NZ512208A - Fruit flavour related genes that control ester formation - Google Patents

Abstract

DNA sequences encoding enzymes involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit are disclosed. The enzymes have alcohol acyl transferase, alcohol dehydrogenase, pyruvate decarboxylase, thiolase, aminotransferase and esterase activities, respectively. The polypeptides having said activities are disclosed. Expression vectors comprising the DNA sequences may be used for regulating ester formation in fruit. Genetically modified plants and microorganisms may be used for producing esters.

Description

512208 WO 00/32789 PCT/NL99/00737 Fruit Flavour Related Genes And Use Thereof The invention disclosed herein relates to strawberry (fragaria ananassa) and lemon (citrus limon) fruit derived 5 genes and enzymes specifically involved in the formation of aliphatic and/ or aromatic esters and other aroma and flavour compounds in fruits. More specifically it relates to a process for improving natural volatile fruit flavours by the control of one or more than one gene implicated in 10 that process.

Background of the invention Like most natural products the chemical composition of strawberry aroma and flavour is quite complex [Zabe-takis and Holden, (1997); J. Sci. Food. Agric 74: 421-434]. 15 Over 300 compounds have been identified which may contribute to flavour/aroma. Qualitatively, the major components of strawberry flavour and aroma may be grouped into several chemical classes including acids, aldehydes, ketones, alcohols, esters, and lactones. Other groups include 20 sulphur compounds, acetals, furans, phenols and even traces of epoxides, and hydrocarbons. Compounds produced by these groups, whilst often present at low levels, may have a significant impact on the overall flavour of strawberry.

In general, fruit flavour compounds develop 25 during ripening when the metabolism of the fruit changes to catabolism. Esters are proposed to be qualitatively and quantitatively the most important class of volatiles produced and are a key component of strawberry flavour. Seven volatile esters, ethyl hexanoate, methyl hexanoate, 3 0 ethyl butanoate, methyl butanoate, hexyl acetate, ethyl propionate and 2-hexenyl-acetate were reported to contribute largely to the aroma associated with the fruit. However, more then a 100 types of esters have been reported during the years to be identified in strawberry volatile analysis 35 [Zabetakis and Holden, (1997); J. Sci. Food. Agric 74: 421-434] .

♦ WO 00/32789 PCT/NL99/00737 2 In lemon, the acetate esters derived from cyclic terpene alcohols are used frequently as flavour, fragrance, scent and aroma components. The lower fatty acid esters of acyclic terpene alcohols (geraniol, linalool, and citronel-5 lol) are the acetates (linalyl acetate, geranyl acetate) which are the main flavour components of lemon peel oils. These are used for obtaining citrus notes [Bauer, K and Garbe, D, (1985) . Common fragrance and flavour materials. Preparation, properties and uses. VCH Publishers, Florida, 10 USA]. Formates, propionates, butyrates occur less frequently. In addition to the cyclic and acyclic terpene esters, lemon is a rich source for the ester methyl anthranillate, which is a methyl ester of o-aminobenzoic acid. This compound is known to be a major constituent of citrus limon 15 fruit and flowers and can also be found in the leaves.

Primary metabolites which include sugars, fatty acids, nucleotides and amino acids have been proposed to serve as precursors for the production of secondary metabolites such as volatile compounds. Sugars in the fruit 20 in addition to acting as a carbon source, act as precursors for the flavour compound furanones [Zabetakis and Holden, (1996); Plant Cell Tissue and Organ Culture 45:25-29]. Amino acid metabolism leads to the formation of various aldehydes, acids, carbonyls, aliphatic and branched-chain 25 alcohols, some of which serve as aroma and flavour components. The changes in amino acid content during maturation of fruits in relation to fruit flavour formation, have been studied in banana, tomato and strawberry. The amino acids valine, leucine and isoleucine have been proposed to serve 30 as flavour precursors in banana fruit, and are metabolised to branched chain alcohols, 2-propanol, isoamyl alcohol, and 2-methylbutanol, respectively [Drawert and Berger, (1981); Bioflavour '81; Schreier, P., Ed.; de Gruyter: Berlin]. The conversion of alanine, leucine and valine to 35 flavour compounds has also been demonstrated in tomato [Yu et al., (1968); Phytochemistry 6: 1457-1465]. In strawberry, alanine was proposed to be the main free amino acid metabolised to flavour compounds. This was based mainly on the dramatic decrease in its content just before the formation of volatile aroma and flavour compounds commenced in the fruit [Perez et al., (1992); J.Agric. Food Chem. 40: 5 2232-2235] . In addition feeding alanine to strawberry cultures resulted in the formation of several esters such as methyl and ethyl hexanoate which are important constituents of strawberry volatiles [Drawert and Berger, (1981) ; Bioflavour '81; Schreier, P., Ed.; de Gruyter: Berlin]. 10 Like sugars and amino acids the fatty acids in strawberry play an important role in ester formation. In mango fruit there have been reports that fatty acids are actively metabolised during ripening. Changes in fatty acids and triglycerides have been associated with changes in aroma 15 and flavour during mango ripening [Gholop AS and Bandyopha-day C, J Am. Oil Chem. Soc.,52: 514-516, 1975]. Therefore fatty acid related enzymes may have a role in the production of flavour volatiles. Thiolase is the last enzyme in the 6-oxidation of fatty acids. It catalyses the thiolytic 20 cleavage involving a molecule of CoA. The products of this reaction are acetyl-CoA and acyl-CoA derivatives containing two carbon atoms less than the original acyl-CoA molecule that underwent oxidation. The acyl-CoA formed in the cleavage reaction may be utilised at the final stage of the 25 biosynthetic pathway for ester formation in fruit. Therefore the profile of fatty acid and amino acid precursors found in each fruit, along with the specificity of the enzymes in the biosynthetic pathway/process leading to ester formation could have a key role in determining the types of esters 30 formed.

