WO2008119735A1

WO2008119735A1 - Method of producing hydroxy fatty acids

Info

Publication number: WO2008119735A1
Application number: PCT/EP2008/053647
Authority: WO
Inventors: Ivo Feussner; Ellen Hornung; Alena Liavonchanka
Original assignee: Georg-August-Universität Göttingen
Priority date: 2007-04-02
Filing date: 2008-03-27
Publication date: 2008-10-09

Abstract

The present invention relates to methods for producing hydroxy fatty acids. In particular, this invention relates to the production of hydroxy fatty acids by use of a fatty acid hydratase from Streptococcus pyogenes. The inventive fatty acid hydratase is useful for the production of hydroxy fatty acids on a large scale, and for further processing into flavor ingredients and/or fragrance ingredients and/or lubricants, surfactants, detergents, coatings, paints, biopolymers and/or plasticizers.

Description

Method of producing hydroxy fatty acids

Fatty acids differ from each other in the length and in the number and position of the double bonds. Natural fatty acids usually consist of an even number of carbon atoms and are unbranched. Fatty acids with one or more double bonds are called unsaturated fatty acids, with the double bond usually being in the cώ-confϊguration.

Fats and oils have gained considerable interest in the last years, because they are some of the most important renewable raw materials for the chemical industry. To adapt the fatty acids to the different uses, modifications have been introduced to the fatty acids. While most of the modifications were earlier directed to the carboxyl group of the fatty acid, in recent years also the alkyl chain of the fatty acids has been modified to obtain important starting materials for fine chemical industry.

One of these modification reactions is the addition of water to the double bonds of unsaturated fatty acids, leading to the formation of hydroxy fatty acids. Hydroxy fatty acids can be used as lubricants, surfactants and plasticizers; as components in detergent, coating and paint industries; and in the synthesis of resins. For example, hydroxy fatty acids cover a certain percentage of the market of bioplastics, which market is estimated to become 300.000 tons until the year 2010 in the European Union. Furthermore, short-chain hydroxy fatty acids and lactones made from these fatty acids can be used as flavor ingredients and/or fragrance ingredients. The lactones possess various sensory properties with mainly fruity and fatty characteristics, which make them interesting food additives. One of the main products derived from aroma biotechnology is γ-decalactone which can be obtained by biotransformation of the long-chain hydroxy fatty acid precursor by yeast cells.

Both a chemical reaction and microbes can be used for the hydration reaction. The chemical addition of water has the disadvantage that it is neither regioselective nor stereoselective. The lack of regioselectivity means that all carbon atoms of the double bonds are hydrated with essentially the same efficiency, leading to the formation of a mixture of hydrated products which have to be separated from each other after completion of the reaction. The term "stereoselectivity" means that of a compound present in both a cis and a trans configuration, only one of these configurations of the compound is modified. In contrast, microbial hydration is both regioselective and stereoselective. Microbial water attachment to unsaturated fatty acids was observed with Pseudomonas species, as well as with the bacterial genera Nocardia, Rhodococcus, Corynebacterium and Micrococcus (Wallen et al. (1962) Arch. Biochem. Biophys. 99: 249-253; Koritala et al. (1989) Appl. Microbiol. Biotechnol. 32: 299-304; Seo et al. (1981) Agric. Biol. Chem. 45: 2025-2030; Blank et al. (1991) Agric. Biol. Chem. 55: 2651-2652). Furthermore, hydroxy fatty acids could also be obtained with the yeast Saccharomyces cerevisiae (El-Sharkawy et al. (1992) Appl. Environ. Microbiol. 58: 2116-2122). However, use of this microorganism does not involve the use of a purified enzyme, but rather the use of a cell extract. Furthermore, hydroxy fatty acids are only produced in low and middle yield. Thus, there is a strong interest in identifying and cloning of prokaryotic fatty acid hydratases which produce hydroxy fatty acids in high yields and without the formation of by-products.

It is therefore an object of the present invention to provide a prokaryotic fatty acid hydratase which facilitates the production of hydroxy fatty acids in high yields without the formation of by-products. Furthermore, it is an object of the present invention to provide the nucleotide sequence of a fatty acid hydratrase which enables transgenic over-expression of the hydratase in suitable cells such as bacterial or fungal cells, which further enhances the yield of the hydroxy fatty acids produced by the enzyme.

The above-mentioned objects of the invention are solved by the method according to claim 1. Preferred embodiments are represented by the subject-matter of the dependent claims.

The aim of the present inventors was to characterize a 67-kDa protein from Streptococcus pyogenes which was previously described as a myo sin-cross-reactive antigen present only in pathogenic groups of Streptococci (KiI et al. (1994) Infect. Immun. 62: 2440-2449). This protein has some sequence similarity to previously described fatty acid double bond isomerases. However, surprisingly, the inventors observed that hydoxy fatty acids are the only reaction products when the protein is incubated together with unsaturated fatty acids. Therefore, this protein has the activity of a fatty acid hydratase instead of the activity of an isomerase. The protein of the present invention has regioselectivity, as it only hydrates the carbon atom at position 10 of the fatty acids and not the carbon atom at position 9 of the fatty acids, leading to the production of 10-hydroxy fatty acids (see Table 1) and as only substrates possessing a double bond in the cώ-confϊguration are hydrated by the enzyme.

The fatty acid hydratase of the present invention catalyses the nucleophilic addition of a water molecule to an unsaturated fatty acid which has a double bond between carbon atoms 9 and 10 which leads to the formation of 10-hydroxy fatty acids. The present invention relates to a method for producing hydroxy fatty acids from unsaturated fatty acids by use of a protein having the enzymatic activity of a fatty acid hydratase wherein the protein is encoded by a nucleic acid sequence selected from the group consisting of: a) nucleic acid sequences comprising a nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQ ID NO: 1, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, b) nucleic acid sequences comprising the nucleotide sequence depicted in

SEQ ID NO: 2, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, c) nucleic acid sequences comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of said nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, d) nucleic acid sequences comprising a nucleotide sequence which shows at least 40 % identity to the nucleotide sequence from a), b) or c), or comprising fragments of said nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase. The present invention further relates to a protein having the enzymatic activity of a fatty acid hydratase and the amino acid sequence depicted in SEQ ID No. 1 or an amino acid sequence which is at least 50% or 60%, preferably at least 70%, 75% or 80%, more preferably at least 85% or 90% and most preferably at least 95%, 97% or 99% identical to the amino acid sequence depicted in SEQ ID NO. 1.

The term "unsaturated fatty acid" refers to a fatty acid which contains at least one double bond. Examples for unsaturated fatty acids include lauroleic acid (C 12:1), myristoleic acid (C14:l), palmitoleic acid (C16:l), oleic acid (C18:l), linoleic acid (C18:2), alpha- lino lenic acid (C18:3), arachidonic acid (20:4), eicosapentaenoic acid (C20:5), erucic acid (C22:l) and docosahexaenoic acid (C22:6).

"Hydroxy fatty acids" are formed from the unsaturated fatty acids by hydration, i.e. water addition to the double bond, which means that one carbon atom of the double bond contains a hydroxy group and one carbon atom of the double bond contains a hydrogen atom after the hydration reaction.

The fatty acid hydratase of the present invention specifically produces 10-hydroxy fatty acids in which the hydroxy group is located on carbon atom 10 of the fatty acid. For example, 10-hydroxyhexadecanoic acid is produced from palmitoleic acid, 10- hydroxyoctadecanoic acid is produced from oleic acid, 10-hydroxy-(9Z)-octadec-9- enoic acid is produced from linoleic acid and 10-hydroxy- 12Z, 15Z- octadeca-12,15- dienoic acid is produced from α-linolenic acid.

The usual numbering of the carbon atoms within the fatty acid molecules is used, starting from the carbon atom of the carboxy group of the fatty acid. The unsaturated fatty acids which are to be reacted to the hydroxy fatty acids may be used in pure form or in the form of their natural precursors which include, for example, natural oils and fats from different organisms containing a considerable amount of the fatty acid which is to be converted by the fatty acid hydratase to the corresponding hydroxy fatty acid. Examples of natural oils and fats include obtusiloba oil, evening primrose seed oil, soybean oil, corn oil, safflower oil, wheat germ oil, rice oil, sesame oil, rapeseed oil, olive oil, linseed oil, milk fat, suet, lard, egg yolk oil, fish oil, seaweed, algae, filamentous fungi, ferns and protozoa. Hydrolysates of natural oils and fats can be obtained by treating natural oils and fats with an enzyme such as a hydrolase, for example a lipase. The type of natural precursor to be used depends on the type of fatty acid which is to be reacted with the fatty acid hydratase. For example, linseed oil may be used as a natural precursor of linolenic acid, while sunflower oil may be used as a natural precursor of linoleic acid.

