DNA sequences, identical to the sequences of the new putative class I diterpene synthases SsTps1132 (SEQ ID NO:3) Four contigs were found. The previously identified contig 1610 (SEQ ID NO:45) and three additional contigs (53 to 96 bp in length, SEQ ID NO:70 to 72), not previously identified as a fragment of a diterpene synthase. Blastx search of these three sequences showed no homology to known protein sequences. The failure to discover homology for these contigs is due to the short lengths of these fragments and the low homology of SsTps1132 with the diterpene synthases present in the databases.

Example 8

(0180) Heterologous expression of S. sclarea class I diterpene synthases in E. coli

(0181) To determine an enzymatic activity for SsTps1132 (SEQ ID NO:3), recombinant proteins were expressed in E. coli. The full-length cDNAs were inserted into the pet101/D-TOPO vector using the pET101 Champion Directional TOPO Expression Kit.

(0182) Two constructs were prepared: one to express the full-length protein and one to express a truncated protein, based on the prediction of the chloroplast transit peptide. The full-length SsTps1132 open reading frame (SEQ ID NO:6) was amplified from the cDNA library using primer pair 1132_start_1 (SEQ ID NO:64) and 1132-stop (SEQ ID NO:61). Primers 1132_start2 (SEQ ID NO:65) and 1132_stop (SEQ ID NO:61) were used to prepare the construct for expression of SsTps1132 (SEQ ID NO:3) with an N-terminal deletion of 50 amino acids. All cDNA amplifications for expression of the expression constructs were performed using Pfu DNA polymerase (Promega), in a final volume of 50μl, containing 5μg of Pfu DNA polymerase 10X buffer, 200μM each of dNTP, 0.4μM each of reverse and reverse primer, 2.9 units of Pfu DNA polymerase and 5µl of 100-fold diluted cDNA (prepared as described herein in Example 1 using the Marathon™ cDNA Amplification Kit (Clontech)). Thermal cycling conditions were as follows: 1.5 min at 95°C; 30 cycles of 45 sec at 95°C, 30 sec at 58°C and 5 min at 72°C; and 10 min at 72°C.

(0183) After ligation into the pET101 vector, several clones were selected for each construct and were sequenced to ensure that no mutations were introduced during PCR amplification. Two constructs were selected: SsTps1132 (SEQ ID NO:6) and 1132-2-5 (SEQ ID NO:73). The alignment of the two amino acid sequences deduced from these constructs is shown in Figure 9.

(0184) Plasmids pET101-SsTps1132 and pET101-1132-2-5 were transferred into E. coli Bl21 (DE3) cells (Novagene, Madison, WI). Single colonies of transformed cells were used to inoculate 5ml of LB medium. After 5 to 6 hours of incubation at 37°C, the cultures were transferred to a 20°C incubator and allowed to equilibrate for 1 hour. Protein expression was then induced by the addition of 1mM IPTG and the culture was incubated overnight at 20°C. The following day, cells were collected by centrifugation, resuspended in 0.1 volume of 50mM MOPSO, pH 7, 10% glycerol and lysed by sonication. Extracts were cleared by centrifugation (30 min at 20,000g) and supernatants containing soluble proteins were used for further experiments. Crude protein extracts were analyzed by SDS-PAGE and compared with protein extracts obtained from cells transformed with empty plasmid pET101. It appeared that deletion of the signal peptide improved heterologous expression in E. coli.

Example 9

(0185) Enzyme activity of recombinant class I diterpene synthases from S. sclarea in E. coli'

(0186) E. coli crude protein extracts, containing the recombinant proteins and prepared as described in Example 8 were used for the characterization of enzymatic activities. Enzymatic assays were performed as described in Example 3. All assays were performed in 50mM MOPSO, pH 7, 10% glycerol, 1mM DTT.