A proposed biosynthetic pathway for volatile ester formation in fruits is illustrated in Figure 1. In fruits the transamination and oxidative decarboxylation of amino acids provides the precursors for volatile aroma compounds 35 such as aldehydes, acids, alcohols, esters and thiols. The transamination reaction is catalysed by aminotransferases. Aminotransferases or transaminases are enzymes promoting the WO 00/32789 PCT/NL99/00737 4 first step in the catabolism of L-amino acids by removing the a-amino groups. As a result of this transamination reaction, the a-amino group is transferred to the a-carbon atom of a-ketoglutarate, leaving behind the a-keto acid 5 analogue of the amino acid. Aminotransferases have been studied in various micro-organisms and some of them have been purified [Gelfand et al, (1997); J. Bacterid. 130:429-440, Lee et al., (1985); J. Gen. Microbiol. 131: 459-467]. No strawberry fruit specific aminotransferase to date has 10 been cloned or characterised.

Ketoacids produced by transamination can be enzyma-tically degraded to the corresponding aldehydes or carboxy-lic acids. The enzyme which can catalyse this reaction is pyruvate decarboxylase. An EST encoding part of pyruvate 15 decarboxylase, the first to be characterised from a strawberry fruit has been mentioned in patent application WO 97/27295. The pyruvate decarboxylase enzyme provides precursors for the biosynthesis of volatile flavour compounds. Alcohol dehydrogenase enzymes have been implicated in the 2 0 interconversion of the aldehyde and alcohol forms of flavour volatiles. The mechanism of ester formation has been shown to be a coenzyme A dependent reaction. Esterification is a result of transacylation from acyl-coenzyme A to an alcohol. The enzyme is therefore termed as alcohol acyl transferase 25 (AAT) and plays a major role in the biosynthesis of volatile esters.

It is still unclear whether esterase enzymes (Fig 1) , are involved in the hydrolysis of esters or in their synthesis [Mauricio et al., (1993) J. Agric. Food Chem. 41, 30 2086-2091].

Ester formation by micro-organisms has been studied in most detail and has been cited in a number of publications [Harada et al. , (1985); Plant Cell Physiol. 26(6): 1067-1074], Fujii et al., (1996); Yeast 12:593-598, Fujii et al., 35 (1994); Applied and Enviro. Microbiology 8:2786-2792 ]. Alcohol acyl transferases (AAT's) have been identified from both fungi [Yoshihide et al. , (1978); AGRIC. Biol. Chem. 42 (2):269-274] , and yeast [Fujii et al. , (1994); Applied and WO 00/32789 PCT/NL99/00737 •• Enviro. Microbiology 8:2786-2792 ] and have been the subject of patent applications (EP 0 574 941 A2). The AAT from the fungi Neurospora sp. which was purified to homogeneity acts on various acyl Coenzyme A containing more than a four 5 carbon linear chain but not on acetyl coenzyme A as described for the yeast enzyme [Yamauchi et al. , (1989); Agric. Biol. Chem. 53(6) 1551-1556] . In Cladosporium cladosporioi-des No. 9, AAT was partially purified and was described to form acetate esters like the yeast enzyme [Yamakawa et al., 10 (1978); Agric. Biol. Chem. 42 (2):269-274] . So it is clear that the type of esters formed is dependant on the substrate specificity of the ester forming enzyme to alcohols, and the availability of acyl-CoAs. For example the yeast Saccha-romyces cerevisiae (sake yeast) and brewer's yeast (bottom 15 fermenting yeast) AAT's show high affinity to acetyl-CoA and the alcohols ethanol and isoamyl alcohol, giving rise to the formation of acetate esters. These esters play an important role in determining flavour characteristics of beer.

