WO2008119735A1 - Procédé de fabrication d'acide gras hydroxy - Google Patents

Procédé de fabrication d'acide gras hydroxy Download PDF

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WO2008119735A1
WO2008119735A1 PCT/EP2008/053647 EP2008053647W WO2008119735A1 WO 2008119735 A1 WO2008119735 A1 WO 2008119735A1 EP 2008053647 W EP2008053647 W EP 2008053647W WO 2008119735 A1 WO2008119735 A1 WO 2008119735A1
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fatty acids
protein
fatty acid
nucleic acid
cell
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PCT/EP2008/053647
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English (en)
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Ivo Feussner
Ellen Hornung
Alena Liavonchanka
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Georg-August-Universität Göttingen
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Publication of WO2008119735A1 publication Critical patent/WO2008119735A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/205—Heterocyclic compounds
    • A23L27/2052—Heterocyclic compounds having oxygen or sulfur as the only hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/0069—Heterocyclic compounds
    • C11B9/0073—Heterocyclic compounds containing only O or S as heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to methods for producing hydroxy fatty acids.
  • 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.
  • 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 c ⁇ -conf ⁇ guration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • microbial hydration is both regioselective and stereoselective.
  • hydroxy fatty acids could also be obtained with the yeast Saccharomyces cerevisiae (El-Sharkawy et al. (1992) Appl. Environ. Microbiol. 58: 2116-2122).
  • use of this microorganism does not involve the use of a purified enzyme, but rather the use of a cell extract.
  • hydroxy fatty acids are only produced in low and middle yield.
  • prokaryotic fatty acid hydratases which produce hydroxy fatty acids in high yields and without the formation of by-products.
  • 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.
  • 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 c ⁇ -conf ⁇ guration 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
  • 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
  • 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.
  • unsaturated fatty acid refers to a fatty acid which contains at least one double bond.
  • 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.
  • 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
  • 10-hydroxy- 12Z, 15Z- octadeca-12,15- dienoic acid is produced from ⁇ -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.
  • 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.
  • linseed oil may be used as a natural precursor of linolenic acid
  • 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.
  • 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.
  • transgenic cells e.g. E.coli
  • E.coli E.coli
  • cell lyses kits such as Cellutic® of Sigma
  • the lysate may be directly added to a solution of fatty acids.
  • 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 ⁇ l of soluble extract prepared in this way is typically incubated with 5 to 60 ⁇ g, preferably 10 to 50 ⁇ g and most preferably 20- 30 ⁇ g 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.
  • 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.
  • 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.
  • an affinity tag such as cellulose binding protein, maltose binding protein, glutathione- S -transferase or a His- tag.
  • affinity tag may be removed by the action of a protease such as factor X.
  • affinity tags provide increased activity of the recombinant fusion protein due to improved protein folding/solubility, etc.
  • affinity 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.
  • 10 ⁇ g of purified protein is incubated with 10 to 50 ⁇ g, preferably 20 to 40 ⁇ g and most preferably 30 ⁇ g 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.
  • wild type is to be understood as the respective non genetically modified parent organism.
  • fragments of DNA 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.
  • fragments of the protein 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.
  • 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.
  • nucleic acid (molecule) 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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. Russia, FL).
  • reverse transcriptase e.g. Moloney-MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku Amerika, Inc., St. Russia, 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.
  • 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).
  • 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.
  • 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 30 0 C for short molecules (i.e.
  • stringent conditions can comprise the addition of agents, such as formamide, which destabilize the hybrids.
  • agents such as formamide, which destabilize the hybrids.
  • nucleotide sequences which are at least 60% homologous to each other usually remain hybridized to each other.
  • 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.
  • SSC sodium chloride/sodium citrate
  • 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).
  • the temperature under standard conditions is about 42°C.
  • the hybridization conditions for DNA:DNA hybrids are for example 0.1 x SSC and 20 0 C to 45°C, preferably 30 0 C to 45°C.