The fatty acid to be converted by the fatty acid hydratase of the present invention has a length of between 12 and 22 carbon atoms, i.e. 12, 14, 16, 18, 20 or 22 carbon atoms, preferably a length of between 12 and 18 carbon atoms, i.e. 12, 14, 16 or 18 carbon atoms. Most preferably the fatty acid to be converted by the enzyme of the present invention is oleic acid which has one double bond between carbon atoms 9 and 10 or its natural precursor, yielding upon hydration 10-hydroxy octadecanoic acid.

The production of hydroxy fatty acids by the fatty acid hydratase of the present invention may take place both in vitro and in vivo.

For example, transgenic cells (e.g. E.coli) may be lysed, e.g. by sonifϊcation, osmotic shock or by the use of cell lyses kits such as Cellutic® of Sigma and then the lysate may be directly added to a solution of fatty acids. For this, the cell pellet has to be resuspended in triple volume of lysis buffer, treated with ultrasound if necessary, and centrifuged at 20.000 g for 10 minutes. 100 μl of soluble extract prepared in this way is typically incubated with 5 to 60 μg, preferably 10 to 50 μg and most preferably 20- 30 μg of fatty acid in 1 ml of an appropriate buffer, as described in the Examples. After 1 hour incubation around 60% of substrate was converted to 10-hydroxy- octadecenoic acid when linoleic acid was used as a substrate, based on GC-MS data.

The fatty acid hydratase may also be purified from a transgenic organism by general methods for extraction and purification of a protein. For example, an enzyme can be extracted from the cells using a homogenizer or glass beads, or by ammonia dissolution, the enzyme method, etc. and then purified by means of filtration, centrifugation, salting-out, precipitation with an organic solvent, immune precipitation, etc. as well as dialysis, ultrafiltration, gel filtration, electrophoresis, chromatography using an adsorbent, an affinity adsorbent or molecular seeds, liquid- phase partitition, ion exchange, batch method and crystallization, either alone or in combination.

Preferably, the nucleotide sequence coding for the fatty acid hydratase is cloned into a vector which contains a nucleotide sequence coding for an affinity tag such as cellulose binding protein, maltose binding protein, glutathione- S -transferase or a His- tag. After transcription and translation of the DNA, a fusion protein of the tag and the fatty acid hydratase is formed which can then be easily purified by means of affinity chromatography.

Vectors and methods which are useful for the production and purification of the fusion protein are well known to those skilled in the art. After purification, the affinity tag may be removed by the action of a protease such as factor X. However, such affinity tags provide increased activity of the recombinant fusion protein due to improved protein folding/solubility, etc. These and other tags also allow the immobilization of the fusion protein on cheap matrices such as amylose, cellulose, which can be used in reactors for the stabilization and recycling of the recombinant enzyme. One example for purifying fatty acid hydratase by means of a His-tag and a corresponding affinity chromatography column is given below.

Typically, 10 μg of purified protein is incubated with 10 to 50 μg, preferably 20 to 40 μg and most preferably 30 μg of fatty acid in an appropriate buffer.

The method according to the invention for expressing the fatty acid hydratase in a transgenic organism causes an increase of the fatty acid hydratase content in a transgenic organism, e.g. a transgenic plant and/or plant cell or a transgenic bacterial cell or a transgenic yeast cell as compared to the corresponding wild type cell. This increase in content is at least 5%, preferably at least 20%, also preferably at least 50%, especially preferably at least 100%, also especially preferably at least by the factor of 5, particularly preferably at least by the factor of 10, also particularly preferably at least by the factor of 50, and most preferably at least by the factor of 100.

According to the invention, the term "wild type" is to be understood as the respective non genetically modified parent organism.

The term "fragments of DNA," as it is used herein, is to be understood as DNA segments encoding a protein having the activity of a fatty acid hydratase, wherein the proteins encoded by the DNA segments essentially have the same fatty acid hydratase activity as the proteins encoded by the complete DNA sequence, and the production of hydroxy fatty acids can be achieved with these fragments. The DNA fragment has at least 10% or 20%, preferably 30 or 40%, more preferably 50% or 60%, even more preferably 70% or 75% and most preferably 80%, 85%, 90% or 95% of the length of the complete DNA sequence.

The term "fragments of the protein," as it is used herein, indicates protein segments having the activity of the specific fatty acid hydratase according to the invention, wherein the protein segments essentially have the same fatty acid hydratase activity as the full length protein, and the production of hydroxy fatty acids can be achieved with these fragments. The protein fragment has at least 10% or 20%, preferably 30 or 40%, more preferably 50% or 60%, even more preferably 70% or 75% and most preferably 80%, 85%, 90% or 95% of the length of the complete protein sequence.

For producing hydroxy fatty acids according to the method of the present invention, also proteins having an amino acid sequence identity of at least 50% or 60%, preferably at least 70%, 75% or 80%, more preferably at least 85% or 90% and most preferably at least 95%, 97% or 99% to the amino acid sequence depicted in SEQ ID NO. 1 can be used.

"Essentially the same enzymatic activity" of the fatty acid hydratase used in the method according to the invention means that the enzymatic activity of the fragment is still at least 50%, preferably at least 60%, especially preferably at least 70%, and particularly preferably at least 80%, and most preferably at least 90% or 95% as compared to the enzymes encoded by the nucleic acid sequence with SEQ ID No. 2. Fatty acid hydratase enzymes with essentially the same enzymatic activity are thus also capable of producing hydroxy fatty acids in transgenic bacterial cells, transgenic yeast cells or transgenic plant cells. The fatty acid hydratase activity can be determined by incubating the purified enzyme or extracts from host cells or a host organism with an unsaturated fatty acid substrate such as oleic acid under appropriate conditions and analysis of the reaction products, e.g. by GC-MS analysis. Details about enzyme activity assays and analysis of the reaction products are given below in the Examples.

The term "nucleic acid (molecule)", as it is used herein, comprises furthermore in a preferred embodiment the untranslated sequence located at the 3' and at the 5' end of the encoding gene region: at least 500, preferably 200, especially preferably 100 nucleotides of the sequence upstream of the 5' end of the encoding region, and at least 100, preferably 50, especially preferably 20 nucleotides of the sequence downstream of the 3' end of the encoding gene region.

An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural repository of nucleic acids. An "isolated" nucleic acid preferably has no sequences naturally flanking the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (e.g. sequences located at the 5' and 3' ends of the nucleic acid). In various embodiments the isolated fatty acid hydratase molecule can contain, for example, less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0,5 kb, or 0,1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates. All nucleic acid molecules mentioned herein can be e.g. RNA, DNA, or cDNA.

The nucleic acid molecules used in the method, such as a nucleic acid molecule with a nucleotide sequence of SEQ ID No. 2 or a part thereof, can be isolated using standard molecular biological techniques and the sequence information provided herein. With the aid of comparison algorithms, which can be found e.g. on the NCBI homepage under http://www.ncbi.nlm.nih.gov, a homologous sequence, for example, or homologous, conserved sequence regions can also be identified on DNA or amino acid level. Essential parts of this sequence, or the entire homologous sequence can be used as a hybridization probe using standard hybridization techniques (as described in Sambrook et al, vide supra) for isolating additional nucleic acid sequences from other organisms which are useful in the method by means of screening of cDNA and/or genomic libraries. Moreover, a nucleic acid molecule comprising a complete sequence of SEQ ID No. 2 or a part thereof, can be isolated by means of a polymerase chain reaction, using oligonucleotide primers on the basis of the sequences given herein or parts thereof (e.g. a nucleic acid molecule comprising the complete sequence or a part thereof can be isolated by means of polymerase chain reaction using oligonucleotide primers which have been generated based on this same sequence). For example, mRNA can be isolated from cells (e.g. by means of the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294 - 5299), and cDNA can be produced from it by means of reverse transcriptase (e.g. Moloney-MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku Amerika, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification by means of polymerase chain reaction can be generated based on the sequence shown in SEQ ID No. 2, or with the aid of the amino acid sequence shown in SEQ ID No. 1. A nucleic acid according to the invention can be amplified by using cDNA, or alternatively by using genomic DNA as a template, as well as suitable oligonucleotide primers by means of standard PCR amplification techniques. The nucleic acid amplified in this way can be cloned in a suitable vector and characterized by means of DNA sequence analysis. Oligonucleotides corresponding to a fatty acid hydratase nucleotide sequence can be produced by standard synthesis methods, such as an automatic DNA synthesizer. The fatty acid hydratase activity of the proteins encoded by these nucleic acid sequences can then be determined by the enzyme assays described below in the Examples.