In plants ester formation has been studied both in 20 fruit and flowers. Melon, banana and strawberry AAT's have been investigated using crude fruit extracts. The formation of esters from aldehydes that were incubated with whole strawberry fruit has been reported [Yamashita, et al. , (1989); Agr. Biol. Chem. 39 (12) 2303-2307] . Analysis of the 25 substrate specificity of the enzymes from the various sources, revealed differences in their affinity to acyl-CoAs and alcohols [Ueda et al. , (1992); Nippon Shokukin Kogyo Gakkaishi 39 (2): 183-187, Perez et al. , (1996); J. AgricFood Chem. 44: 3286-3290, Harada et al. , (1985); Plant Cell 30 Physiol. 26(6) : 1067-1074] . Maximum activity for the strawberry (AAT) was obtained using acetyl CoA and hexyl alcohol as substrates, and acetyl-CoA and butyl alcohol is the preferred substrate for the banana enzyme. A clear correlation could be observed between substrate preference and 35 volatile esters present in both fruits. The purification method and some properties of the strawberry AATase enzyme have been reported. However, to date no sequence of the peptide or of the gene has been disclosed. In Clarkia WO 00/32789 PCT/NL99/00737 6 breweri the ester benzylacetate is an important constituent of flower scent. The purification of the protein and the gene AAT encoding it has been reported. The flower AAT enzyme was not reported to have affinity to aliphatic 5 alcohols. The enzyme has high affinity to the aromatic alcohols such as benzyl alcohol and cinnamyl alcohol [Duda-reva et al., (1998); The Plant Journal 14(3) 297-304]. Genes coding for flower aromatic acyl transferases, that acylate plant pigments, causing changes in colour tone have been the 10 subject of a recent patent application [EP 0810287] . Recently, an aromatic amino transferase enzyme has been purified from lactic acid bacteria and was shown to initiate the conversion of amino acids to cheese flavour compounds [Yvon et al., (1997); Applied and Environmental Microbiology 414-15 419] .

Although volatile esters are qualitatively and quantitatively one of the most important classes of volatile compounds in fruit flavour and aroma, there are very few reports concerning the biochemical aspects of ester forma-20 tion in fruits. An understanding of the precursors and characterisation of enzymes involved in the pathways leading to the formation of flavours in fruit is essential for the production of natural flavours. Plant derived flavour components alone represent a world-wide market of 1.5 25 billion dollars. Presently, the main ways to produce plant flavour compounds is by the synthetic route. Synthetic organic chemicals constitute more than 80-90% (by weight and value) of the raw materials used in flavour and fragrance formulations. Problems often exist concerning production. 30 Extraction from intact plants and conventional fermentation are currently providing alternative routes for the commercial production of flavour and aroma chemicals. However, the demand for natural flavours by the consumer has been steadily increasing, and often demand outstrips supply. In many 35 cases sought after flavour compounds preclude isolation. Unlike other fruit flavours, no single chemical or class of chemicals in particular are associated with strawberry flavour. To date no strawberry fruit specific aminotransfe- 7 rase, pyruvate decarboxylase, thiolase, alcohol dehydrogenase, or acyl-transferase enzymes at the route of this invention, involved directly or in prpviding precursors for ester formation, hence flavour compounds,, has been isolated and 5 characterized. The genes and their peptides at the route of this invention are involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit and can therefore provide a novel method for the in-vivo and in-vitro biotechnology production of bio-flavours, natural 10 flavour chemicals by recombinant means.