  • the hybridization conditions for DNA:RNA hybrids are for example 0.1 x SSC and 30 0 C 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.
  • 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%.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • non-natural, synthetic (“artificial") methods such as a mutagenization.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hydroxy fatty acids can also be reacted to lactones which are ubiquitous volatile flavors.
  • 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.
  • ⁇ -dodecalactone is formed from 10-hydroxy octadecanoic acid, which is the hydration product of oleic acid.
  • ⁇ -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.
  • Yarrowia lipolytica is added to the hydroxy fatty acids.
  • 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 ⁇ -oxidation reaction in order to enhance the yield of the lactone.
  • 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.
  • 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.
  • 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.
  • 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.
  • these regulatory sequences are sequences to which inductors or repressors bind, thereby regulating the expression of the nucleic acid.
  • 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 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.
  • additional useful sequences such as additional regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequences.
  • synthetic promoters only or in addition.
  • 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.
  • 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.
  • 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.
  • the person of average skill in the art is able to isolate additional suitable promoters using routine methods.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • certain vectors can control the expression of genes to which they are functionally linked. These vectors are called here "expression vectors.”
  • 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.
  • the invention is also intended to comprise other types of expression vectors, such as viral vectors which fulfill similar functions.
  • 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.
  • 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
  • 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 Pi 5 , phage SPOl P 26 , pSOD, EFTu, EFTs, GroEL, MetZ (last 5 from C. glutamicum), which are used preferably in bacteria.
  • promoters such as cos-, tac-, trp-, tet-, trp-, tet-, lpp-, lac-, lpp-lac-, laclq-, T7-, T5-, T3-, gal-, trc-, ara
  • 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.
  • 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, ⁇ gtl l, or pBdCl, Streptomyces pIJlOl, pIJ364, pIJ702, or pIJ361, Bacillus pUBl 10, pC194, or pBD214, Corynebacterium pSA77, or pAJ667.
  • the pBR series such as pBR322
  • the pUC series such as pUC18, or pUC19
  • Ml 13mp series pKC30, pRep4,
  • the expression vector represents a yeast expression vector or a bacillovirus expression vector.
  • the fatty acid hydratase can be expressed in single-celled plant cells (such as algae), see Falciatore et al., 1999, Marine
  • 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.
  • 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).
  • translation enhancers e.g. the overdrive sequence containing the 5' untranslated leader sequence of the tobacco mosaic virus, which increases the protein/RNA ratio
  • 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.
  • 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.
  • the fatty acid hydratase sequence 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.
  • the primer should be selected so that the amplificate comprises the entire codogenic sequence from the start codon to the stop codon.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • standard conditions refers to the cultivation of a microorganism in a standard medium.
  • the temperature, pH and incubation time can vary as described below.
  • 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 1 Z 2 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.
  • 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
  • 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.
  • 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 NH 4 Cl or (NFLi) 2 SO 4 , NH 4 OH, 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.
  • 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 105 0 C 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 NH 4 OH during growth.
  • 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.
  • the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles.
  • 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.
  • 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.
  • 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.
  • 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.
  • C. glutamicum vectors can be found in the Handbook of Corynebacterium 2005 Eggeling, L. Bott, M., eds., CRC press USA.
  • 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.
  • 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.
  • 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.
  • the protein expression vector is a yeast expression vector.
  • yeast expression vector examples include pYepSecl (Baldari, et al. (1987) Embo J.
  • 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.
  • 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.
  • transformation and transfection techniques
  • transfection means 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.
  • 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
  • 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.
  • a gene that encodes a selectable marker is generally introduced into the host cells along with the gene of interest.
  • 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.
  • an essential gene for growth on a minimal medium such as serA
  • a minimal medium such as serA
  • 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).
  • 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.
  • selection 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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
  • 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
  • the PCR parameters were initial 94 0 C denaturation for 2 minutes followed by 25 cycles of 94 0 C, denaturation for 30 seconds, 53 0 C annealing for 30 seconds and 72 0 C elongation for 1 minute. In the end a final 72 0 C elongation phase of 5 minutes was added.