In the context of this invention the term "hybridization under stringent conditions" means that the hybridization is performed in vitro under conditions stringent enough to ensure a specific hybridization. Stringent in vitro hybridization conditions are known to the person skilled in the art, and can be found in the literature (e.g. Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3^rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY). The term "specific hybridization" refers to the fact that a molecule preferably binds to a certain nucleic acid sequence, the target sequence, under stringent conditions, if the target sequence is part of a complex mixture of, for example, DNA or RNA molecules, but does not bind, or at least to a considerably lesser degree, to other sequences.

Stringent conditions depend on the circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected so that the hybridization temperature is approximately 5°C below the melting point (T_m) for the specific sequence at a defined ionic strength and a defined pH value. T_m is the temperature (at a defined pH value, a defined ionic strength and a defined nucleic acid concentration) at which 50% of the molecules complementary to the target sequence hybridize to the target sequence in the equilibrium state. Typically, stringent conditions are those in which the salt concentration is at least about 0.01 to 1.0 M of sodium ion concentration (or the concentration of another salt) at a pH of between 7.0 and 8.3 and the temperature is at least 30⁰C for short molecules (i.e. for example 10 to 50 nucleotides). Furthermore, stringent conditions can comprise the addition of agents, such as formamide, which destabilize the hybrids. In hybridization under stringent conditions as used herein, nucleotide sequences which are at least 60% homologous to each other usually remain hybridized to each other. Preferably, the stringent conditions are selected in such a way that sequences which are homologous to each other by at least about 65%, preferably at least about 70%, and especially preferably at least about 75%, or more, usually remain hybridized to each other. A preferred, non-limiting example for stringent hybridization conditions are hybridizations in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washing steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. The temperature ranges, for example, under standard hybridization conditions depending on the type of nucleic acid, between 42°C and 58°C in an aqueous buffer at a concentration of 0.1 to 5 x SSC (pH 7.2).

If an organic solvent, e.g. 50% formamide, is present in the above-mentioned buffer, the temperature under standard conditions is about 42°C. Preferably, the hybridization conditions for DNA:DNA hybrids are for example 0.1 x SSC and 20⁰C to 45°C, preferably 30⁰C to 45°C. Preferably, the hybridization conditions for DNA:RNA hybrids are for example 0.1 x SSC and 30⁰C to 55°C, preferably between 45°C to 55°C. The hybridization temperatures mentioned above are determined for example for a nucleic acid having a length of about 100 base pairs and a G/C content of 50% in the absence of formamide. The person skilled in the art knows how the required hybridization conditions can be determined using the above mentioned, or the following, textbooks: Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), Hames und Higgins (publisher) 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (publisher) 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

The term "sequence identity" in terms of the invention denotes identity, i.e. the same nucleotides in the same 5 '-3' sequence, across the entire sequence of the nucleic acid sequence depicted in SEQ ID No. 2, of at least 40%, 50%, 60%, 70%, 75%, 80%, preferably of at least 85%, especially preferred of at least 90%, 93% and most preferred of at least 95%, 97%, 99%.

The sequence identity is determined by means of a number of programs which are based on various algorithms. The algorithms of Needleman and Wunsch, or of Smith and Waterman, provide particularly reliable results. For the sequence comparisons, the program PiIeUp (Feng and Doolittle (1987) J. MoI. Evolution 25: 351 - 360; Higgins et al. (1989) CABIOS 5: 151 - 153), or the programs Gap and Best Fit (Needleman and Wunsch (1970) J. MoI. Biol. 48: 443 - 453, and Smith and Waterman (1981) Adv. Appl. Math. 2: 482 - 489) were used, which are contained in the GCG software package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA).

The sequence identity values stated above in percent were determined using the program GAP across the entire sequence region with the following settings: gap weight: 50, length weight: 3, average match: 10,000 and average mismatch: 0.000.

Unless stated otherwise, these settings were used as the standard settings for sequence comparisons.

Nucleic acid sequences deviating from the nucleic acid sequence given in SEQ ID No.2 can, for example, be created by the insertion of one or several nucleotide substitutions, additions, or deletions in a nucleotide sequence of SEQ ID No. 2, so that proteins are created into which one or more amino acid substitutions, additions, or deletions were inserted as compared to the sequence stated in SEQ ID No. 2.

Mutations can be inserted in one of the sequences of SEQ ID No. 2 using standard techniques, such as site-specific mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are generated at one or several of the predicted nonessential amino acid residues, that is at amino acid residues which do not influence the enzymatic activity of the fatty acid hydratase. In a "conservative amino acid substitution" an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the area of expertise. These families comprise amino acids having basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid and glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as alanine, valine, leucine, iso leucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (such as threonine, valine, iso leucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan). A predicted nonessential amino acid residue in the fatty acid hydratase used according to the invention will thus preferably be replaced by another amino acid residue from the same family of side chains. Alternatively, in another embodiment the mutations may be inserted randomly across the entire sequence or a part of it encoding the fatty acid hydratase, for example, by means of saturation mutagenesis, and the resulting mutants may be screened for the fatty acid hydratase activity by recombinantly expressing the encoded protein, in order to identify mutants which have retained the fatty acid hydratase activity. The fatty acid hydratase activity of the protein can, for example, be determined using the assays described herein.

In terms of the invention, "transgenic" or "recombinant" means, with regard to e.g. a nucleic acid sequence, an expression cassette (= gene construct), or a vector containing the nucleic acid sequence according to the invention, or an organism transformed with the respective nucleic acid sequences, expression cassettes, or vectors, all of those constructs being produced by means of genetic technologies, that either a) the nucleic acid sequence according to the invention, or b) a genetic control sequence functionally linked to the nucleic acid sequence according to the invention, such as a promoter, or c) a) and b)

is not in its natural genetic environment, or has been modified by genetic techniques, the modification being, for example, a substitution, addition, deletion, inversion, or insertion of one or several nucleotide residues. Natural genetic environment means the natural genomic, or chromosomal locus in the parental organism, or the existence in a genomic library. In case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably at least partially conserved. The environment flanks the nucleic acid sequence at least on one side, and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, particularly preferably at least 5000 bp. A naturally occurring expression cassette - such as the naturally occurring combination of the natural promoter of the fatty acid hydratase with the respective fatty acid hydratase genes - becomes a transgenic expression cassette when it is modified by means of non-natural, synthetic ("artificial") methods, such as a mutagenization. Corresponding methods are described in US 5,565,350 or WO 00/15815, for example.

In terms of the invention, the term transgenic plant, or plant cell, or transgenic bacterial cell or transgenic yeast cell means, as described above, that the nucleic acids used in the method are not at their natural site in the genome of the plant, or the plant cell or transgenic bacterial cell or transgenic yeast cell, whereby the nucleic acids can be homologously or heterologously expressed. However, transgenic also means that although the nucleic acids according to the invention are located at their natural site in the genome of an organism, the sequence has been changed compared to the natural sequence, and/or the regulatory sequences of the natural sequences have been modified. Preferably, transgenic is to be understood as the expression of the nucleic acids according to the invention at a non-natural site in the genome, i.e. the nucleic acids are homologously, or preferably heterologously expressed.

It is obvious to the person skilled in the art that the nucleic acid sequence used for the production of the transgenic plant, or plant cell, or transgenic bacterial cell or transgenic yeast cell which encodes a fatty acid hydratase, may have to be adjusted to the organism specific codon usage. The codon usage can be determined with computer analyses of other known genes of the selected organism.

Furthermore, the method of the present invention further comprises recovering the produced hydroxy fatty acids and/or using the produced hydroxy fatty acids for the production of flavor ingredients and/or fragrance ingredients and/or - using the produced hydroxy fatty acids as lubricant, surfactant or plasticizer and/or using the produced hydroxy fatty acids for the production of lubricants, surfactants, detergents, coatings, paints, biopolymers and/or plasticizers.

The hydroxy fatty acids may be recovered from the transgenic organism or the in vitro reaction mixture by extraction and adsorption techniques which are well known to the person skilled in the art. The short-chain hydroxy fatty acids can be used as a fragrance or flavor ingredient.

The hydroxy fatty acids can also be reacted to lactones which are ubiquitous volatile flavors. Generally, lactones are produced by the beta-oxidation of hydroxy fatty acids. The position and stereo-chemical orientation of the hydroxy group in the hydroxy fatty acids determine which particular lactone will be produced. For example, γ-dodecalactone is formed from 10-hydroxy octadecanoic acid, which is the hydration product of oleic acid. Usually, β-oxidation is performed by fermentation in the presence of a fungus or a yeast species such as Aspergillus oryzae, Geotrichium klebahnii, Yarrowia lipolytica, and Hansenula saturnus. Preferably, Yarrowia lipolytica is added to the hydroxy fatty acids. However, the process of beta- oxidation often does not lead to the production of the lactone directly, but to the production of the direct precursor of the lactone, which is then spontaneously lactonized under acid conditions. Therefore, it is preferred to adjust the pH of the culture to a pH between 2 and 5 after the β-oxidation reaction in order to enhance the yield of the lactone.

