BRPI0906166B1 - 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 a non-human organism or cell transformed to harbor the same nucleic acid, are also parts of the present invention.

Description

technical field

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 a non-human organism or a cell transformed to harbor the same nucleic acid, are also parts of the present invention.

state of the art

Sclareol is a member of the terpenoid or terpene family, comprising a high amount of natural products. 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 thousands 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.

The biosynthetic production of terpenes involves enzymes called terpene synthases. These enzymes convert a precursor into one or more terpene products. Most often, the precursor is an acyclic terpene precursor, and in particular, many diterpene synthases catalyze the cyclization of the acyclic precursor geranylgeranyl pyrophosphate. However, in some special cases, terpene synthases catalyze the transformation of an already cyclic molecule into one or more terpene products.

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 similar to that of monoterpenes and sesquiterpenes, as it is initiated by ionization of the disphosphate ester function of GGPP, followed by reaction of the resulting carbocation 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.

Genes and cDNAs encoding diterpene synthases from 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 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 is found, in which the second aspartate residue is involved as a proton donor.

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.

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.

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.

Several diterpene synthases have already been identified. In particular, US 7,238,514 discloses various diterpene synthases, the nucleic acids which encode them, as well as unicellular organisms, transformed to express each of these synthases, along with a GGPP synthase, thereby producing diterpenes in vivo. However, no method for the biosynthetic production of sclareol, using a polypeptide, which has a sclareol synthesizing activity, as provided herein, is specifically disclosed in that patent. The amino acid and nucleotide sequences disclosed in the patent are very different from the sequences of the present invention. Among the diterpene synthases described in that document, the closest to the polypeptides of the present invention are an mRNA from Cucumis sativus for an ent-kaurene synthase, created by SEQ ID NO:389 in US 7,238,514 and an mRNA from Cucurbita maxima for an ent-kaurene synthase B, created by SEQ ID NO:395 in US 7,238,514 and accession number AAB39482.1. These polypeptides and the one referred to in the invention only share 32% of identity. Furthermore, there is no suggestion in this prior art document that the described diterpene synthases are useful for sclareol production.

Terpene synthases, which have a certain percentage of sequence identity with the sequences of the present invention, were also found in the sequence data banks. However, the percentage of identification between the known diterpene synthases and the polypeptides of the invention is very low. Synthases closest to those 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 (accession number 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 genetic family similar to rice kaurene synthase, 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-marked 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 polypeptide of the invention. The potential ability of any of these sequences to catalyze sclareol production is never mentioned in the state of the art.

In addition to the difference between the sequences themselves, it also has to be pointed out that the structure and properties of ent-kaurene and ent-cassadiene are very different from those of sclareol. In particular, 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.

A state of the art document specifically relates to a sclareol synthase (Banthorpe, Brown and Morris, Partial purification of farnesyl pyrophosphate: Drimenol cyclase and geranylgeranyl pyrophosphate: Sclareol cyclase, using cell culture as a source material, 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.

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

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 diterpenes while having little residue, a more energy and resource efficient process and at the same time reducing dependency on fossil fuels. It is an additional objective to provide enzymes capable of synthesizing sclareol, which is useful as perfumery and/or aroma components. Abbreviations Used bp base pair kb kilo base BSA bovine serum albumin DNA deoxyribonucleic acid cDNA DNA complementary dT deoxy thymine dNTP deoxy nucleotide triphosphate DTT dithiothreitol GC gas chromatography GGPP Geranylgeranyl pyrophosphate IPTG isopropyl-D-thiogalactopyranoside LB lysogeny broth LPP labdenediol diphosphate MOPSO sulfonic acid 3-(N-morpholino)-2-hydroxypropane MS mass spectrometer ORF open reading frame PCR polymerase chain reaction RMCE recombinase-mediated cassette exchange RT-PCR polymerase chain reaction via reverse transcription 3'-/5' -RACE rapid amplification of 3' and 5' ends of cDNA RNA ribonucleic acid mRNA ribonucleic acid messenger nt nucleotide RNase ribonuclease RuBisCO ribulose-1,5-bisphosphate carboxylase SDS-PAGE polyacrylamide gel electrophoresis - SDS SsLPPs Salvia sclarea labdenediol diphosphate- syntheses UTR Untranslated Region

Description of the invention

The present invention provides a method for biosynthetically producing sclareol in an economical, reliable and reproducible manner.

An object of the present invention is therefore a method of producing sclareol comprising a) contacting labdenediol diphosphate (LPP) with at least one polypeptide having a sclareol synthesizing activity and comprising an amino acid sequence of at least 50 % identical to SEQ ID NO:1; and b) optionally isolating the sclareol produced in step a).

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

Sclareol and LPP are defined by the manner of their formulas, as depicted in Figure 1.

As a "sclareol synthase" or as a "polypeptide having a sclareol synthesizing activity", we refer here to a polypeptide capable of catalyzing the synthesis of sclareol starting from (LPP). The ability of a polypeptide to catalyze sclareol synthesis can be confirmed by performing the enzymatic assay as detailed in the Examples.

In accordance with the present invention, polypeptides must also include truncated polypeptides, provided that they retain their sclareol synthesizing activity as defined above and that they share at least the defined percentage of identification with the corresponding fragment of SEQ ID NO:1.

According to a preferred embodiment, the sclareol production method comprises contacting LPP with a polypeptide, having a sclareol synthesizing activity and comprising an amino acid sequence of at least 55%, 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. According to a more preferred embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO:1. In an even more preferred embodiment, said polypeptide consists of SEQ ID NO:1.

According to a preferred embodiment, the sclareol synthase is a truncated polypeptide, comprising an amino acid sequence at least 50% identical to SEQ ID NO:102. Preferably, the polypeptide comprises at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 70%, preferably at least 75%, preferably at least 75% amino acid sequence. 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:96. In another preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO:96. In a more preferred embodiment, the polypeptide consists of SEQ ID NO:96.

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.

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.

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.