Summary of invention The object of the present invention is to disclose the genes/peptides involved in the process of volatile ester production, particularly aliphatic esters although not 15 exclusively so, hence fruit flavour and aroma. This is the first time to our knowledge, that the genes/peptides sequences from an entire pathway to the production of volatile esters in fruit, in particularly strawberry, have been disclosed. The nucleotide and polypeptide sequences descri-20 bed in this application can serve as a tool for a vast number of applications related to ester formation and the biotechnological engineering of natural and artificial fruit flavours for the food industry. The invention is based on the identification of genes which encode proteins central to 25 the pathway leading to volatile ester formation in fruit. DNA sequences which encode these proteins have been cloned and characterised. The nucleic acid/peptide sequences may be used in expression systems, for industrial application and /or to modify plants with the goal to produce natural and/or 30 synthetic flavours. 3 I OCT 2003 - RECFM/cp , 7a In particular the invention provides a nucleic acid sequence selected from: (a) An isolated nucleic acid sequence encoding a protein comprising the amino acid sequences LxxxYPxxGR, wherein x constitutes any amino acid sequence, and said protein having alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit. (b) An isolated nucleic acid sequence encoding a protein comprising the amino acid sequence LxxxYPxxGR and either of WTNFFNPLDFGW or a 70% homologue of this peptide and PSRVxxVTxFLxKxLI, wherein x constitutes any amino acid sequence, and said protein having alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

An isolated nucleic acid sequence encoding a protein which has at least 80% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 3B, said protein or a fragment thereof has aminotransferase activity and is involved in the biosynthetic pathway for aliphatic and /or aromatic ester production in fruit.

(A) An isolated nucleic acid sequence encoding a protein which has at least 90% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 4B, said protein or a fragment thereof has thiolase activity and is intellectual property Office OF M.Z 3 I OCT 2003 received 7b involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit. (e) An isolated nucleic acid sequence encoding a protein which has at least 90% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 5B, said protein or a fragment thereof has pyruvate decarboxylase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit. (0 An isolated nucleic acid sequence encoding a protein which has at least 75% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 6B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

(®) An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 7B, 8B, 9B and 10B, or which has at least; i) 70% homology with the amino acid sequence of a 326 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 7B, or ii) 75% homology with the amino acid sequence of a 278 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 8B, or iii) 70% homology with the amino acid sequence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 9B, or INTELk!9TUAL PROPERTY OFRCF OF I\|.Z 31 OCT 2003 _received 7c iv) 80% homology with the amino acid sequence of a 188 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 10B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 11B, or which has at least 75% homology with the amino acid sequence of a 181 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 11B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 12B and 13B, or which has at least; 70% homology with the amino acid sequence of a 176 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 12B, or 70% homology with the amino acid sequence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 13B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 15B, or which has at least 80% homology with the amino acid sequence SEQ ID NO: 15B, said protein or a fragment thereof has esterase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

^'NTEL^ I UAL pR0pERTy UFF/CB OF N.Z 3 I OCT 2003 RECPl\/Pn 7d Definitions of Terms ^Nucleic acid' sequence as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic PROPERTY I OFRCE OF N.z i 31 OCT 2003 .received 8 • origin which may be single- or double-stranded, and represents the sense or antisense strand. Similarly, 'amino acid sequence' as used herein refers to peptide or protein sequence.

The term 'agonist' , as used herein, refers to a molecule which, when bound to a polypeptide causes a change in the polypeptide which modifies the activity of the polypeptide. Agonist may include proteins, nucleic acids, carbohydrates, or any other molecule which binds to the 10 polypeptide.

The term 'antagonist' or 'inhibitor' as used herein, refer to a molecule which when bound to a polypeptide, blocks or modulates the biological or immunological activity of the polypeptide. Antagonists or inhibitors may 15 include proteins, nucleic acids, carbohydrates, or any other molecules which bind to the polypeptide.

'Stringency' typically occurs in the range from about Tm-5°C (5°C below the Tm of the probe) to about 2 0°C to 25°C below Tm. As will be understood by those skilled in the 20 art, a stringent hybridisation can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.

The term 'hybridisation' as used herein shall include 'any process' by which a strand of nucleic acid 25 joins with a complementary strand through base pairing' (Coombs J, (1994) Directory of biotechnology, Stockton press, New York NY).

A 'deletion' is defined as a change in either nucleotide or amino acid sequence in which one or more 30 nucleotides or amino acid residues, respectively, are absent.

An 'insertion' or 'addition' is that change in nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, 35 respectively, as compared to the naturally occurring polypeptide (s) . 9 A 'substitution' results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

As used herein, the term 'substantially purified' refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and that are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. The definition 'substantially homologous' means that a particular subject sequence, for example a mutant sequence, varies from the reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and the subject sequence.

A 'variant' of a polypeptide as outlined in the first and second aspects of this invention is defined as an amino acid sequence that is different by one or more amino acid 'substitutions'. A variant may have 'conservative' changes, wherein a substituted amino acid has similar structural or chemical properties eg replacement of leucine with isoleucine. More rarely a variant may have 'non-conservative' changes, eg replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted, without abolishing biological or immunological activity may be found using computer programmes well known in the art, for example, DNAStar software.