An overview of the production of lactones from hydroxy fatty acids is given in Krings and Berger (1998) Appl. Microbiol. Biotechnol. 49: 1-8; Wache et al. (2003) Appl. Microbiol. Biotechnol. 61 : 393-404 and in EP 0 578 388.

After their insertion into a bacterial cell, yeast cell, plant cell, or plant, the nucleic acids used in the method can either be located on a separate plasmid, or advantageously be integrated into the genome of the host cell. In the case of integration into the genome, the integration can occur randomly, or by means of such a recombination that the native gene is replaced by the inserted copy, which causes the modulation of cellular fatty acid hydratase expression, or by using a gene in trans so that the gene is functionally linked to a functional expression unit which contains at least one sequence ensuring the expression of a gene, and at least one sequence ensuring the polyadenylation of a functionally transcribed gene.

In addition to the nucleic acid sequence for the fatty acid hydratase to be transferred, the recombinant nucleic acid molecules which are used for the expression of the fatty acid hydratase comprise further regulatory elements. Which precise regulatory elements these vectors have to contain depends in each case on the process in which these vectors are to be used. The person skilled in the art and familiar with the various methods mentioned above for the production of transgenic plants in which the expression of a protein is increased, knows which regulatory and other elements these vectors must contain.

Typically, the regulatory elements which are part of the vectors are such that allow for the transcription and, if desired, for the translation in the plant cell, bacterial cell or yeast cell. Depending on the organism selected, this can mean, for example, that the gene is only expressed and/or overexpressed after induction, or that it is expressed and/or overexpressed immediately. For example, these regulatory sequences are sequences to which inductors or repressors bind, thereby regulating the expression of the nucleic acid. In addition to these new regulatory sequences, or instead of these sequences, the natural regulation of the sequences upstream of the actual structural genes may still be existent, and may possibly have been genetically modified so that the natural regulation is disabled, and the expression of the genes is increased. The recombinant nucleic acid molecule, however, can also be constructed in a simpler manner, i.e. no additional regulation signals are inserted upstream of the nucleic acid sequence, and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence has been mutated in such a manner that regulation no longer occurs, and/or gene expression is increased. To increase the activity, these altered promoters can also be inserted singly, in the form of partial sequences, upstream of the natural gene. Furthermore, the gene construct can also advantageously contain one or more so-called enhancer sequences which are functionally linked to the promoter, and which allow an increased expression of the nucleic acid sequence. Additional useful sequences, such as additional regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequences. In principle, it is possible to use any of the natural promoters with their regulation sequences for the method according to the invention. However, it is also possible and advantageous to use synthetic promoters only or in addition.

For the expression in plant cells, the promoters can be either constitutive, inducible, tissue-specific or development-specific promoters. The choice of promoter, as well as of other regulatory sequences, determines the regional and temporal expression pattern, and therefore also the fatty acid hydratase expression in transgenic plants.

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter as well as other constitutive promoters described in WO 99/43838 and US patent No 6,072,050; the CaMV 35S promoter (Odell et al. (1985) Nature 313: 810 - 812); the actin promoter (McElroy et al. (1990) Plant Cell 2:163 - 171); the ubiquitin promoter (Christensen et al. (1989) Plant MoI. Biol. 12: 619 - 632 and Christensen et al. (1992) Plant MoI. Biol. 18: 675 - 689); the pEMU promoter (Last et al. (1991) Theor. Appl. Genet. 81 : 581 - 588); the MAS promoter (Velten et al. (1984) EMBO J. 3: 2723 - 2730); the ALS promoter (US application No 08/409,297), and similar promoters. When using a constitutive promoter, a cell- specific or tissue-specific expression can also be achieved by inhibiting the gene expression in the cells, or tissues, in which they are not desired, e.g. by forming antibodies which bind the gene product and thus prevent its activity, or by suitable inhibitors which act in these cells.

The fatty acid hydratase gene may also be expressed under control of an inducible promoter, and especially preferably by a pathogen-inducible promoter or a wound- inducible promoter. Suitable promoters are well-known to the person skilled in the art. Chemically regulated promoters can be used in order to regulate the expression of a gene in a plant by the use of an exogenous chemical regulator (see review in Gatz (1997) Annu. Rev. Plant Physiol. Plant MoI. Biol, 48: 89-108). Chemically inducible promoters are particularly suited whenever it is desired that the gene expression occurs in a time specific manner. Examples for this include the In2-2 promoter of corn, which is activated by benzenesulfonamide, the GST promoter of corn, which is induced by hydrophobic electrophilic compounds, and the PR- Ia promoter of tobacco, which is activated by salicylic acid. Other chemically regulated promoters include steroid responsive promoters (see, for example, the glucocorticoid- inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421 - 10425 and McNellis et al. (1998) Plant J. 14 (2): 247 - 257), ethanol- inducible and tetracycline-inducible promoters (see, for example, Gatz et al. (1991) MoI. Gen. Genet. 227: 229 - 237; US patent Nos. 5,814,618 and 5,789,156).

The person skilled in the art knows that the use of inducible promoters allows the production of plants, or plant cells, which express only transiently the sequences according to the invention. Situations, in which a transient resistance is desirable, are known to the person skilled in the art. Furthermore, the person skilled in the art also knows that transient expression, and thereby transient resistance can be achieved by the use of vectors non-stab Iy replicating in plant cells, and carrying the respective sequences for the expression of the fatty acid hydratase.

Tissue-specific promoters can also be used in order to achieve increased fatty acid hydratase expression within a certain plant tissue. Suitable tissue-specific promoters are, for instance, those allowing leaf-specific, epidermis-specific, fruit-specific and mesophyll-specific expression. Suitable tissue-specific promoters are well-known to the person skilled in the art. Furthermore, the person of average skill in the art is able to isolate additional suitable promoters using routine methods. Thus, the person skilled in the art using current molecular biological methods, such as hybridization experiments, or DNA protein binding studies is able to identify e.g. additional epidermis-specific regulatory nucleic acid elements. Here, for example, the desired tissue is isolated in a first step from the desired organism from which the regulatory sequences are to be isolated, the entire poly(A)⁺ RNA is isolated from it, and a cDNA library is created. In a second step, using cDNA clones which are based on poly(A)⁺ RNA molecules from another tissue, those clones are identified from the first bank by means of hybridization whose corresponding po Iy(A)⁺ RNA molecules merely accumulate in the desired tissue. Then, using the cDNAs thus identified, promotors are isolated which have tissue-specific regulatory elements. Additional PCR based methods for the isolation of suitable tissue-specific promoters are also available to the person skilled in the art.

The vectors according to the invention can additionally comprise e.g. enhancer elements as regulatory elements. They may also contain resistance genes, replication signals, and additional DNA regions, which enable propagation of the vectors in bacteria, such as E. coli. The regulatory elements also comprise sequences which effect a stabilization of the vectors in the host cells. Such regulatory elements particularly comprise sequences facilitating stable integration of the vector into the host genome of the plant, or an autonomous replication of the vector in the plant cells. Such regulatory elements are known to the person skilled in the art.

The so-called termination sequences are sequences which ensure the proper termination of transcription, or translation. If the transferred nucleic acids are to be translated, the termination sequences are typically stop codons and respective regulatory sequences; if the transferred nucleic acids are only to be transcribed, they are generally po Iy(A) sequences.

As used herein, the term "vector" relates to a nucleic acid molecule which can transport another nucleic acid, to which it is bound, into a cell. A vector type is a "plasmid" representing a circular double stranded DNA loop, into which additional DNA segments can be ligated. Another vector type is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they have been inserted (e.g. bacterial vectors with a bacterial replication origin). Other vectors are advantageously integrated into the genome of a host cell when inserted in the host cell, and thereby replicated together with the host genome. Also, certain vectors can control the expression of genes to which they are functionally linked. These vectors are called here "expression vectors." Usually, expression vectors suitable for DNA recombination techniques are of the plasmid type. In the present description

"plasmid" and "vector" can be used interchangeably, since the plasmid is the vector type most often used. However, the invention is also intended to comprise other types of expression vectors, such as viral vectors which fulfill similar functions. Furthermore, the term "vector" is also intended to comprise other vectors known to the person skilled in the art, such as phages, viruses, such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.