The polypeptides, either in an isolated form or together with other proteins, for example 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, DTT, BSA 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.

LPP 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 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.

LPP can be obtained by contacting GGPP with an isolated LPP synthase. Examples 1 to 3 below show a method for isolating an LPP synthase encoding Salvia sclarea cDNA, a method for heterologous expression of said cDNA in E. coli, a method for purifying the LPP synthase thus produced and a method for the in vitro production of LPP, using the isolated LPP-synthase.

According to another preferred embodiment, the sclareol production method is carried out in vivo. In that case, step a) of the method described above comprises culturing a non-human organism or cell, capable of producing LPP and transformed to express a polypeptide, having a sclareol synthesizing activity and comprising an amino acid sequence, at least , 70% identical to SEQ ID NO.1 under conditions leading to sclareol production.

According to a more preferred configuration, the method further comprises, before step a), the transformation of a non-human organism or cell, capable of producing LPP with at least one nucleic acid, which encodes a polypeptide, having a sclareol synthesis activity and comprising an amino acid sequence at least 70% identical to SEQ ID NO:1, such that said organism expresses said polypeptide.

According to a preferred embodiment, the nucleic acid used to transform the organism or host cell comprises a nucleotide 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 SEQ ID NO:2 or the complement thereof. In another preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:2 or the complement thereof. In a more preferred embodiment, the nucleic acid consists of SEQ ID NO:2 or the complement thereof.

According to a further preferred embodiment, the nucleic acid used to transform the organism or host cell comprises a nucleotide sequence of at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 60% 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% and even more preferably at least 98% identical to SEQ ID NO:93 or the complement thereof. In another preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:93 or the complement thereof. In a more preferred embodiment, the nucleic acid consists of SEQ ID NO:93 or the complement thereof.

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

The organism or cell is to "express" a polypeptide, provided that the organism or cell is transformed to host a nucleic acid, which encodes said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the organism or in the host cell. The term "express" includes "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 dedicated to such transformed non-human organisms or cells as specific objects of the present invention and in the Examples.

A particular organism or cell is intended to be "capable of producing LPP" when it naturally produces LPP or when it does not naturally produce LPP but produces GGPP (or is transformed) and is transformed to express an LPP synthase, or prior to the production of LPP. transformation with a nucleic acid encoding a sclareol synthase or together with said nucleic acid. Organisms or cells transformed to produce a greater amount of LPP than the naturally occurring organism or cell are also encompassed by "organisms or cells capable of producing LPP". In a preferred embodiment, the organism accumulates LPP naturally or is transformed to accumulate LPP.

Methods for transforming organisms so that they express an LPP synthase may be known in the art for transforming a host organism. These methods are discussed in more detail later, and a specific example of expression of an LPP synthase in E. coli is provided in Example 2. Methods for transforming an organism to produce GGPP are also 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.

To carry out the invention in vivo, the organism or host cell is cultured 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.

Non-human 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 organism used to carry out the invention in vivo is a plant, prokaryote or fungus.

Any plant, prokaryote or fungus can be used to carry out the method of the invention in vivo. 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.

In a more preferred embodiment, the non-human organism is a microorganism. 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.

Most of these organisms do not naturally produce LPP. To be suitable for carrying out the method of the invention, these organisms have to be transformed to produce said precursor. They can then be transformed prior to modification with the nucleic acid, which encodes the polypeptide, having a sclareol synthesizing activity, or simultaneously, as explained above.

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.

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.

An important tool for carrying out the method of the invention is the polypeptide itself. A polypeptide having a sclareol synthesis activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 is therefore another object of the present invention.

According to a preferred embodiment, the sclareol synthase comprises an amino acid sequence of at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 70% 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. In another preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO:1. In a more preferred embodiment, the polypeptide consists of SEQ ID NO:1.

According to another preferred embodiment of the invention, the polypeptide is derived from Salvia sclarea.

As used herein, the terms "sclareol synthase" or "polypeptide having a sclareol synthesizing activity" refer to a genus of polypeptides or peptide fragments that includes the amino acid sequences identified herein, as well as truncated polypeptides or variants , assuming that they retain their sclareol synthesis activity as defined above and that they share at least the defined percentage identification with the corresponding fragment of SEQ ID NO:1.

In a preferred embodiment, the sclareol synthase comprises an amino acid sequence at least 50% identical to SEQ ID NO:96. Preferably, the sclareol synthase 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:96. In another preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO:96. In a more preferred embodiment, the polypeptide consists of SEQ ID NO:96.

Examples of variant polypeptides are naturally occurring proteins that result from alternative mRNA splicing 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 mutating a nucleic acid of the invention, as described later, are also encompassed by the invention.

The nucleic acid encoding the polypeptide having a sclareol synthesis activity, as defined above, is a necessary tool for modifying non-human organisms or cells, proposed to be used when the method is carried out in vivo. A nucleic acid encoding a polypeptide, as defined in any of the above configurations, is therefore another object of the invention.

In a preferred embodiment, the nucleic acid comprises a nucleotide sequence that is at least 50% identical to SEQ ID NO:2 or the complement thereof. According to a more preferred embodiment, said nucleic acid 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:2 or the complement thereof. In a preferred embodiment, the nucleic acid comprises a nucleotide sequence identical to SEQ ID NO:2 or the complement thereof. According to an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:2 or the complement thereof.

According to another preferred embodiment of the invention, the nucleic acid is derived from Salvia sclarea.

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. Particularly useful truncated nucleic acids are nucleic acids at least 70% identical to SEQ ID NO:93 or the complement thereof.

Nucleic acids obtained by mutations of SEQ ID NO:2 or the complement thereof are also covered by the invention, provided that they share, at least, the defined percentage of identification with the corresponding fragment of SEQ ID NO:2 and that encode polypeptides having a sclareol synthesizing activity 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. Variant nucleic acids can be prepared in order to adapt their 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.