  • the resulting DNA fragment was digested with Nhel and Notl (10 U each enzyme, overnight at 37 0 C) 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 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 ⁇ g/ml kanamycin at 37 0 C until OD 600 ⁇ 0.6-0.8.
  • Isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM.
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • Fractionated cell lysates were analyzed by SDS PAGE (4.8 % stacking gel, 12 % separating gel). For this 30 ⁇ l 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 0 C for 5 minutes and 20 ⁇ l 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.
  • 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). Ni 2+ -affinity chromatography was performed using Akta prime purification system and a HisTrap HP column (GE healthcare, Great Britain) at 12 0 C.
  • buffer A 0.1 M sodium phosphate pH 7.1, 0.1 M NaCl
  • 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 ⁇ l 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 temperature gradient was 130 0 C for 1 min, 130-250 0 C at 8 K/min and 250 0 C for 6 min.
  • 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- ⁇ Z.lSZ-octadeca- ⁇ .lS-dienoic acid

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Abstract

La présente invention concerne des procédés de fabrication d'acides gras hydroxy. Cette invention concerne notamment la fabrication d'acides gras hydroxy en utilisant une hydratase d'acide gras issue de Streptococcus pyogenes. L'hydratase d'acide gras selon l'invention est utile pour la fabrication d'acides gras hydroxy à grande échelle et pour une transformation ultérieure en ingrédients de saveur et/ou en ingrédients de fragrance et/ou en lubrifiants, tensioactifs, détergents, revêtements, peintures, biopolymères et/ou plastifiants.
PCT/EP2008/053647 2007-04-02 2008-03-27 Procédé de fabrication d'acide gras hydroxy WO2008119735A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2336341A1 (fr) 2009-12-21 2011-06-22 Philippe Marliere Procédé pour la production d'un alcène comprenant l'étape de conversion d'un alcool par une étape de déshydratation enzymatique.
EP2706117A1 (fr) 2012-09-07 2014-03-12 Basf Se Procédé de préparation de l'acide sébacique
WO2014152434A2 (fr) 2013-03-15 2014-09-25 Genomatica, Inc. Micro-organismes et procédés de production de butadiène et de composés associés par assimilation de formiate
WO2015157719A1 (fr) * 2014-04-10 2015-10-15 REG Life Sciences, LLC Voies semi-synthétiques pour obtenir des composés organiques
US9181566B2 (en) 2011-12-30 2015-11-10 Butamax Advanced Biofuels Llc Genetic switches for butanol production
WO2016004334A1 (fr) 2014-07-03 2016-01-07 Genomatica, Inc. Micro-organismes pour la production de composés en 4c-5 c avec une insaturation et procédés associés
WO2016044713A1 (fr) 2014-09-18 2016-03-24 Genomatica, Inc. Organismes microbiens non naturels présentant une meilleure efficacité énergétique
US10487342B2 (en) 2014-07-11 2019-11-26 Genomatica, Inc. Microorganisms and methods for the production of butadiene using acetyl-CoA
DE102019110921A1 (de) * 2019-04-26 2020-10-29 Fuchs Petrolub Se Schmierfette umfassend Metallseifen und Metallkomplexseifen auf Basis von R-10-Hydroxyoctadecansäure
US10920252B2 (en) 2015-03-26 2021-02-16 Stichling Wageningen Research; Technische Universiteit Delft Production of fatty acid estolides
WO2021028560A1 (fr) 2019-08-14 2021-02-18 B.R.A.I.N. Biotechnology Research And Information Network Ag Nouvelles oléate hydratases
WO2022238489A1 (fr) 2021-05-12 2022-11-17 Ab Enzymes Gmbh Préparations d'huile fermentées

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EP2336341A1 (fr) 2009-12-21 2011-06-22 Philippe Marliere Procédé pour la production d'un alcène comprenant l'étape de conversion d'un alcool par une étape de déshydratation enzymatique.