In a recombinant expression vector, the term "operatively linked thereto" means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in such a way that the expression of the nucleotide sequence is possible, and that both sequences are linked to each other in such a way so as to fulfil the predicted function ascribed to the sequence. The term "regulatory sequence" is intended to comprise promoters, enhancers, and other expression control elements (e.g. terminator sequences, polyadenylation signals). These regulation sequences are described e.g. in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or in Gruber and Crosby, in: Methods in Plant Molecular Biology and

Biotechnology, CRC Press, Boca Raton, Florida, publisher: Glick and Thompson, Chapter 7, 89-108. Regulatory sequences comprise those sequences which regulate the constitutive expression of a nucleotide sequence in many types of host cells, and those sequences which regulate the direct expression of the nucleotide sequence only in certain host cells under certain conditions. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-, tet-, lpp-, lac-, lpp-lac-, laclq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SP02,phage lambdaPR, phage lambdaPL, phage SPOl Pi₅, phage SPOl P₂₆, pSOD, EFTu, EFTs, GroEL, MetZ (last 5 from C. glutamicum), which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADCl, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28, ADH and ENO2. The person skilled in the art knows that the design of the expression vector can depend on factors, such as the choice of the host cell to be transformed, the desired extent of the protein expression, etc.

The recombinant expression vectors used for the expression of the fatty acid hydratase can be active in both prokaryotic and eukaryotic cells. This is advantageous, since intermediate steps of the vector construction are often performed for the sake of simplicity in microorganisms. These cloning vectors contain a replication signal for the respective microorganism, and a marker gene for the selection of successfully transformed bacterial cells. Suitable vectors for expression in prokaryotic organisms are known to the person skilled in the art; they include e.g. E. coli pEXP5-NT/TOPO, pMAL series, pLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as pUC18, or pUC19, the Ml 13mp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-Bl, λgtl l, or pBdCl, Streptomyces pIJlOl, pIJ364, pIJ702, or pIJ361, Bacillus pUBl 10, pC194, or pBD214, Corynebacterium pSA77, or pAJ667.

In another embodiment the expression vector represents a yeast expression vector or a bacillovirus expression vector.

The above named vectors provide only a small overview of possible suitable vectors. Additional plasmids are known to the person skilled in the art and are described in e.g.: Cloning Vectors (publisher Pouwels, P.H. et al. Elsevier, Amsterdam, New

York-Oxford, 1985). For additional suitable expression systems for prokaryotic and eukaryotic cells see chapters 15 and 16 of Sambrook and Russell, vide supra.

In another embodiment of the method, the fatty acid hydratase can be expressed in single-celled plant cells (such as algae), see Falciatore et al., 1999, Marine

Biotechnology 1 (3):239-251 and the literature cited therein, and in plant cells of higher plants (e.g. spermatophytes, such as crop plants). Examples for plant expression vectors comprise those extensively described in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992), Plant MoI. Biol. 20: 1195-1197; and Bevan, M.W. (1984), Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1, Engineering and Utilization, publisher: Kung and R. Wu, Academic Press, 1993, S. 15-38.

Since the expression of plant genes is frequently not limited to the transcription level, a plant expression cassette preferably contains, in addition to the elements described above, other functionally linked sequences such as translation enhancers, e.g. the overdrive sequence containing the 5' untranslated leader sequence of the tobacco mosaic virus, which increases the protein/RNA ratio (Gallie et al. (1987) Nucl. Acids Research 15: 8693-8711).

The gene to be expressed must, as described above, be functionally linked to a suitable promoter which regulates the gene expression in a time specific, cell specific or tissue specific manner. Suitable promoters have already been described above.

Other preferred sequences for the use in the functional connection in gene expression cassettes are targeting sequences which are required for the targeting of the gene product into the respective cell compartment (see an overview in Kermode (1996) Crit. Rev. Plant Sci. 15 (4): 285-423 and the literature cited therein), such as into the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, elaio somes, peroxisomes, and other compartments of plant cells.

In order to insert the fatty acid hydratase sequence into the expression vectors, it is advantageously subjected to an amplification and ligation in the known manner. Preferably, one proceeds in accordance with the protocol of the Pfu DNA polymerase, or a Pfu/Taq DNA polymerase mixture. The primers are selected in accordance with the sequence to be amplified. Advantageously, the primer should be selected so that the amplificate comprises the entire codogenic sequence from the start codon to the stop codon. Advantageously, the amplificate is analyzed subsequent to the amplification. For example, the analysis can be made in respect of quality and quantity after gel electrophoretic separation. The amplificate can then be purified according to a standard protocol (e.g. Qiagen). An aliquot of the purified amplificate is then available for the subsequent cloning. Suitable cloning vectors are generally known to the person skilled in the art. These particularly include vectors which are replicable in microbial systems, i.e. especially vectors which allow for an efficient cloning in bacteria, yeasts or fungi, and which allow for the stable transformation of plants. Especially worth mentioning are various binary and co-integrated vector systems suitable for the T-DNA mediated transformation of plants. Such vector systems are usually characterized in that they contain at least the vir-genes needed for the agrobacteria mediated transformation, as well as the T-DNA limiting sequences (T-DNA border). Preferably, these vector systems also comprise further cis regulatory regions, such as promoters and terminators and/or selection markers used to identify the respective transformed organisms. While vir genes and T-DNA sequences are arranged on the same vector in co-integrated vector systems, binary systems are based on at least two vectors, one of which carries vir genes, but no T-DNA, and a second carries T-DNA, but no vir genes. The latter vectors are thus relatively small, easy to manipulate and replicable both in E. coli as well as in agrobacterium. These binary vectors include vectors of the series pBIB-HYG, pPZP, pBecks, pGreen. According to the invention, Binl9, pBHOl, pBinAR, pGPTV and pCAMBIA are preferred. An overview of binary vectors and their use is provided by Hellens et al. (2000) Trends in Plant Science 5, 446-451.

For the preparation of the vector, the vectors can initially be linearized by means of restriction endonuclease(s) and then enzymatically modified in any suitable way. The vector is then purified and an aliquot is used for cloning. During cloning the enzymatically cut and if necessary purified amplificate is linked to similarly prepared vector fragments by means of a ligase. A certain nucleic acid construct, or vector construct, or plasmid construct, may have one, or even several, codogenic gene regions. Preferably, the codogenic gene regions in these constructs are functionally linked to regulatory sequences. The regulatory sequences especially include plant sequences, such as the promoters and terminators described above. The constructs can be advantageously cultivated in microorganisms, especially in E. coli and Agrobacterium tumefaciens, in a suitable medium, and stably propagated under selection conditions. The cells are then harvested and lysed and the plasmid is extracted therefrom. This allows a transfer of heterologous DNA into plants or microorganisms .

With the advantageous use of cloning vectors, the nucleic acids used in the method according to the invention, the nucleic acids and the nucleic acid constructs according to the invention can be inserted into organisms, such as microorganisms, or plants, and used for plant transformation, just as those published and cited in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapters 6/7, p. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1, Engineering and Utilization, publisher: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al, Techniques for Gene Transfer, in: Transgenic Plants, Bd. 1, Engineering and Utilization, publisher: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus (1991) Annu. Rev. Plant Physiol. Plant Molec. Biol. 42: 205-225. The nucleic acids used in the method, the nucleic acids and nucleic acid constructs, and/or vectors according to the invention, can therefore be used for the genetic modification of a broad spectrum of organisms, preferably of bacterial cells, but also of yeast cells or plant cells.

There are plurality of known techniques available for inserting DNA into a plant host cell, and the person skilled in the art will have no difficulty in finding the most suitable method in each case. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, the fusion of protoplasts, the direct gene transfer of isolated DNA into protoplasts, the electroporation of DNA, the insertion of DNA by means of the biolistic method, as well as other possibilities. Both stable and transient transformants can be generated in this manner.

The transformed cells grow within the plant in the usual way (also see McCormick et al. (1986), Plant Cell Reports 5, 81-84). The resulting plants can be cultivated normally, and interbred with plants having the same transformed genetic code, or a different genetic code. The resulting hybrid individuals possess the corresponding phenotypical characteristics.

Two or more generations should be cultivated in order to ensure that the phenotypical characteristic is stably retained and transmitted. Also, seeds should be harvested in order to ensure that the respective phenotype, or other characteristic have been retained.

Likewise, transgenic lines can be identified according to conventional methods which lines are homozygous for the new nucleic acid molecules, and their phenotypical behavior with regard to a present, or absent pathogen responsiveness can be analyzed and compared to the behaviour of hemizygous lines.

Of course, the plant cells containing the nucleic acid molecules according to the invention may also be further cultivated in the form of a cell culture (including protoplasts, calli, suspension cultures, and the like).

According to the invention, the term transgenic plant comprises the plant in its entirety, as well as all parts of the plant in which the expression of the fatty acid hydratase proteins according to the invention is increased. This includes all parts of the plant and plant organs, such as leaf, stem, seed, root, tubers, anthers, fibers, root hair, stalk, embryos, calli, cotelydons, petioles, crop material, plant tissue, reproductive tissue, cell cultures derived from the transgenic plant, and/or which can be used to produce the transgenic plant.