According to a further preferred embodiment, the nucleic acid is a truncated nucleic acid, comprising 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 95 % and even more preferably at least 98% identical to SEQ ID NO:93 or the complement thereof. In another preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO:93 or the complement thereof. In a more preferred embodiment, the nucleic acid consists of SEQ ID NO:93 or the complement thereof.

Generally speaking, the nucleic acid of the invention can be isolated using a passively parallel sequencing approach, which is extensively developed in Examples 5 and 6. The first step of this method is the overall sequencing of the cDNA library. The cDNA library is first fragmented by nebulization. The fragments are then amplified by PCR and the sequencing reaction is performed. Short sequences of 35 bases called “reads” are obtained. The “reads” are reassembled into contiguous sequences (“contigs”), using software with defined minimum extent of overlap and percentage of homology settings. The "reads" and "contigs" are then searched for sequence identification with known enzymes of the same type. Based on these homologies, reads and contigs are selected and used to synthesize primers in order to perform full length sclareol synthase PCR amplification.

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 at least one nucleic acid, according to any embodiment of the invention. This vector is therefore also an object of the present invention.

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, expression vectors include 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.

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 methods of producing or creating polypeptides having a sclareol synthesis activity as disclosed further below.

Recombinant non-human organisms and cells, transformed to harbor at least one nucleic acid of the invention so that they heterologously express or overexpress at least one polypeptide of the invention are also very useful tools for carrying out the method of the invention. These non-human organisms and cells are therefore another object of the present invention.

Non-human organisms of the invention can be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human organism of the invention is a plant, prokaryote or fungus. Said organism may be any plant, prokaryote or fungus. 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.

In a more preferred embodiment, the non-human organism is a microorganism. According to an even more preferred embodiment, said microorganism is a bacterium or yeast and more preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

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. Preferred larger eukaryotic cells are plant cells or fungal cells.

The term "transformed" refers to the fact that the host has been genetically engineered to encompass one, two or more copies of any nucleic acids of the invention. Preferably, the term "transformed" relates to hosts that heterologously express the polypeptides of the invention, as well as overexpress them. Accordingly, in one embodiment, the present invention provides a transformed organism in which the polypeptide of the invention is expressed in greater amounts than in the same non-transformed organism.

There are several models 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., I 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.

Methods of transforming host organisms or cells to harbor transgenic nucleic acids, such as those of the present invention, 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. .

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 switching (RMCE), site-specific viral chromosome insertion, adenovirus, and pronuclear injection.

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 sclareol synthesizing activity. Therefore, the invention provides a method of producing at least one polypeptide having a sclareol synthesis activity comprising a) culturing a non-human organism or cell, transformed with the expression vector of the invention, in a way that harbors a nucleic acid, according to the invention and express or overexpress a polypeptide, encoded by said nucleic acid and having a sclareol synthesizing activity; b) isolating the polypeptide having a sclareol synthesizing activity from the cultured non-human organism or cell in step a).

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, in a way that it houses at least , a nucleic acid, according to the invention and expresses or overexpresses at least one polypeptide encoded by said nucleic acid.

Transformation and cultivation of the non-human 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.

A "polypeptide variant", as referred to herein, means a polypeptide having a sclareol synthesizing activity and being substantially homologous to a native polypeptide, but having an amino acid sequence different from that encoded by any of the nucleic acid sequences of the invention due to one or more deletions, insertions or substitutions.

Variants can 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 substituting one aliphatic residue for another, such as Lie, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. See Zubay, Biochemistry, 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.

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 can be used to obtain desired elevated or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product delivery. In addition, variants can be prepared to have at least one modified property, for example, a higher affinity for the substrate, an improved specificity for the production of one or more desired compounds, a different product distribution, a different enzyme activity. , an increase in enzyme reaction rate, greater 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. As stated above, the invention provides isolated and purified, recombinant and non-recombinant, polypeptides such as from Salvia sclarea. 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 natural sclareol synthases. Alterations to the native amino acid sequence can be performed by any of a number of conventional methods.

Polypeptide variants, resulting from a fusion of additional peptide sequences at the amino- and carboxyl-terminal ends of the polypeptides of the invention, can be used to enhance expression of the polypeptides, be useful in purifying the protein, or improve the enzymatic activity of the polypeptide in a desired environment or system of expression. 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.

Therefore, in one embodiment, the present invention provides a method for preparing a variant polypeptide having a sclareol synthesizing activity and comprising the steps of: (a) selecting a nucleic acid according to any of the embodiments set forth 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).

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): 10747-1075. In short, DNA shuffling refers to a process of random recombination of known sequences in vitro, involving at least two nucleic acids selected for recombination. For example, mutations can be introduced at particular 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 detection. 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.

Accordingly, SEQ ID NO:2 or 93 can be recombined with any other diterpene synthase encoding nucleic acids, for example isolated from an organism other than Salvia sclarea. 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.

In step (d), the polypeptide obtained in step (c) is evaluated for at least one modified property, for example, a desired modified enzyme 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.

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.

In one embodiment, the present invention provides a method for preparing a nucleic acid encoding a variant polypeptide having a sclareol synthesizing activity, the method comprising steps (a)-(e), disclosed above and further comprising step in:

All publications mentioned 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

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

Figure 2: Putative biosynthesis of sclareol from LPP, which is catalyzed by SsTps1132 (SEQ ID NO:1).

Figure 3: Alignment of the amino acid sequences of class I diterpene synthase-like fragments with the sequence of Oriza sativa stemodeno synthase (Accession No. AAZ76733).

Figure 4: Alignment of the deduced amino acid sequence of SsTps1132 (SEQ ID NO:1) and SsTps1137 (SEQ ID NO:86) with amino acid sequences of diterpene synthases selected from the database.

Figure 5: Alignment of the amino acid sequences of SsTps1132 (SEQ ID NO:1) and 1132-2-5 (SEQ ID NO:96), which were heterologously expressed in E. coli.