The term 'biologically active' refers to a polypeptide as outlined in the first and second aspects of the invention, having structural, regulatory, or biochemical function of their naturally occurring counterparts. Likewise, 'immunologically active' defines the capability of the natural, recombinant or synthetic polypeptide, as outlined in the first and second aspects of the invention, or any oligopeptide thereof, to induce a specific immune response PCT/N L99/00737 in an appropriate animal or cells and to bind with specific antibodies.

The term 'natural products' are those products that are obtained directly from plants and sometimes from animal 5 sources by physical procedures. 'Nature identical compounds' are produced synthetically but are chemically identical to their 'natural counterparts'. Artificial flavour substances are compounds that have not been identified in plant or animal products for human consumption. 'Nature-identical' 10 aroma substances are with few exceptions the only synthetic compounds used in flavours in addition to 'natural products' .

The definition 'host cell' refers to a cell in which an alien process is executed by bio-interaction, 15 irrespective of the cell belongs to a unicellular, multicellular, a differentiated organism or to an artificial cell, cell culture or protoplast. The definition 'host cell' in the context of this invention is to encompass the definition 'plant cell'.

'Plant cell' by definition is meant by any self- propagating cell bounded by a semi permeable membrane and containing one or more plastids. Such a cell requires a cell wall if further propagation is required. 'Plant cell', as used herein, includes without limitation, seeds, suspension 25 cultures, embryos, meristematic regions, callous tissues, protoplasts, leaves, roots, shoots, gametophytes, sporophy-tes, pollen and microspores.

With the definition 'transformed cell' or 'transgenic cell' is meant a cell (or ancestor of said cell) into 3 0 which by means of recombinant DNA techniques, DNA encoding the target polypeptide can be introduced.

The definition 'micro-organisms' refers to microscopic organisms, such as Archaea, Bacteria, Cyanobacteria, Microalgae, Fungi, Yeast, Viruses, Protozoa, Rotifers, 35 Nematodes, Micro-Crustaceans, Micro-Molluscs, Micro-Shellfish, Micro-insects etc.

The definition 'plant(s)' refers to eukaryotic, autotrophic organisms. They are characterised by direct 11 usage of solar energy for their primary metabolism, their permanent cell wall and in case of multicellular individuals their open unlimited growth. In case of heterotrophic plants, the organisms are in an evolutionary context essen-5 tially derived from autotrophic plants in their structure and metabolism.

'Dicotyledons' (and all scientific equivalents referring to the same group of plants) form one of the two divisions of the flowering plants or angiospermae in which 10 the embryo has two or more free or fused cotyledons.

*Monocotyledons' (and all scientific equivalents referring to the same group of plants) form one of the two divisions of the flowering plants or angiospermae in which the embryo has one cotyledon.

'Angiospermae' or flowering plants are seed plants characterised by flowers as specialised organs of plant reproduction and by carpels covering the ovaries. Gymno-spermae are seed plants characterised by strobili as specialised organs for plant repoduction and by naked sporophylls 20 bearing the male or female reproductive organs. 'Ornamental' plants are plants that are primarily in cultivation for their habitus, special shape, (flower, foliage or otherwise) colour or other characteristics which contribute to human well being indoor as cut flowers or pot plants or outdoors 25 in the man made landscape. 'Vegetables' are plants that are purposely bred or selected for human consumption of foliage, tubers, stems, fruits, flowers of parts of them and that need an intensive cultivation regime. 'Arable crops' are purposely bred or selected for human objectivity's (ranging 3 0 from direct or indirect consumption, feed or industrial applications such as fibres) .

The definition 'process' is the development of, a method(ology) concerning, the progress of a series of activities in a certain context. 'Biological' process is a 35 process based on metabolic activity in organisms, or essentially derived from that by biochemical, biophysical, physiological, ecological or genetic means. 'Industrial' 12 process is an economically feasible process with high added value, aiming at bulk production.

The definition 'homology' refers to the basic similarity of a particular structure in different organisms, 5 usually as result from the descent from a common ancestor.

The definition 'promoter' is intended as a nucleotide sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render tissue-specific gene expression; such elements may be 10 located in the 5' or 3' regions of the native gene.

The definition 'operably linked' is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) 15 are bound to the regulatory sequence(s).

The definition 'fruit' (botanically) is the ripened ovary of a plant and its contents. The definition 'fruit' (agronomically linguistically) is the ripened ovary and its contents together with any structure with which they are 20 combined, as in case of strawberry or apple, the receptacle.

The definition 'climacteric' is pointing at the phase of increased respiration found at fruit ripening and at senescence. The definition 'non-climacteric' is pointing at no such phase being present.