The plants used for the method according to the invention can in principle be any plant which is suitable for the synthesis and modification of fatty acids. Preferably, it is a monocotyledonous or dicotyledonous agricultural plant, a food plant or a fodder plant.

Examples pf monocotyledonous plants are plants belonging to the genera Avena (oat), Triticum (wheat), Secale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn), and the like.

Dicotyledonous agricultural plants comprise, inter alia, cotton, leguminous plants such as pulses, and especially alfalfa, soy bean, oilseed rape, canola, tomato, sugar beet, potato, sunflower, ornamental plants as well as trees. Additional agricultural plants can comprise fruit (especially apples, pears, cherries, grapes, citrus, pineapples, and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal as well as in medical plants Rauwolfia and Digitalis. Especially preferred are the cereals wheat, rye, oats, barley, rice, corn, and millet, as well as the dicotyledonous plants sugar beet, oilseed rape, soy, tomato, potato, and tobacco. Additional agricultural plants can be gathered from US patent No. 6,137,030.

Preferred plants are marigold, sunflower, Arabidopsis, tobacco, red pepper, soy, tomato, eggplant, peppers, carrot, potato, corn, lettuce and types of cabbage, cereals, alfalfa, oats, barley, rye, wheat, triticale, millet, rice, alfalfa, flax, cotton, hemp, Brassicaceae, such as oilseed rape or canola, sugar beet, sugarcane, nut and wine species, or trees, such as aspen or yew tree. Depending on the vector system used, transgenic plants can also be generated according to the invention, in which the nucleic acids to be transferred are contained in the plant cell, or the plant, as an independently replicating system. The vectors used for the transfer of the plants must then possess the corresponding DNA sequences which facilitate the replication of the plasmids used for the transfer within the cell.

The specific expression of the fatty acid hydratase protein in the plants, or plant cells or bacterial cells or yeast cells according to the invention can be proven and tracked by means of common molecular biological and biochemical methods. The person skilled in the art knows these techniques and is easily able to select suitable detection methods, such as a Northern Blot analysis for the detection of fatty acid hydratase- specific RNA, or for the determination of the amount of accumulation of fatty acid hydratase-specific RNA, or a Southern Blot, or PCR, analysis for the detection of DNA sequences encoding the fatty acid hydratase. The probe or primer sequences used for this purpose can either be identical to the sequence given in SEQ ID No. 2, or show some slight differences to this sequence.

Further preferred host organisms for use in the method according to the present invention are fungi such as for example Mortierella, Saprolegnia or Pythium, bacteria such as the ones of the genus Escherichia, yeasts such as Saccharomyces, cyanobacteria, ciliates, algae or protozoa such as dino flagellates such as Crypthecodinium. Industrially used suitable microorganisms include, but are not limited to Gram negative bacteria such as E. coli, Gram positive bacteria such B. subtilis, fungi such as A. niger, A. nidulans, N. crassa; yeasts such as S. cerevisiae, K. lactis, H. polymorpha, P. pastoris, Y. lipolytica; actinomycetes such as Streptomyces sp. Several of these microorganisms are described in the 'Manual of Industrial Microbiology and Biotechnology' (editors in chief: A. L. Demain, J.E. Davies; ASM Press).

Further suitable host cells can be derived from: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Suitable expression strains, e.g. with a lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.

If microorganisms, for instance bacteria such as Escherichia, yeasts such as

Saccharomyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Traustochytrium, algae such as Isochrysis, Phaeodactylum, Chlamydomonas, Volvox or Crypthecodinium are used in the method according to the present invention, these organisms are preferably grown under standard conditions in a fermentation process in manner known to the expert.

The term "standard conditions" refers to the cultivation of a microorganism in a standard medium. The temperature, pH and incubation time can vary as described below.

The standard culture conditions for each microorganism used can be taken from the textbooks, such as Sambrook and Russell, Molecular Cloning - A laboratory manual, Cold Spring Harbour Laboratory Press, 3^rd edition (2001).

E.g., E. coli and C. glutamicum strains are routinely grown in MB or LB and BHI broth (Follettie, M. T. et al. (1993) J. Bacteriol. 175: 4096-4103, Difco Becton Dickinson). Usual standard minimal media for E. coli are M9 and modified MCGC (Yoshihama et al. (1985) J. Bacteriol. 162: 591-507; Liebl et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210. ). Other suitable standard media for the cultivation of bacteria include NZCYM, SOB, TB, CG12 ¹Z₂ and YT.

"Standard media" within the meaning of the present invention are intended to include all media which are suitable for the cultivation of microorganisms. Both enriched and minimal media are comprised with minimal media being preferred.

"Minimal media" are media that contain only the minimal necessities for the growth of wild-type cells, i.e. inorganic salts, a carbon source and water.

In contrast, "enriched media" are designed to fulfil all growth requirements of a specific microorganism, i.e. in addition to the contents of the minimal media they contain for example growth factors.

Antibiotics may be added to the standard media in the following amounts

(micrograms per milliliter): ampicillin, 50; kanamycin, 25; nalidixic acid, 25 to allow for the selection of transformed strains.

Suitable media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, lactose, maltose, sucrose, raffinose, starch, dextrin, rhamnose, inositol, xylose, arabinose, pyruvic acid or cellulose may serve as very good carbon sources.

It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH₄Cl or (NFLi)₂SO₄, NH₄OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract, peptone, malt extract, casein hydrolyzate and others.

Inorganic salt compounds which may be included in the media include the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P. M.

Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (brain heart infusion, DIFCO) or others.

All medium components should be sterilized, either by heat (20 minutes at 1.5 bar and 121°C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. AIl media components may be present at the beginning of growth, or they can optionally be added continuously or batch wise. Culture conditions are defined separately for each experiment.

The temperature should be is usually in a range between 15°C and 45°C, but the range may be higher, up to 105⁰C for thermophilic organisms. The temperature can be kept constant or can be altered during the experiment. The pH of the medium may be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of an acid or base, such as acetic acid, sulfuric acid, phosphoric acid, NaOH, KOH or NH₄OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml or 250 shake flasks are used, filled with about 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude about 25 mm) using a speed-range of about 100-300 'rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.

If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested.

The preparation of standard media used for the cultivation of bacteria usually does not involve the addition of single amino acids. Instead, in enriched media for use under standard culture conditions a mixture of amino acids such as peptone or trypton is added.

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.

Fusion vectors add a number of amino acids to a protein encoded by the inserted nucleic acid sequence, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by providing a ligand for affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pQE (Qiagen), pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40), pMAL (New England Bio labs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively.

Examples for C. glutamicum vectors can be found in the Handbook of Corynebacterium 2005 Eggeling, L. Bott, M., eds., CRC press USA.

Examples of suitable inducible non- fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69: 301-315), pLG338, pACYC184, pBR322,pUC18, pUC19, pKC30, pRep4,pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290,pIN- III 113-Bl, egtll, pBdCl, and pET Hd (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York ISBN 0 444 904018). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET Hd vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7gnl). This viral polymerase is supplied by host strains BL21 (DE3) or HMS 174 (DE3) from a resident X prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJlOl, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUBl 10, pC194, or pBD214 are suitable for transformation of Bacillus species.

Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBLl, pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018). In another embodiment, the protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast (S. cerevisiae) include pYepSecl (Baldari, et al. (1987) Embo J. 6: 229-234), 2i, pAG-1, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York (ISBN 0 444 904018).

For the purposes of the present invention, an operative link is understood to be the sequential arrangement of promoter, coding sequence, terminator and, optionally, further regulatory elements in such a way that each of the regulatory elements can fulfill its function, according to its determination, when expressing the coding sequence.

Vector DNA can be introduced into prokaryotic via conventional transformation or transfection techniques. As used herein, the terms "transformation" and

"transfection", "conjugation" and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e. g., linear DNA or RNA (e. g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning : A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003), and other laboratory manuals.

In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as, but not limited to, G418, hygromycin , kanamycine, tetracycline, neomycineampicillin (and other pencillins), cephalosporins, fluoroquinones, naladixic a id, chloramphenicol, spectinomyin, ertythromycin, streptomycin and methotrexate. Other selectable markers include wild type genes that can complement mutated versions of the equivalent gene in a host or starting strain. For example, an essential gene for growth on a minimal medium, such as serA, can be mutated or deleted from the genome of a C. glutamicum starting or host strain of the invention as described herein above to create a serine auxotroph. Then, a vector containing a wild type or other functional copy of a serA gene can be used to select for transformants or integrants. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the above-mentioned modified nucleic acid sequences or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e. g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

When plasmids without an origin of replication and two different marker genes are used, it is also possible to generate marker- free strains which have part of the insert inserted into the genome. This is achieved by two consecutive events of homologous recombination (see also Becker et al., Applied and Environmental Microbiology, 71 (12), p. 8587-8596). In another embodiment, recombinant microorganisms can be produced which contain selection systems which allow for regulated expression of the introduced gene. For example, inclusion of one of the above-mentioned nucleic acid sequences on a vector placing it under control of the lac operon permits expression of the gene only in the presence of IPTG. Such regulatory systems are well known in the art.