Figure 6: GC analysis of the products obtained after incubation of the different recombinant 1132 proteins with LPP. Crude protein extracts of E. coli expressing the recombinant SsTps1132 and 1132-2-5 proteins (SEQ ID NO:1 and 96) were incubated with LPP in a final volume of 1mL of 50mM MOPSO, pH 7, supplemented with 15mM of MgCl2 and 1mM DTT.

Figure 7: CG-MS analysis of LPP generated products by recombinant 1132-2-5 protein. (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 (SEQ ID NO:93). (B) Mass spectrum of the peak at retention time of 14.3. (C) Mass spectrum of an authentic sclareol standard.

Figure 8: GC analysis of products obtained after co-incubation of recombinant proteins SsTps1132 and 1132-2-5 (SEQ ID NO:1 and 96) with recombinant protein SsLPPs3 (SEQ ID NO:24) in the presence of GGPP.

Invention Specific Embodiments or Examples

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

Example 1 Isolation of LPP-synthase, which encode Salvia sclarea DNAs by a PCR approach. A. Extraction of plant matter and RNA.

Developing flower buds of Salvia sclarea (1.5 to 2 cm 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

PCR were performed using the DT3F reverse primer (5-GAYRTNGAYGAYACNGCNATGG-3' (SEQ ID NO:3)) and the DT4R reverse primer (5'-GTYTTNCCNAKCCANACRTCRYYT-3' (SEQ ID NO:4)). The PCR mix contained 0.4μM of each primer, 300μM of each dNTPs, 5μL of 10X HotStarTaq® DNA polymerase buffer (Qiagen), 2μL of cDNA dissolved in 100 fold, 0.5μL of HotStarTaq® 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 cDNAs 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, 403-410, 1990). A 354 bp sequence called FN23 (SEQ ID NO:5) was obtained. This DNA fragment had the expected size and showed sequence homology to diterpene synthases.

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

Specific oligonucleotides for the FN23 sequence (SEQ ID NO:5) were created. FN23-F1 (3-GCACGGATACGACGTCGATCCAAATGTAC-5' (SEQ ID NO:6)), FN23-F2 (3-GGGCTGCTCAACTAAGATTTCCAGGAG-5' (SEQ ID NO:7)) and FN23-F3 (5'-GGGTGATATCCGACCACTTATTTGATGAG-5' ( SEQ ID NO:8)). These primers were used in RT-PCR in combination with oligodT primers, extended with an adapter sequence (5'-AATTCGGTACCCGGGATCC(T)I7-3') (SEQ ID NO:9). 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 primer adapterP (5'- AATTCGGTACCCGGGATCC-3' (SEQ ID NO: 10)) in combination with the same or FN23-specific primers harbored. This PCR approach yielded a 1271 bp cDNA fragment (FN30 (SEQ ID NO: 11)) having a perfect 192 bp overlap with the FN23 fragment (SEQ ID NO:5) and containing the 3' end, including the codon of interruption and the 3' uncoding sequence of the corresponding cDNA.

For amplification of the 5' end of the cDNA, antisense oligonucleotides, specific for FN23 were created: FN23-R1 (5-CATGGCATCTTCAACCCCAGCTTTATCTCATC-3' (SEQ ID NO: 12)), FN23-R2 (5- GTGGTCGGATATCACCCATTCTTTCTTGAAGTCG-3' ( SEQ ID NO:13)), FN23-R3 (5-CATTGGAGATGCAGACTCGACCGATTGACC-3' (SEQ ID NO:14)). 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:15) having a perfect 227 bp overlap with FN23 (SEQ ID NO:5). Comparison with known diterpene synthase sequences revealed that the fragment FN40 (SEQ ID NO: 15) contained the translation initiation codon and an 87 bp clear region. Assembly of the three cDNA fragments (FN23, FN30 and FN40 (SEQ ID NO: 5, 11 and 15) provided a full length cDNA sequence (SaTpsl) of 2655 bp (SEQ ID NO: 16) with an open reading frame of 2355 bp coding for a protein of residues 785 (SEQ ID NO: 17).

Example 2 Heterologous expression of S. sclarea LLP synthase in E. coli.

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:16) was PCR amplified from the cDNA library with the reverse and reverse primers SaTps-Nde (3 - TACTGACATATGACTTCTGTAAATTTGAGCAGAGCACC-5' (SEQ ID NO : 18)) and SaTps-Kpn (3'-TTGGTACCTCATACAACCGGTCGAAAGAGTACTTTG-5' (SEQ ID NO: 19)), 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 Ndel site at position 1614 of the open reading frame, this amplification was performed in two steps by overlap extension PCR (Horton et al, Gene 77, 61-68, 1989), using the SaTps-Nde (SEQ ID NO:18) and SaTps-Kpn (SEQ ID NO:19) primers in combination with the Satps-mutlf (5-GTTGGAGTGGATCCACATCCAGGAATGGTAC-3' (SEQ ID NO:20)) and Satps-mutlr primers (3'-GTACCATTCCTGCATCTGGATCCACTCCAAC-5' (SEQ ID NO:21)), designed to remove the Ndel 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 Ndel-KpnI fragment into the pETDuet-1 vector.

Sequence analysis of several clones obtained by amplification of the cDNA library with the SaTpsl specific primers showed some variability at several positions in 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.

These variations appeared to occur in a random fashion across 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:22) and SsLPPs9 (SEQ ID NO:23), representing sequence variability, were selected for heterologous expression and enzyme characterization experiments.

Plasmids pETDuet-SsLPPs3 and pETDuet-SsLPPs9 were transferred into E. coli BI21 (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. 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:24 and 25) were clearly detected and the apparent molecular weight estimated at 90 KDa, a value in agreement with the calculated molecular weight of 83 KDa.

Example 3 Purification of Salvia sclarea LPP synthase and enzymatic activities

Plasmids PCR2.1-Topo containing the cDNA of SsLPPs3 and SsLPPs9 (SEQ ID NO:22 and 23) (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, created to express the proteins with an N-terminal hexahistidine tag (His-tail). Purification was performed under native conditions using the ProBond™ 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, the recombinant enzymes SsLPPs3 and SsLPPs9 "His-tail" (SEQ ID NO:97 and 98) could be purified to apparent homogeneity.