(0187) Enzyme activities were first evaluated using a GGPP or LPP substrate, the product of SsLPPs and the presumed intermediate in sclareol biosynthesis (Examples 1 to 4). GGPP was synthesized as described by Keller and Thompson (J. Chromatogr 645(1), 1993, 161-167) and LPP was prepared enzymatically as described in Example 3. Assays were performed in the presence of 10 to 100μM substrate, 15mM MgCl2 and 0.1 to 0.5mg of crude protein in a total volume of 1mL. Tubes were incubated 4 to 12 hours at 30°C and extracted twice with one volume of pentane. After concentration under nitrogen flow, the extracts were analyzed by GC and GC/MS (using the conditions described in Example 3) and compared with extracts from the control protein assay (obtained from cells transformed with the empty plasmid). With GGPP as substrate, no activity was observed with any of the recombinant proteins (data not shown). With LPP as substrate, activity was observed with SsTps1132 (SEQ ID NO:3) and 1132-2-5 (SEQ ID NO:74) (Figure 10). The enzymes were also active in the absence of MgCl2 and the same product profiles were observed with almost the same overall activity. Product identification was confirmed by matching retention times (Figure 10) and comparing the mass spectrum with the spectrum of an authentic standard (Figure 11). In all assays, a single sclareol peak was observed with no trace of additional product.

(0188) Assays were then performed with coincubation of class II diterpene synthases (SsLPPs3, SEQ ID NO:1; Examples 1-4) and class I diterpene synthases (series 1132, SEQ ID NO:3 and 74). Assays were performed in 50mM MOPSO, pH 7, 10% glycerol, 1mM DTT, 50μM GGPP, with 1mM MgCl2 and in the presence of 50μL of E. coli crude protein extracts, expressing the different constructs. Thus, assays in the presence of 50μL of crude protein extracts containing the recombinant enzyme SsLPPs3 (SEQ ID NO:1) and 50μL of extracts containing SsTps1132 (SEQ ID NO:3) or 1132-2-5 (SEQ ID NO:74 ) were evaluated for the production of diterpene products. Figure 12 shows the GC profiles of extracts from these incubations in the presence of MgCl2. Sclareol was produced with both 1132 constructs (SEQ ID NO:3 and 74), a result resistant to the assay described above with LPP as substrate. No significant difference was observed when omitting MgCl2 from incubations (data not shown).

(0189) In conclusion, SsTps1132 (SEQ ID NO:6) encodes sclareol synthase and catalyzes the conversion of LPP to sclareol.

(0190) Thus, we show that, in Salvia sclarea, sclareol is synthesized from GGPP in two stages by two distinct enzymes, an LPP synthase and a sclareol synthase. We isolated cDNAs encoding each of the two diterpene synthases. LPP synthase, encoded by the SsLPPs3 and SsLPPs9 cDNAs, catalyzes the conversion of GGPP to LPP and contains the characteristic components of class II diterpene synthases. Sclareol synthase, encoded by the SsTps1132 cDNA, catalyzes the conversion of LPP to sclareol and is related to class I diterpene synthases although with some peculiarities, i.e. a large deletion at the N-terminal end.

Example 10

(0191) In vivo production of sclareol in E. coli by coexpression of the two diterpene synthases.

(0192) To evaluate the in vivo production of sclareol in E. coli cells, plasmids and transformed cells are prepared for the coexpression of two diterpene synthases (LPP synthase and sclareol synthase). In addition to the two diterpene synthases, an FPP synthase and a GGPP synthase are also co-expressed to ensure sufficient assembly of GGPP in cells. To further increase carbon flux in the pathway and increase the level of GGPP and subsequently the level of sclareol produced, genes encoding a partial mevalonate pathway are also expressed in the same cells. These last genes encode a mevalonate kinase (mvaK1), a phosphomevalonate kinase (mvaK2), mevalonate diphosphate decarboxylase (MvaD) and an isopentenyl diphosphate isomerase (idi) and convert mevalonate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the two FPP synthase substrates.

(0193) The yeast FPP synthase gene (Accession number J05091) is amplified from genomic DNA of S. cerevisiae using primers FPPy_NcoI (SEQ ID NO:75) and FPPY-Eco (SEQ ID NO:6). Genomic DNA is isolated from S. cerevisiae using the Qiagen RNA/DNA Maxi Kit (Qiagen AG, Basel, Switzerland). PCR is performed with Pfu DNA polymerase (Promega AG, Dubendorf, Switzerland) in a final volume of 50μl containing 0.4μl of each primer, 200μM of dNTPs, 0.5μl of DNA polymerase, 5μl of S genomic DNA . cerevisiae. The PCR cycle conditions are 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; and 10 min at 72°C. The amplified DNA is ligated as a NdeI-Ecorl fragment into the first multiple cloning site (MCS1) of plasmid pACYCDuet-1 providing plasmid pACYCDuet-FPPs, which hosts the FPPs gene under the control of a T7 promoter.