Another aspect of the invention pertains to organisms or host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Besides using transgenic organisms or cells such as bacterial, yeast or plant cells and fungi or algae, the fatty acid hydratase enzyme of the present invention may also be expressed in a cell- free system using a suitable expression vector. The cell free expression is a coupled transcription and translation reaction to produce active recombinant protein in high amounts in vitro and can be obtained commercially from companies such as Invitrogen (Expressway Mini Cell- Free Expression System).

The cloning and characterization of the fatty acid hydratase from Sterptococcus pyogenes will be described below. The following examples should not be construed as limiting. The content of all literature, patent applications, patents, and published patent applications cited in this patent application is incorporated herein by reference. EXAMPLES

1. General methods

Basic molecular biological and biochemical techniques were performed as described in (Ausubel et al., 1993). Genomic DNA was purified by lysing of cells (25 mM Tris/HCl, pH 8.0, 1 % glucose, 10 mM EDTA, 0.01 % lysozyme) followed by extraction with phenol and precipitation with ethanol and 3M sodium acetate. DNA purification was performed using Illustra GFX PCR DNA and gel band purification kit (GE Healthcare, Great Britain), plasmid DNA purification was performed using NucleoSpin Plasmids Kit (Macherey-Nagel, USA) and sequencing was done with the BigDye Terminator vl.l Cycle Sequencing Kit (Applied Biosystems, USA) according to the manufacturer's protocol.

Restriction enzymes and T4 ligase were obtained from MBI Fermentas (Germany), TfI polymerase was from Biozyme (Germany). Chemicals were from Sigma (Germany) and Roth (Germany); solvents (HPLC grade) were from Baker (USA), fatty acids from Cayman Chemicals (USA).

2. Cloning and vector construction for bacterial expression

Genomic DNA of Streptococcus pyogenes was isolated, the complete open reading frame (ORF) of the protein (accession number U09352) was amplified by PCR using the primers 5 '-TGGATCCATGTATTATACTAGTGGTAATTACGAAG-S ' (restriction site BamHI, underlined; SEQ ID No. 3) and

5 '-TGTCGACTACATAAGATTAGCATCTTTGAGCAATTC-S ' (restriction site Sail, underlined; SEQ ID No. 4). The PCR parameters were initial 94 ⁰C denaturation for 2 minutes followed by 25 cycles of 94 ⁰C denaturation for 30 seconds, 53 ⁰C annealing for 30 seconds and 72 ⁰C elongation for 1 minute. In the end a final 72 ⁰C elongation phase of 5 minutes was added. The PCR product was purified and ligated into pGEM-T vector (Promega, Germany) according to the manufacturer's protocol. Ligation products were transformed into Escherichia coli XLl Blue cells (Stratagene, USA). Positive clones were identified by colony PCR using insert-specific primers as above. Plasmid DNA was isolated and insert was sequenced.

The pGEM-T plasmids were used as template for amplification of the ORF by PCR using the primers 5 '- AAAGCTAGC ATGTATTAT ACTAGTGGT AATT ACG-3 ' (restriction site Nhel, underlined; SEQ ID No. 5) and

5 '-AAAGCGGCCGCTTACATAAGATTAGCATCTTTGAGC-S ' (restriction site Notl, underlined; SEQ ID No. 6), the PCR parameters were initial 94 ⁰C denaturation for 2 minutes followed by 25 cycles of 94 ⁰C, denaturation for 30 seconds, 53 ⁰C annealing for 30 seconds and 72 ⁰C elongation for 1 minute. In the end a final 72 ⁰C elongation phase of 5 minutes was added. The resulting DNA fragment was digested with Nhel and Notl (10 U each enzyme, overnight at 37 ⁰C) and purified. PET24a- and pET28a-vectors (Novagen, Germany) were digested using the same restriction enzymes. The fragment was ligated into these vectors resulting in the plasmids pET24a-SpHyd and pET28a-SpHyd, respectively. The ligation products were transformed into Escherichia coli XLl Blue cells. Positive clones were identified by colony PCR using primers above. Plasmid DNA was isolated and purified, insert presence was verified by restriction digestion with Nhel and Notl. Identity of insert with the published sequence was confirmed by sequencing using primers T7 promoter primer, T7 terminator primer and primer 5'- AGAATCTCTAGGAGATC AAACC-3' (SEQ ID No. 7). 3. Protein overproduction in E. coli

The inventive fatty acid hydratase, called SpHyd, was overproduced as N-terminal 6xHis-tag fusion protein and as a non-tagged protein. E. coli BL21 Star, C43 and Rosetta 2 strains were transformed with pET24a-SpHyd and pET28a-SpHyd. The cells were cultivated in LB medium with 25 μg/ml kanamycin at 37 ⁰C until OD₆₀₀ ~ 0.6-0.8. Then Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The cells were grown for 3 hours at 37 ⁰C. Afterwards they were harvested by centrifugation (10 minutes at 3220 g). For protein overproduction analysis cell lysates were obtained by freezing/thawing bacterial pellets in Tris buffer (0.1 M Tris/HCl pH 8.0, 0.15 M NaCl, 1 mM EDTA), incubation with 1 mg/ml lysozyme for 10-15 minutes at room temperature, sonifϊcation at 4 ⁰C (2x 30 seconds, 20 % power, 50 % duty cycle) and centrifugation for 5 minutes at 2000Og. For largescale overproduction E. coli B121 Star cells were grown in 2xYT medium with 25 μg/ml kanamycin at 37 ⁰C until OD₆₀₀ ~ 0.6-0.8. Then IPTG was added to a final concentration of 0.1 mM, the cells were shifted to 16 ⁰C, grown at this temperature over night and harvested by centrifugation (10 minutes at 9100 g).

Fractionated cell lysates were analyzed by SDS PAGE (4.8 % stacking gel, 12 % separating gel). For this 30 μl of the sonicated samples were centrifuged (5 minutes at 20000 g) and pellet and supernatant mixed with loading buffer (Fermentas, Germany). All samples were heated at 96 ⁰C for 5 minutes and 20 μl of each sample was loaded along with Protein Molecular Weight Marker from Fermentas (USA). The gel was run at 40 mA for 1 hour and afterwards stained with Coomassie-G 250.

The result of the overproduction analysis is shown in Figure 1.

4. His-tag fusion protein purification For protein purification cell lysates were obtained by resuspension of the cell pellet in buffer A (0.1 M sodium phosphate pH 7.1, 0.1 M NaCl), freezing/thawing, incubation with 1 g/1 lysozyme and 1 rnM DTT, addition of DNAse I and centrifugation (20 minutes at 73000 g). Ni²⁺-affinity chromatography was performed using Akta prime purification system and a HisTrap HP column (GE healthcare, Great Britain) at 12 ⁰C. The column was equilibrated with 10 column volumes of buffer A, clear supernatant was loaded at 1 ml/min, column washed with 10 column volumes buffer A and protein eluted in buffer B (buffer A with 0.5 M imidazol). Fractions of 1 ml were collected. After elution 10 μl aliquots of peak fractions were tested by SDS PAGE. The protein was concentrated to 15 mg/ml in 50 mM HEPES/NaOH (pH 7.5) by ultrafiltration with Amicon and Microcon concentrators (Millipore, USA). Protein concentration was estimated spectrophotometrically using 8280 of 103.960 M^"1 cm^"1 (estimated theoretically at http ://www.expasy .ch/tools/protparam.html).

The result of the affinity purification is shown in Figure 2.

5. Activity tests

For protein activity tests 20 μg of the corresponding fatty acid and 10 μg of protein were mixed with 1 ml of buffer A (see 4.). The reactions were incubated on shaker for at least 1 hour at 37 ⁰C.