Affinity purified enzymes were incubated 12 hours at 30°C with 200μM GGPP and 1mM DTT in MOPSO, pH 7, 10% glycerol, 1mM DTT. No diterpene products were observed when extracting the incubation medium with pentane 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 and demonstrated the enzymatic formation of labdenediol diphosphate (LPP) as the sole product of GGPP by recombinant diterpene synthase.

GC analysis was performed on an Agilent Series 6890 GC system, 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 1.5mL/min. The initial oven temperature was 100°C (1 min of suspension), followed by a gradient of 10°C/min to 300°C. GC-MS analysis was performed under the same conditions and spectra were recorded on an Agilent 5975 mass detector.

Example 4 PCR approach for homology cloning of class I diterpene synthases (sclareol synthase) from S, sclarea.

The cloning and characterization of SsLPPs3 (SEQ ID NO:24) and SsLPPs9 (SEQ ID NO:25) in Examples 1 to 3 suggest that sclareol biosynthesis in S. sclarea involves two proteins, SsLPPs and a diterpene- class I synthase, sclareol synthase, catalyzing the conversion of LPP to sclareol.

A PCR approach was used in a first attempt to isolate the cDNA sequences of class I diterpene synthases. Oligonucleotides were created, based on the sequences converted in plant diterpene synthases and especially in diterpene synthases that catalyze the cyclization of C2o-diphosphate esters through an ionization mechanism. The sequences with accession numbers BAB19275, AAB39482, AAD30231, AAD34295, CAE05201, BAB12441, AAT49066, CAE05199, AAU05906, BAD17672, AAQ72565, AAL09965, AAK83563, AAS47691 , AAS47690 and AAR13860 were selected from public sequence databases (http:// /www.ncbi.nlm.nih.gov). All of these protein sequences correspond to class I diterpene synthases and contain the DDxxD motif (where x represents any amino acid), characteristic of the ionization-dependent cyclization mechanism in terpene synthases. From the alignment of these sequences, two conserved motifs were first selected in the N-terminal region, used for the creation of sense oligonucleotides. YDT(A/S)WVA and (D/N)GSWG. In the amino acid sequence of SsLPPs (SEQ ID NO: 24 and 25, Examples 1 to 3), these two motifs were also conserved, although with some differences for the first motif (YDTAVIA). Thus, the sequence of SsLPPs was also taken into account for the creation of sense oligonucleotides. From the first motif, three oligonucleotides were created to cover all sequence variations: DiTpsTB_F1, 5'-TATGATACNGCNGTNATDGC-3' (SEQ ID NO:26); DiTpsTB_F2, 5'-TATGACACGGCAGTGATGCC-3' (SEQ ID NO:27); DiTpsTB_F3, 3'-TATGACACGGCAKKGRTNGC-5' (SEQ ID NO:28). From the following motif, two oligonucleotides were created: DiTpsTB_F4, 5'-CAACTGGCTGATGGNTCNTGGGG-3' (SEQ ID NO:29); DiTpsTB_F5, 5-CAACTGGCTGATGGCTCATGGGG-3' (SEQ ID NO:30). The DDxxD motif, located in the C-terminal region of the proteins and involved in binding the diphosphate moiety at the active site, was used to create two antisense oligonucleotides. DiTpsTB_R1, 5'-GATCCTCCAACRTCRWARARRTCRTC-3' (SEQ ID NO:31), DiTpsTB_R2, 5'-GATCCCTCCACGTCGWAGAAGTCGTC-3' (SEQ ID NO:32).

These primers were used for PCR amplification of a Salvia sclarea cDNA library (prepared as described in Example 1). PCRs were performed using Advantage® 2 Polymerase Mixture (Clontech). Each PCR mix contained, in a total volume of 50μL, 5μL of PCR Buffer Advantage® 2, 200μM of dNTPs, 200nM of each oligonucleotide primer, 5μL of cDNA diluted 20 times, 1 μl_ of Advantage Polymerase Mixture®. The following conditions were used for amplifications: 3 minutes of denaturation at 94°C; 15 cycles of 1 minute of denaturation at 94°C, 1 min of annealing at 65°C for the first cycle and minus one degree for each subsequent cycle and extension of 2 minutes at 72°C; 20 cycles of 1 minute of denaturation at 94°C, 1 min of annealing at 58°C and extension of 2 minutes at 72°C; and finally extension for 10 minutes at 72°C. Different PCRs were performed with possible combinations of sense and antisense oligonucleotides. The amplicons were analyzed for expected sizes and for sequence homology to diterpene synthases. Unfortunately, using this PCR approach, no diterpene-related sequence could be obtained.

Example 5 Massively parallel sequencing of a cDNA library from the S.sclarea flower.

Since the classical homology-based cloning approach was unsuccessful in cloning class I diterpene synthase from S. sclarea, we assured to use an approach based on global sequencing of the cDNA library. 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).

Technology and equipment were prepared at Fastens SA (Geneva, Switzerland) and DNA sample preparation and sequencing were performed by Fastens SA. An aliquot (1 µg) 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 Marathon™ cDNA Amplification Kit (Example 1). Genomic Sample (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.

Edena software (Dr David Hernandez, Genomic Research Laboratory, University of Geneva Hospitals, Geneva, Switzerland) 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.

To assess assembly quality, contigs were searched for sequence identification with the DNA sequence of SsLPPs, the class II diterpene synthases, first isolated from the S. sclarea cDNA library (SsLPPs3 (SEQ ID NO: 22), 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 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 (SEQ ID NO:33 to 35) covered only 8.7% of the full-length sequence, albeit with a 99% identification.

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.qov) was the large subunit sequence of ribulose-1,5-bisphosphate carboxylase (RuBisCO) from Salvia sclarea (Access 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 (SEQ ID NO:36 and 37). 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.