The definition 'antisense' RNA is an RNA sequence which is complementary to a sequence of bases in the corresponding mRNA: complementary in the sense that each base (or majority of bases) in the antisense strand (read in the 5' to 3' sense) is capable of pairing with the corresponding 30 base (G with C, A with U) , in the mRNA sequence read in the 5' to 3' sense.

The definition 'sense' RNA is an RNA sequence which is substantially homologous to at least part of the corresponding mRNA sequence.

INTELLECTUAL PROPERTY OFRCE OF M.Z 31 OCT 2003 received 12a The term "antimicrobial" means having an action against microbes, ie bacteria, yeasts and other microorganisms, which action results in death or an impaired reproduction or growth of the microorganism.

The term "cell-free lysate" means a result from cell lysis in which all cells either have lysed and/or from which all intact cells have been removed.

The term "molecular marker" means a recognizable genetic sequence which can be used to identify a genetic locus, a linkage group, or a recombination event.

The term "diagnostic tools" mean tools which can be used in diagnosis. In the case of nucleic acids (such as in claim 34) this means nucleic acids that are used or can help for the detection of other nucleic acids such as in PCR reactions and the like).

The term "nutraceutical" means compounds that would normally be contained within the food or feed and which exert a pharmaceutical effect.

The term "biological control of pests" means antipathogenic use of biological compounds or organisms. This can range from eg the use of organisms which act as predator for pathological organisms, thereby alleviating the pathogenic affect caused by these pests, to use of proteins, or nucleic acids as antimicrobials, pest repellents, etc.

The term "protection of stored products" means preventing detonation of products in storage. Detoriation can occur through microorganisms (fouling) or through environmental conditions (desiccation, denaturation).

Brief description of drawings ^ The present invention may be more fully understood by reference to the following description, when read together with the accompanying drawings: Figure 1. The proposed biosynthetic pathway for the produc-5 tion of esters in fruits.

Figure 2. Volatile ester emission during strawberry fruit development. GC-MS chromatograms (detector response: 100% = 2xl06 total ion counts) of volatiles in-vivo released by strawberry fruits (cv. Elsanta) at different stages of 10 development. Developmental stages: G-green; W-white; T-turning; P- pink; R-red; DR-dark red. The five main volatile esters detected are marked with numbers: 1-methyl hexanoate; 2-hexyl acetate; 3-hexyl butanoate; 4-octyl acetate; 5-octyl butyrate.A GCMS chromatogram of a mature strawberry fruit of 15 the cultivar Elsanta. The analysis was done using the headspace method. Esters are marked with dots.

Figure 3. The expression pattern of strawberry alcohol acyl transferase cDNA (SLE27, SEQ ID N0:1A) in various tissues of the cultivar Elsanta by Northern blot analysis. The Northern 20 blot was hybridised with a full length cDNA fragment of SEQID:1A (SLE27) .

Figure 4. The expression pattern of a lemon alcohol acyl transferase (CLF26, SEQ ID NO:2A) in various tissues of the 25 lemon cultivar Mayer by Northern blot analysis. The Northern blot was hybridised with a full length cDNA fragment of SEQ ID 2A (CLF26).

Figure 5. Expression analysis in various tissues of the strawberry cultivar Elsanta by Northern blots analysis. The 3 0 Northern blots where hybridised with full length cDNA fragments corresponding to (a) a thiolase SEQ ID 4A (SLG150) (b) a pyruvate decarboxylase SEQ ID 5A (SLH51) and (c) an alcohol dehydrogenase SEQ ID 6A (SLB3 9).

TNTBIEC I^L PROPERTY OFFICE OF N.Z 31 OCT 2003 14 Figure 6. Strawberry genomic Southern analysis. 10 lg DNA was digested with restriction enzymes Hindlll, EcoRI, Xbal and Xhol, separated on an agarose gel and blotted onto a nylon filter. The DNA was hybridised with a full length 32P-5 labelled strawberry alcohol acyl transferase cDNA (SLE27) probe, corresponding to SEQ ID 1A and autoradiographed.

Figure 7. A scheme of the pRSET B (Invitrogen) vector used for the expression of various alcohol acyl transferases in E.coli.