(0194) An operator containing the genes encoding mvaKI, mvaK2, MvaD and idi is amplified from Streptococcus pneumoniae genomic DNA (ATCC BAA-334, LGC Standards, Molsheim, France) with primers MVA-up1-start (SEQ ID NO:77) and MVA-up2-interrupt (SEQ ID NO:78). PCR is performed using PfuUltraTM II HS Fusion DNA Polymerase (Stratagene), the composition of the PCR mix being according to the manufacturer's instructions. The thermal cycling condition is 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 is purified on an agarose gel and ligated using the In-FusionTM Dry-Down PCR Cloning Kit (clontech) into the second MCS of plasmid pACYDuet-FPPs digested with NdeI and XhoI providing plasmid pACYCDuet-4506 (Figure 13A). The sequences of the two inserts are completely sequenced to exclude any mutations.

(0195) The CrtE gene from Pantoea agglomerans encoding a GGPP synthase (Accession number M90698) is selected and synthesized with codon optimization (DNA2.0, Menlo Park, CA 94025, USA). The CrtE gene is amplified with CrtE_Nco (SEQ ID NO:79) and CrtE_Bam (SEQ ID NO:80) primers in order to introduce NcoI and BamHI restriction sites at the 5' end and 3' end, respectively. PCR is performed with Pfu DNA polymerase (Promega AG, Dubendorf, Switzerland) under the same conditions as described above. The product of this amplification is ligated into the first MCS of plasmid pETDuet between the Nco I and BamH I restriction sites, yielding plasmid pETDuet-CrtE. Plasmid pETDuet-SsLPPS3-del4 (containing the SsLPPS3 cDNA with a 63 codon deletion (SsLPPS3-del4, SEQ ID NO:31, Example 4) is digested with the restriction enzymes Nde I and Kpn I. The insert is recovered and transferred to the same sites as the pETDuet-CrtE plasmid, resulting in the pETDuet-CrtE-SsLPPS3-del4 plasmid containing both the SsLPPS and CrtE genes under the control of a T7 promoter ( Figure 13B ).

(0196) Plasmid pET101-1132-2-5 (containing the SsTps1132 cDNA with a 50 codon deletion (SsTps1132-2-5, SEQ ID NO:73, Example 8)) is digested with Nco I restriction enzymes and Sac I and insert transferred to the same sites as plasmid pRSDuet-1, yielding plasmid pRSDuet-1132-2-5 (not shown in Figure 13).

(0197) E. coli BL21 Star™(DE3) cells (Invitrogen) can be cotransformed with the 3 plasmids pACYCDuet-4506 (Figure 13A), pETDuet-CrtE-SsLPPS3-del4 (Figure 13B) and pRSDuet-1132-2-5 (each hosting a different replication and resistance gene origin). Transformed cells are selected on carbenicillin (50μg/ml) chloramphenicol (34μg/ml) kanamycin (35μg/ml) LB-agarose plates. Single colonies are used to inoculate 5mL of liquid LB medium, supplemented with the same antibiotics. The culture is incubated overnight at 37°C. The next day, 2mL of TB medium supplemented with the same antibiotics is inoculated with 0.2mL of the overnight culture. After 6 hours of incubation at 37°C, the culture is cooled to 28°C and 1mM IPTG, 2mg/ml mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5N NaOH at a concentration of 1g/ml and incubating the solution for 30 min at 37°C) and 0.2 ml of decane are added to each tube. Cultures are incubated for 48 hours at 28°C. Cultures are then extracted twice with 2 volumes of ethyl acetate, the organic phase is concentrated to 500μL and analyzed by GC-MS as described above in Example 3.

(0198) This example shows that an E. coli cell transformed with LPP synthase and a sclareol synthase, as defined in the present invention, is capable of producing sclareol. The other enzymes with which the E. coli cell is transformed are not essential for sclareol production. In fact, sclareol is also produced when an E. coli cell is transformed with LPP synthase and sclareol synthase alone, but in smaller amounts. The other enzymes with which the E. coli cell is transformed are added for the sole purpose of increasing the amount of precursor available for LPP synthase and sclareol synthase.