All reads (unassembled data) were then queried for sequence identification with the SsLPPs3 sequence (SEQ ID NO:22). 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 the selected fragments with the reference sequence revealed that the SsLPPs3 sequence (SEQ ID NO:22) was covered in full length 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 greater for RuBisCO than for SsLPPs3 (SEQ ID NO:22). For SsLPPs3 (SEQ ID NO:22), 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.

The amount of reads obtained for a given cDNA is proportional to the abundance of its cDNA. Thus, relative abundances can be estimated by dividing the amount of reads obtained for the cDNAs data by their total lengths. Performing this calculation for RuBisCO and SsLPPs3 (SEQ ID NO:22), provided values of 1160 and 260 reads/Kb, respectively, reflecting an abundance greater than 4.5 of the RuBisCO cDNA relative to the SsLPPs cDNAs . RuBisCO is an enzyme involved in primary metabolism in plants and in catalyzing carbon fixation in the Calvin cycle. The higher relative abundances of RuBisCO reflect a greater representation of genes involved in primary metabolisms compared to the gene involved in secondary metabolism such as diterpene synthesis. BLAST search analysis with the contigs showed that other Calvin cycle enzymes (eg, phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase) and primary metabolism were also abundantly represented in the cDNA library used herein. Thus, the cDNA encoding of enzymes involved in secondary metabolism and particularly diterpene biosynthesis were in too low abundance to obtain sufficient coverage and complete reassembly.

Example 6 Extraction of class I diterpene synthase-like sequences from sequencing data.

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.

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:38), showing sequence homology with class I diterpene synthases. The deduced amino acid sequence of this contig contained the motif DDxxD, characteristic of cyclization initiated by prenyl ionization. diphosphates.

A fraction of the raw data, representing approximately 3x105 reads, was then searched for homology with class I diterpene synthases. The reads were searched using the tBlastn algorithm with five selected class I diterpene synthase amino acid sequences ( 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 maximum length 111 bases. These contigs, as well as the remaining isolated reads, were used to search Blastx against a protein database to confirm its identification with class I diterpene synthases. Finally, 5 DNA fragments were retained (SEQ ID NO:39 to 43).

The amino acid sequences were deduced from the selected fragments (SEQ ID NO: 44 to 48) and were aligned with reference diterpene synthase sequences, allowing their relative positioning. Figure 3 shows an alignment of these sequences with a full-length diterpene synthase sequence, the Oriza sativa estemodeno synthase (Morrone et al, 2006; NCBI Accession Nos AAZ76733) collected as a reference.

Example 7 PCR amplification of full-length class I diterpene synthase cDNAs.

A set of reverse and reverse oligonucleotides was deduced from DNA sequences related to diterpene synthases selected from sequencing the S. sclarea cDNA library (Example 6). These primers were used in combination with cDNA adapter primers in 375' 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.

Using the Cont250-Fwd primer (SEQ ID NO:49), a 547 bp DNA sequence (1130Cont250, SEQ ID NO:81) 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:51) and Cont147_fw2 (SEQ ID NO:52), we obtained a 1473 bp sequence (1132Cont147, SEQ ID NO:82), containing the 3' end and 1293 bp of the coding region of a diterpene synthase cDNA. The primer Cont224_fw (SEQ ID NO:57) provided a 207 bp DNA fragment (1137Cont224, SEQ ID NO:83), encoding the C-terminal 43 amino acids of a diterpene synthase with a sequence distinct from 1132Cont147 (SEQ ID NO :82). The primers Cont147_revl (SEQ ID NO:53) and Cont147_rev2 (SEQ ID NO:54) allowed the amplification of a 464 bp DNA fragment (1134Cont147, SEQ ID NO:84). 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 (SEQ ID NO:22 and 23). PCR with the other primers deduced from the DNA sequences related to diterpene synthases (primers cont224-rev (SEQ ID NO:58), cont250-rev (SEQ ID NQ:50), cont33-fw1 (SEQ ID NO:55) and cont33-rev (SEQ ID NO:56)) did not provide sequences related to diterpene synthase.

From the only sequence containing an obvious translation initiation region of a diterpene synthase (1130Cont250, SEQ ID NO:81), sense oligonucleotides were deduced from the 5' untranslated region (UTR) (1130-fwl (SEQ ID NO:59) and 1130-fw2 (SEQ ID NO:60) and the 5' end of the open reading frame (ORF) (1130-fw3, SEQ ID NO:61). distinct synthases (1132Cont147 (SEQ ID NO:82) and 1137Cont224 (SEQ ID NO:83)), reverse sense primers were deduced from the 3' UTR (1132-rev1 (SEQ ID NO:65) and 1137-rev1 (SEQ ID NO: :62)) or the 3' end of the open reading frame (1132-rev2 (SEQ ID NO:64) and 1137-rev2 (SEQ ID NO:63)).PCRs were performed with different combinations of these reverse and reverse primers. Combining primers deduced from sequence 1130Cont250 (SEQ ID NO:81) with primers deduced from sequence 1137Cont224 (SEQ ID NO:83) produced a 2388 bp fragment (SEQ ID NO:85), which encodes a protein of 795 5 amino acids (SsTps1137, SEQ ID NO:86)). Comparison with published sequences showed homologies with class I diterpene synthases and particularly ent-kaurene synthases B. The highest homology was with an uncharacterized protein from Vitis vinifera (NCBI Accession No. CAO64942, 59% ID), an ent -kaureen synthase from Cucumis sativus (NCBI Accession Nos BAB19275, 54% ID), an ent-kaurene synthase from Lactuca sativa (NCBI Accession Nos BAB12441, 54% ID). The amino acid sequence SsTps1137 (SEQ ID NO:86) contained a DDFFD motif typical of ionization-dependent terpene synthases (class I) and did not contain the characteristic class II motif.