Figure 8. Verification of ester formation by the SLE27 (SAAT), SEQ ID NO: IB protein using GC-MS. GC-MS chromato-grams (detector response: 100% = 2xl06 total ion counts) of volatiles produced with the incubation conditions as described below. (A) Butanol and butyl acetate standards. (B) SAAT 15 protein + butanol + acetyl-CoA. (C) As in B, protein absent. (D) As in B, butanol absent. (E) As in B, acetyl-CoA absent. (F) GFP protein + butanol + acetyl-CoA. (G) Empty pRSET B vector elute + butanol + acetyl-CoA. Other visible peaks are impurities from the butanol substrate. 2 0 Figure 9. Western blot loaded with protein corresponding to four different treatments carried out on the pellet, supernatant and elute from a Ni-NTA column after expression of strawberry alcohol acyl transferase (SLE 27, SEQ ID N0:1A) in E. coli. As a control for the experiment the Green 25 Fluorescent Protein (GFP) was partially purified in the same way and the elute from one treatment was loaded on the gel. The blot was hybridised with a commercial antibody (Clon-tech) raised against an epitope fused at the N- terminus of the recombinant protein.

Figure 10. Gene expression profiles of strawberry alcohol acyl transferase (SLE 27, SEQ ID NO: 1A)), alcohol dehydrogenase (SLF138, SEQ ID NO: 11A), pyruvate decarboxylase (SLH 51, SEQ ID NO: 5A) , alcohol dehydrogenase (SLG16, SEQ ID NO: 12A) and amino transferase {SLF96, SEQ ID NO: 3A) . Ex pression ratios were monitered during fruit development and ripening in the red stage vs green fruit and red fruit v.s turning fruit.

Figure 11. SLE27 (SAAT), SEQ ID NO: IB (SAAT) - induced [14C] -acetyl-CoA incorporation in esters. (A) Flame ionization detector (FID)-signal of unlabelled standards of 1, hexyla-cetate; 2, 1-hexanol; 3, octylacetate and 4, 1-octanol. (B) and (C): Radio-GC chromatograms of labelled products formed by the SAAT protein from 0.1 mM [14C]-acetyl-CoA and alcohols with (B) 2 mM 1-octanol or (C) 2 mM 1-hexanol.

Figure 12.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the strawberry alcohol acyl transferase (SLE27, SEQ ID N0:1A). (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with 1-butanol.

Figure 13.

Verification of ester formation using GC-MS A) E. coli cell cultures expressing the melon alcohol acyl transferase (MAY 5, SEQ ID NO:23 (A). (B) E. coli cell cultures expressing the empty vector cassette. No alcohol supplements.

Figure 14.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the apple alcohol acyl transferase (MAY 3, SEQ ID NO:18A). (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with 1-butanol.

Figure 15.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the citrus alcohol acyl transferase (CLF26: SEQ ID N0:2A). (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with 1-butanol. 16 Figure 16.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the banana alcohol acyl transferase (MAY 2, SEQ ID NO:17A). (B) E. coli cell cultures expressing the 5 empty vector cassette. No alcohol supplements.

Figure 17.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the apple alcohol acyl transferase (MAY3, SEQ ID NO:18A). (B) E. coli cell cultures expressing 10 the empty vector cassette. Both cultures supplemented with isoamylalcohol, n-butanol, cis-2-hexen-l-ol and isopropanol.

Figure 18.

Verification of ester formation using GC-MS (A) E. coli cell cultures expressing the strawberry Vesca alcohol acyl 15 transferase (SUN1, SEQ ID NO:16A). (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with isoamylalcohol, n-butanol, cis-2-hexen-l-ol and isopropanol.

Detailed description of the invention According to a first aspect the invention provides an insolated DNA sequence encoding (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: IB or SEQ ID NO: 2B, or (b) a polypeptide having at least 3 0% homology 25 with the amino acid sequence SEQ ID NO: IB or at least 4 0% homology with the amino acid sequence SEQ ID NO: 2B, or (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide 30 has alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and /or aromatic ester production in fruit. 17 Preferably, the DNA sequence encodes a polypeptide having at least 50% homology, more preferably at least 70% homology, with any of the amino acid sequences SEQ ID NO: IB or SEQ ID NO: 2B, or a fragment thereof.

With reference to the nucleic acid sequence the invention provides an isolated DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 1A or SEQ ID NO: 2A which encodes a polypeptide having alcohol acyl transferase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 1A or SEQ ID NO: 2A, or (c) which has at least 25% homology with any of the sequences of (a) or (b), or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) .

Preferably the DNA sequence has a nucleic acid sequence (c) which has at least 40% homology, more preferably at least 60% homology, with any of the sequences of (a) or (b), or (d) which is capable of hybridising under strin gent conditions to said sequence (c).