Example 11

(0199) In vivo production of sclareol in E. coli by expression of a fusion protein containing an LPP synthase and a sclareol synthase

(0200) The same approach is used for the production of sclareol with a fusion protein containing LPP synthase and sclareol synthase linked together in a single polypeptide. A plasmid is prepared containing a new cDNA comprising the sequence SsLLPS3-del4 (SEQ ID NO:31) in the 5' part and sequence 1132-2-5 (SEQ ID NO:73) in the 3' part (SEQ ID NO:87 ). In this fusion cDNA, the cDNA stop codon of SsLPPS3-del4 and the cDNA start codon of 1132-2-5 are deleted. The two cDNAs are linked by a 15 bp sequence (SEQ ID NO:81) that encodes a peptide (SEQ ID NO:82), which constitutes the linker between the two protein domains. The amino acid sequence of the encoded fusion polypeptide is provided in SEQ ID NO:88.

(0201) The cDNA of SsLPPS3-del4 (SEQ ID NO:31) is reamplified from plasmid pETDuet-SsLPPS3-del4 using primers Sa3del4-fusion-inf1 (SEQ ID NO:83) and Sa3del-fusion-inf2 (SEQ ID NO :84). The first primer is created to add a 15bp sequence at the 5' end, complementary to the Nde I region of plasmid pETDuet-1. The second primer is created to remove the stop codon and add the sequence encoding the linker peptide. The 1132-2-5 cDNA (SEQ ID NO:73) is reamplified from plasmid pET101-1132-2-5 with primers 1132-fusion-inf1 (SEQ ID NO:85) and 1132-inf2 (SEQ ID NO: 86). Thus for SsLPPS-del4, these primers are created to add the linker sequence at the 3' end and a 15bp sequence, complementary to the sequence of the Kpn I region of plasmid pETDuet-1. PCR is performed with Pfu DNA polymerase (Promega AG, Dubendorf, Switzerland) under the same conditions as described above in Example 10. Plasmid pETDuet-CrtE-SsLPPS3-del4 is digested with Nde I and Kpn I enzymes and DNA of linear plasmid is purified from the insert. The two PCR products and the linear plasmid are combined and ligated together using In-Fusion™ Dry-Down PCR Cloning Kit (clontech). The plasmid obtained, pETDuet-CrtE-fusion-SsLPPS-1132 (Figure 13C) is DNA controlled by insert sequencing.

(0202) To evaluate the production of sclareol from GGPP by this enzyme, E. coli BL21 Star™(DE3) cells (Invitrogen) can be cotransformed with plasmids pACYCDuet-4506 (Figure 13A) and pETDuet-CrtE-SsLPPS-1132 -fusion (Figure 13C). Transformed cells are selected on carbenicillin (50μg/ml) chloramphenicol (34μg/ml) LB-agarose plates. Culture, induction, mevalonate supplementation, extraction and analysis are performed as described in Example 10.

(0203) This example shows that an E. coli cell transformed to express a fusion protein with an LPP synthase domain and a sclareol synthase domain, as defined in the present invention, is capable of producing sclareol. The other enzymes with which the E. coli cell is transformed are not essential for sclareol production. In fact, sclareol is also produced when an E. coli cell is transformed with LPP synthase and sclareol synthase alone, but in much smaller amounts. The other enzymes with which the E. coli cell is transformed are added for the sole purpose of increasing the amount of precursor available for LPP synthase and sclareol synthase.

Claims (4)

1. METHOD FOR PRODUCING SCLAREOL, characterized in that it comprises a) incubating geranyl geranyl pyrophosphate (GGPP) with an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, under conditions effective to form labdenediol diphosphate ( LPP); b) incubating the LPP produced in step a) with an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3 under conditions effective to produce sclareol, thereby producing sclareol. 2. METHOD FOR PRODUCING SCLAREOL, according to claim 1, CHARACTERIZED by said polypeptide having an LPP synthesis activity comprising an amino acid sequence SEQ ID NO: 1 or 2 and a sclareol synthesis activity comprising an amino acid sequence SEQ ID NO:1. 3. METHOD FOR PRODUCING SCLAREOL, according to claim 2, CHARACTERIZED by said polypeptide having an LPP synthesis activity comprising SEQ ID NO: 1 or 2 and wherein said polypeptide having a sclareol synthesis activity comprising SEQ ID NO:3. 4. METHOD FOR PRODUCING SCLAREOL, according to claim 3, CHARACTERIZED by said polypeptide having an LPP synthesis activity consisting of SEQ ID NO: 1 or 2 and wherein said polypeptide has a sclareol synthesis activity consisting of the SEQ ID NO:3.