The combination of the same reverse primers with the reverse primers deduced from 15 1132Cont147 (SEQ ID NO:82) did not allow the amplification of any fragment, confirming that these two sequences did not arise from the same cDNA. A 5'RACE approach was then used to identify the 5' end of the ORF, corresponding to sequence 1132Cont147 (SEQ ID NO:82). Using primers 1132_race1 (SEQ ID NO:67) and 1132_race2 (SEQ ID NO:68), a 20 bp 536 sequence (1132RACE, SEQ ID NO:87) was obtained, which overlapped by 41 bases with the 1132Cont147 fragment (SEQ ID NO:82). This RACE product was identical to the previously obtained sequence 1134Cont147 (SEQ ID NO:84) and no extension at the 5' end was observed. As previously noted, this sequence had homology to diterpene synthases, but appeared shorter by at least 200 25 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:82) and to identify the true translation initiation codon. Several sets of oligonucleotides (1132_race3 to 1132_race9, SEQ ID NO:69 to 75) 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:84) was actually the start codon of the corresponding diterpene synthase gene. The nucleotide sequence of this putative diterpene synthase (named SsTps1132, SEQ ID NO:2) was reconstructed from sequences 1132Cont147 (SEQ ID NO:82) and 1132RACE (SEQ ID NO:87). Taking the first ATG, the 1728 bp ORF of SsTps1132 (SEQ ID NO:2) encoded a protein of 575 amino acids (SEQ ID NO:1). 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), 37% identification.

Surprisingly, the identification between SsTps1137 (SEQ ID NO:86) and SsTps1132 (SEQ ID NO:1) proteins was only 30% and these sequences shared only 21 to 23% identification with class II SsLPPs, primarily isolated from S. sclarea (SEQ ID NO;24 and 25, Examples 1-3). An alignment of these two proteins with selected diterpene synthase sequences is shown in Figure 4. The alignment shows that SsTps1132 (SEQ ID NO:1) is truncated at the N-terminal end by 150 to 240 amino acids compared to other diterpene synthases. synthases. 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 chloroplast transit peptide in each protein sequence. For SsTps1137 (SEQ ID NO:86) and SsTps1132 (SEQ ID NO:1), chloroplast transit peptides of 22 and 51 amino acids, respectively, were predicted, supporting a chloroplast localization of both proteins.

Searching all reads for sequences identical to the DNA sequences of 5 SsTps1137 (SEQ ID NO:85) and SsTps1132 (SEQ ID NO:2) yielded only 24 reads for SsTps1137 and 425 reads for SsTps1132. This difference in the amount of reads generated from each transcript reflects a significant difference in expression levels. Based on the relative amount of reads obtained for each transcript, it can be estimated that the expression level of 10 SsTps1132 (220 reads/Kb) was similar to the expression level of SsLPPs (260 reads/Kb) and that SsTps1137 was expressed at a much lower level (10 reads/Kb). With the hypothesis that enzyme catalytic steps in the same metabolic pathway are generally expressed at a similar level, one can speculate that SsTps1132 (SEQ ID NO:1) rather than SsTps1137 (SEQ ID NO:86) 15 is involved in the same metabolic pathway as SsLPPs.

Contigs generated with the Edena software (Example 5) were searched for DNA sequences identical to the sequences of these two new putative class I diterpene synthases. For SsTps1137 (SEQ ID NO:85), no contig was found consistent with the presumed low level of expression of this enzyme. For SsTps1132 (SEQ ID NO:2), 4 contigs were found. The previously identified contig 1610 (SEQ ID NO:38) and three additional contigs (53 to 96 bp in length) (SEQ ID NO:88 to 90), 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 25 to discover homology for these contigs is due to the short lengths of these fragments and the low homology of SsTps1132 (SEQ ID NO:1) with the diterpene synthases present in the databases.

The observation of an N-terminal deletion of SsTps1132 (SEQ ID NO:1) compared to other diterpene synthases also further explains why the PCR approach first employed was unsuccessful. In fact, the reverse primers were created from conserved regions present in the first 150 amino acids of diterpene synthases, a region absent in SsTps1132 (SEQ ID NO:1). The SsTps1137 sequence (SEQ ID NO:86) contains the conserved motifs used to create the primers and the corresponding DNA sequences are complementary to the primer sequences. Presumably, the amplification of SsTps1137 (SEQ ID NO:85) was unsuccessful in the PCR approach due to the low abundance of this transcript.

Example 8 Heterologous expression of S. sclarea class I diterpene synthases in E. coli.

To determine an enzymatic activity for SsTps1137 (SEQ ID NO:86) and SsTps1132 (SEQ ID NO:1), recombinant proteins were expressed in E. coli. The full-length cDNAs were inserted into the pET101/D-TOPQ vector using the pET101 Champion Directional TOPO Expression Kit.

For each enzyme, two constructs were prepared: one to express the full-length protein and one to express a truncated protein, based on the chloroplast transit peptide prediction. The full-length SsTps1137 (SEQ ID NO:85) and SsTps1132 (SEQ ID NO:2) open reading frames were amplified from the cDNA library using primer pairs 1137-start (SEQ ID NO:78) with 1137 -interrupt (SEQ ID NO:80) and 1132-start1 (Seq ID No 76) with 1132-interrupt (SEQ ID NO:66), respectively. Primers 1137_start2 (SEQ ID NO:79) and 1137_stop (SEQ ID NO:80) were used to amplify a truncated 72 bp version of SsTps1137, created to express the protein with 24 amino acids deleted at the N-terminal end. , primers 1132_start2 (SEQ ID NO:77) and 1132_stop (SEQ ID NO:66) were used to amplify a truncated version of SsTps1132, created to express the protein with a 50 amino acid deletion at the N-terminus. All cDNA amplifications expression constructs were performed using Pfu DNA polymerase (Promega), in a final volume of 50μl, containing 5μg of 10X Pfu DNA polymerase 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.