6. Extraction and analysis of lipids

Following incubation, 1 ml of a 1 :1 mixture of methanol/chloroform and 5 μl 50 % acetic acid were added to reactions and briefly centrifuged at 15000 g for phase separation (Bligh and Dyer (1959) Ca, J. Biochem. Physiol. 37: 911-917). Then the lower phase was transferred into a glass tube and dried under nitrogen stream. 400 μl methanol and 7 μl trimethylsilyldiazomethan were added to the dry samples and incubated for 30 minutes at room temperature for converting all fatty acids into their corresponding methyl esters. Then 5 μl of 50 % acetic acid was added to stop the reactions, solvent dried and sample redissolved in 5 μl acetonitrile. Afterwards 1 μl N,O-bis(trimethylsilyl)trifluoroacetamide was added to modify hydroxy groups into trimethylsilyl (TMS) derivatives. GC-MS analysis was carried out using an Agilent 5973 Network mass selective detector connected to an Agilent 6890 gas chromatograph equipped with a capillary DB-23 column (30 m x 0.25 mm; 0.25 Am coating thickness; J and W Scientific, Agilent, Germany). Helium was used as carrier gas (1 ml/min). The temperature gradient was 130 ⁰C for 1 min, 130-250 ⁰C at 8 K/min and 250 ⁰C for 6 min. An electron energy of 70 eV, an ion source temperature of 230 ⁰C, and a temperature of 260 ⁰C for the transfer line was used.

The products of the hydration reaction with different fatty acids as analyzed by GC- MS are shown in Figure 3 and Table 1. The results of this experiment show that the expected hydrated products are produced from the unsaturated fatty acids. The formation of two by-products in case of the hydration of linoleic acid seems to be an experimental artifact, since it only occurs in some buffers, while it does not occur in other buffers.

Table 1 : Main and by-products of SpHyd after reaction with different mono- and polyenoic fatty acids. RT - retention time; M⁺ - molecular ion; 10-HOE - 10- hydroxy-(9Z)-octadec-9-enoic acid; 10-HO - 10-hydroxyoctadecanoic acid; 10,13- diHO - lOJS-dihydroxyoctadecanoic acid; 10-HH - 10-hydroxyhexadecanoic acid; 10-HODE - lO-hydroxy-πZ.lSZ-octadeca-π.lS-dienoic acid

DESCRIPTION OF THE FIGURES

Figure 1 :

Overproduction analysis of recombinant SpHyd. MW - protein molecular weight marker; unind. - uninduced cells; SpHyd if- insoluble fraction, pET24a-SpHyd; pET28 sn - supernatant B121 transformed with pET28a; SpHyd sn - supernatant, pET24a-SpHyd; SpHyd-N sn - supernatant, pET28a-SpHyd.

Figure 2:

SDS-PAGE analysis after affinity chromatography of N-terminal 6xHis-tagged

SpHyd. MW - protein molecular weight marker; sn - supernatant; fr.- tested fraction.

Figure 3 :

GC chromatograms of SpHyd products. Colour code: black - control reaction, red - fatty acid with SpHyd; a - linoleic acid (18:2^Δ9Z'^12Z); b - palmitoleic acid (16:1^Δ9Z); c

- oleic acid (18:1^Δ9Z) d - α-linolenic acid (18:3^Δ9Z'^12Z' ^15Z).

Claims

C L A I M S

1. Method for producing hydroxy fatty acids from unsaturated fatty acids by use of a protein having the enzymatic activity of a fatty acid hydratase wherein the protein is encoded by a nucleic acid sequence selected from the group consisting of: a) nucleic acid sequences comprising a nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQ ID NO: 1, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, b) nucleic acid sequences comprising the nucleotide sequence depicted in SEQ ID NO: 2, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, c) nucleic acid sequences comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of said nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase, d) nucleic acid sequences comprising a nucleotide sequence which shows at least 40 % identity to the nucleotide sequence from a), b) or c), or comprising fragments of said nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a fatty acid hydratase.

2. Method according to claim 1, wherein the protein originates from Streptococcus pyogenes.

3. Method according to claim 1 or 2, wherein the unsaturated fatty acid comprises a double bond between carbon atom 9 and carbon atom 10.

4. Method according to any of the preceding claims, wherein the produced hydroxy fatty acids are 10-hydroxy fatty acids.

5. Method according to any of the preceding claims, wherein the fatty acids have a length of between 12 and 22 carbon atoms.

6. Method according to any of the preceding claims, wherein the production of the hydroxy fatty acids occurs in vitro.

7. Method according to claim 6, wherein the hydroxy fatty acids are produced by use of a purified fatty acid hydratase protein.

8. Method according to claim 7, wherein the fatty acid hydratase is purified by means of an affinity tag.

9. Method according to claim 8, wherein the affinity tag is selected from the group consisting of maltose binding domain, cellulose binding domain, protein A, histidine and glutathione- S -transferase.

10. Method according to any of claims 7 to 9, wherein the fatty acid hydratase is purified from a transgenic organism or transgenic cell.

11. Method according to any of claims 1 to 5, wherein the hydroxy fatty acids are produced in a transgenic organism, transgenic cell or in an extract from said organism or cell.

12. The method according to claim 10 or 11, wherein the organism or cell is a bacterium, a fungus, a yeast, a plant or plant cell.

13. The method according to any of claims 10 to 12, wherein the organism is a Gram negative bacterium such as E. coli, a Gram positive bacterium such as B. subtilis, a fungus such as A. niger, A. nidulans, N. crassa; a yeast such as S. cerevisiae, K. lactis, H. polymorpha, P. pastoris, Y. lipolytica, etc; or an actinomycetes such as Streptomyces sp.

14. Method according to any of claims 10 to 13, wherein the organism has been transformed with a recombinant nucleic acid molecule comprising the following elements: regulatory sequences of a promoter which is active in a target cell, operatively linked thereto a nucleic acid sequence as defined in claim 1 , and optionally operatively linked thereto regulatory sequences which can serve as transcription, termination and/or polyadenylation signals in the target cell.

15. Method according to claim 14, wherein the recombinant nucleic acid molecule further comprises a nucleic acid sequence coding for an affinity tag.

16. Method according to claim 15, wherein the affinity tag is selected from the group consisting of maltose binding domain, cellulose binding domain, protein A, histidine and glutathione- S -transferase.

17. Method according to any of the preceding claims, further comprising: recovering the produced hydroxy fatty acids and/or - using the produced hydroxy fatty acids for the production of flavor ingredients and/or fragrance ingredients and/or using the produced hydroxy fatty acids as lubricant, surfactant or plasticizer and/or using the produced hydroxy fatty acids for the production of lubricants, surfactants, detergents, coatings, paints, biopolymers and/or plasticizers.

18. Method according to claim 17, wherein the produced flavor and/or fragrance ingredients are lactones.

19. Method according to claim 18, wherein the lactones are produced from the hydroxy fatty acids by β-oxidation.

20. Method of producing 10-hydroxy fatty acids in vitro, comprising: providing a protein having the enzymatic activity of a fatty acid hydratase from Streptococcus pyogenes or producing said protein in a transgenic organism or cell; contacting said protein with an unsaturated fatty acid or its natural precursor under suitable reaction conditions; and recovering the produced 10-hydroxy fatty acid.

21. Method according to claim 20, wherein the unsaturated fatty acid is oleic acid and the produced 10-hydroxy fatty acid is 10-hydroxy octadecanoic acid.

22. Method of producing 10-hydroxy fatty acids in transgenic bacterial cells or transgenic yeast cells or transgenic fungi or transgenic plant cells or transgenic plants, comprising: a) transferring a recombinant nucleic acid molecule comprising the following elements regulatory sequences of a promoter which is active in a target cell, operatively linked thereto a nucleic acid sequence encoding a protein having the enzymatic activity of a fatty acid hydratase as defined in claim 1, and optionally operatively linked thereto regulatory sequences which can serve as transcription, termination and/or polyadenylation signals in the target cell, to bacterial cells or yeast cells or fungi or plant cells or plants; b) producing hydroxy fatty acids by expression of the nucleic acid sequence in the transgenic cells, optionally with adding a suitable unsaturated fatty acid; and c) obtaining the produced hydroxy fatty acids from these cells, plants or media wherein the cells or plants are cultivated during the production step.

23. Method of producing flavor ingredients and/or fragrance ingredients in vitro, comprising: providing a protein having the enzymatic activity of a fatty acid hydratase from Streptococcus pyogenes or producing said protein in a transgenic organism or cell; contacting said protein with an unsaturated fatty acid or its natural precursor under suitable reaction conditions to produce hydroxy fatty acids; and oxidizing the produced hydroxy fatty acids to the corresponding lactones.

24. Method according to claim 23, wherein the hydroxy fatty acids are oxidized by Yarrowia lipolytica.

25. Method according to claim 23 or 24, further comprising lowering the pH after the oxidation.

26. Transgenic bacterial cell or yeast cell or fungus or plant cell containing a recombinant nucleic acid molecule comprising the following elements: regulatory sequences of a promoter which is active in a target cell, operatively linked thereto a nucleic acid sequence as defined in claim 2, and optionally operatively linked thereto regulatory sequences which can serve as transcription, termination and/or polyadenylation signals in the target cell, with the proviso that the bacterial cell is not an E. coli cell.