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US9834798B2 (en) 2012-09-07 2017-12-05 Basf Se Process for preparing sebacic acid
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WO2014037328A1 (fr) 2012-09-07 2014-03-13 Basf Se Procédé de préparation d'acide sébacique
WO2014152434A2 (fr) 2013-03-15 2014-09-25 Genomatica, Inc. Micro-organismes et procédés de production de butadiène et de composés associés par assimilation de formiate
JP2021180678A (ja) * 2014-04-10 2021-11-25 ジェノマティカ， インコーポレイテッド 有機化合物の半合成経路
KR102493892B1 (ko) * 2014-04-10 2023-01-31 게노마티카 인코포레이티드 유기 화합물에 대한 반합성 경로
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JP2017512485A (ja) * 2014-04-10 2017-05-25 アールイージー ライフ サイエンシズ リミテッド ライアビリティ カンパニー 有機化合物の半合成経路
WO2015157719A1 (fr) * 2014-04-10 2015-10-15 REG Life Sciences, LLC Voies semi-synthétiques pour obtenir des composés organiques
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US11008597B2 (en) 2014-04-10 2021-05-18 Genomatica, Inc. Chemo-enzymatic process
AU2015243186C1 (en) * 2014-04-10 2020-04-02 Genomatica, Inc. Semisynthetic routes to organic compounds
JP2020110174A (ja) * 2014-04-10 2020-07-27 アールイージー ライフ サイエンシズ リミテッド ライアビリティ カンパニー 有機化合物の半合成経路
JP7266646B2 (ja) 2014-04-10 2023-04-28 ジェノマティカ， インコーポレイテッド 有機化合物の半合成経路
EP3739057A1 (fr) * 2014-04-10 2020-11-18 Genomatica, Inc. Voies semi-synthétiques pour obtenir des composés organiques
AU2019232831B2 (en) * 2014-04-10 2021-04-29 Genomatica, Inc. Semisynthetic routes to organic compounds
WO2016004334A1 (fr) 2014-07-03 2016-01-07 Genomatica, Inc. Micro-organismes pour la production de composés en 4c-5 c avec une insaturation et procédés associés
US10487342B2 (en) 2014-07-11 2019-11-26 Genomatica, Inc. Microorganisms and methods for the production of butadiene using acetyl-CoA
US11371063B2 (en) 2014-07-11 2022-06-28 Genomatica, Inc. Microorganisms and methods for the production of butadiene using acetyl-coA
WO2016044713A1 (fr) 2014-09-18 2016-03-24 Genomatica, Inc. Organismes microbiens non naturels présentant une meilleure efficacité énergétique
EP3741865A1 (fr) 2014-09-18 2020-11-25 Genomatica, Inc. Organismes microbiens non naturels dotés d'une efficacité énergétique améliorée
US10920252B2 (en) 2015-03-26 2021-02-16 Stichling Wageningen Research; Technische Universiteit Delft Production of fatty acid estolides
US11414684B2 (en) 2015-03-26 2022-08-16 Stichting Wageningen Research Production of fatty acid estolides
US11512330B2 (en) 2015-03-26 2022-11-29 Stichting Wageningen Research Production of fatty acid estolides
US11591537B2 (en) 2019-04-26 2023-02-28 Fuchs Petrolub Se Lubricating grease comprising metal soaps and metal complex soaps based on R-10-hydroxyoctadecanoic acid
DE102019110921A1 (de) * 2019-04-26 2020-10-29 Fuchs Petrolub Se Schmierfette umfassend Metallseifen und Metallkomplexseifen auf Basis von R-10-Hydroxyoctadecansäure
WO2021028560A1 (fr) 2019-08-14 2021-02-18 B.R.A.I.N. Biotechnology Research And Information Network Ag Nouvelles oléate hydratases
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