After ligation into the pET101 vector, several clones were selected for each construct and sequenced to ensure that no mutations were introduced during PCR amplification. For SsTps1137, the two constructs 1137-B12 (SEQ ID NO:91) and 1137-2-B12 (SEQ ID NO:92) were selected, containing the SsTps1137 cDNA, respectively, with and without the signal peptide (sequences of corresponding polypeptides are SEQ ID NO:94 and 95). For SsTps1132, two constructs were selected: one with the complete sequence of SsTps1132 (SEQ ID NO:2) and one construct without signal peptide (1132-2-5, SEQ ID NO:93). The alignment of the two amino acid sequences (SEQ ID NO:1 and 96) deduced from these constructs is shown in figure 5.

Plasmids pET101-1137-B12, pET101-1137-2-B12, pET101-SsTps1132 and pET101-1132-2-5 were transferred into E. coli B121 (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. Crude protein extracts were analyzed by SDS-PAGE and compared with protein extracts obtained from cells transformed with empty pET101 plasmid.

Example 9 Enzyme activity of recombinant class I diterpene synthases from S. sclarea in E. coli.

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

Enzymatic activities were first evaluated using a GGPP or LPP substrate, the product of SsLPPs (SEQ ID NO:22) and the presumed intermediate in sclareol biosynthesis (Examples 1 to 3). 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 100pM substrate, 15mM MgCI2 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 a substrate, no activity was observed with the protein extracts containing SsTps1137 recombinant proteins, but with SsTps1132, activity was observed with both SsTps1132 and 1132-2-5 (SEQ ID NO:1 and 96) (Figure 6). The enzymes were also active in the absence of MgCl2 and the same product profiles were observed with approximately the same overall activity. Product identification was confirmed by matching retention times (Figure 6) and comparing the mass spectrum with the spectrum of an authentic standard (Figure 7). In all assays, a single sclareol peak was observed with no trace of additional product.

Assays were then performed with co-incubation of class II diterpene synthases (SsLPPs3, SEQ ID NO:24; Examples 1-3) and class I diterpene synthases (1132 series, SEQ ID NO:1 and 96). Assays were performed in 50mM MOPSO, pH 7, 10% glycerol, 1mM DTT, 50pM 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:24) and 50μL of extracts containing SsTps1132 (SEQ ID NO:1) or 1132-2-5 (SEQ ID NO:96 ) were evaluated for the production of diterpene products. Figure 8 shows the GC profiles of extracts from these incubations in the presence of MgCl2. Sclareol was produced with both 1132 constructs (SEQ ID NO: 1 and 96) (Figure 8), a result consistent with the assay described above with LPP as substrate. No significant difference was observed when omitting MgCl2 from incubations (data not shown).

In conclusion, SsTps1132 (SEQ ID NO:2) encodes sclareol synthase (SEQ ID NO:1) and catalyzes the conversion of LPP to sclareol.

Claims (12)

1. METHOD FOR PRODUCING SCLAREOL, characterized by comprising a) contacting labdenediol diphosphate (LPP) with a recombinant polypeptide having a sclareol synthesis activity and comprising an amino acid sequence as set out in SEQ ID NO: 1 or SEQ ID NO: 1. 96; and b) optionally isolating the sclareol produced in step a). 2. METHOD, according to claim 1, characterized by step a) or b) being carried out through the cultivation of a microorganism capable of producing LPP and transformed to express the said recombinant polypeptide under conditions that lead to the production of sclareol. 3. METHOD, according to claim 2, characterized by further comprising, before the cultivation step, transforming the microorganism capable of producing LPP with a recombinant nucleic acid encoding said recombinant polypeptide, so that said organism expresses said polypeptide . 4. METHOD, according to claim 2 or 3, characterized in that said microorganism is a prokaryote or a fungus. 5. METHOD, according to claim 2 or 3, characterized in that said microorganism is a bacterium or yeast. 6. METHOD, according to claim 5, characterized in that said bacteria are E. coli and said yeast is Saccharomyces cerevisiae. 7. EXPRESSION VECTOR, characterized by comprising a) a nucleic acid encoding a polypeptide having a sclareol synthesis activity and comprising an amino acid sequence as shown in SEQ ID NO: 1, and b) at least one regulatory sequence operably linked to said nucleic acid, the regulatory sequence of which controls transcription, translation initiation or termination, such as a transcription promoter, operator or enhancer, or an mRNA ribosome binding site, and, c) optionally, at least one selectable marker. 8. EXPRESSION VECTOR, according to claim 7, characterized in that it is in the form of a viral vector, a bacteriophage or a plasmid. 9. TRANSFORMED MICROORGANISM, a) with the expression vector of claim 7 or 8, or b) harboring a nucleic acid encoding a polypeptide having a sclareol synthesis activity and comprising an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 96, or c) harboring a nucleic acid as set forth in SEQ ID NO: 2 or SEQ ID NO: 93; characterized in that the microorganism heterologously expresses or overexpresses said polypeptide having a sclareol synthesizing activity and wherein the microorganism is a bacterium or yeast. 10. MICROORGANISM, according to claim 9, characterized in that said bacteria are E. coli and said yeast is Saccharomyces cerevisiae. 11. METHOD FOR PRODUCING AT LEAST ONE RECOMBINANT POLYPEPTIDE HAVING SCLAREOL SYNTASE ACTIVITY, characterized in that it comprises: a) cultivating the microorganism of claim 9 or 10, so that it expresses or overexpresses said polypeptide having a sclareol synthesis activity; b) isolating the polypeptide having a sclareol synthesizing activity from the cultured microorganism in step a). 12. METHOD, according to claim 11, characterized in that it further comprises, before step a), transforming a microorganism with a) the expression vector of claim 7 or 8, or b) a nucleic acid encoding a polypeptide having a sclareol synthesis activity and comprising an amino acid sequence as set out in SEQ ID NO: 1, or c) a nucleic acid as set out in SEQ ID NO: 2, in order for the microorganism to express or overexpress said polypeptide having a sclareol synthesis activity. sclareol synthesis.