According to a second aspect the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO: IB or SEQ ID NO: 2B, or (b) having at least 3 0% homology with the amino acid sequence SEQ ID NO: IB or at least 40% homology with the amino acid sequence SEQ ID NO: 2B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has alcohol acyl transferase activity and is involved in the biosynthetic pathway for ali PCT/N L99/00737 18 phatic and/or aromatic ester production in fruit.

Preferably, the polypeptide has at least 50% homology, more preferably at least 70% homology, with any of 5 the amino acid sequences SEQ ID NO: IB or SEQ ID NO: 2B, or a fragment thereof.

It may be advantageous to produce nucleotide sequences according to the first aspect of the invention as defined above or derivatives thereof possessing a substanti-10 ally different codon usage. It is known by those skilled in the art that as a result of degeneracy of the genetic code, a multitude of gene sequences, some bearing minimal homology to the nucleotide sequences of any known and any naturally occurring genes may be produced. The invention contemplates 15 each and every possible variation of the nucleotide sequences that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring gene 20 sequences, and all such variations are to be considered as being specifically disclosed. In addition the nucleotide sequences of the invention may be used in molecular biology techniques that have not been developed, providing the new techniques rely on properties of nucleotide sequences that 25 are currently known, including but are not limited to such properties such as the triplet genetic code and specific base pair interactions.

Altered nucleic acid sequences of this invention include deletions, insertions, substitutions of different 30 nucleotides resulting in the polynucleotides that encode the same or are functionally equivalent. Deliberate amino acid substitution may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, and/or the amphipathetic nature of the residues as long as the biolo-35 gical activity of the polypeptide is retained. Included in the scope of the present invention are alleles of the polypeptides according to the second aspect of the invention as defined above. As used herein, an 'allele' or 'allelic WO 00/32789 PCT/NL99/00737 19 sequence' is an alternative form of the polypeptides described above. Alleles result from a mutation, eg a change in the nucleic acid sequence, and generally produce altered mRNA or polypeptide whose structure or function may or may 5 not be altered. Any given polypeptide may have none, or more allelic forms. Common allelic changes that give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, 10 one or more times in a given sequence.

It is envisaged that the polynucleotide sequences of the present invention can be used as probes for the isolation of similar sequences from the strawberry genome and lemon genome. By using as a probe a cDNA of the gene 15 sequences of the invention it would be possible by those skilled in the art to obtain comparable gene sequences from various tissues, particularly fruit tissues, from climacteric and non climacteric plants. One aspect of the invention is to provide for hybridisation or PCR probes which are 20 capable of detecting polynucleotide sequences, including genomic sequence(s), encoding the polypeptides of the invention, or closely related molecules. The specificity of the probe, whether it is made from a highly specific region, eg 10 unique nucleotides in the 5' regulatory region, or a 25 less specific region, e.g. in the 3' region, and the stringency of the hybridisation or amplification (maximal, high, intermediate, low) will determine whether the probe identifies only naturally occurring sequence(s) encoding the polypeptide, allele's or related sequences. 30 Additional alcohol acyltransferase sequences from melon, strawberry, vesca, banana, apple, mango and lemon were obtained using primers. These additional sequences are covered by the first aspect of invention. The nucleic acid sequences are shown in SEQ ID NO: 16A to 23A, respectively. 35 The amino acid sequences are shown in SEQ ID NO:16B to 23B, respectively. Apart from the applications as disclosed herein said sequences can be used in combination for the synthesis of a broader range of esters via recombinant means or via transgenic approaches and/or for the synthesis of new flavours.

Probes may also be used for the detection of related sequences and preferably contain at least 50% of any 5 of the nucleotides from any one of the gene encoding sequences according to the present invention. For gene sequences of the present invention, the hybridisation probes may be derived from the nucleotide sequence, or from genomic sequence including promoter, enhancer element and introns. 10 Hybridisation probes may be labelled by a variety of reporter groups, including radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Other means for producing specific hybridisation 15 probes for encoding gene sequences of the invention include the cloning of nucleic acid sequences into suitable vectors for the production of mRNA probes. Such vector are known in the art and are commercially available and may be used to synthesise RNA probes in-vitro by means of the addition of 2 0 the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriately radioactively labeled nucleotides.

According to a third aspect the invention provides a recombinant expression vector comprising a coding sequence which is operably linked to a promoter sequence capable of 25 directing expression of said coding sequence in a host cell for said vector, and a transcription termination sequence, in which the coding sequence is a DNA sequence according to the invention.

In a further embodiment the invention provides a 30 replicative cloning vector comprising an isolated DNA sequence of the invention and a replicon operative in a host cell for said vector.

Methods which are well known to those skilled in the art can be used to construct expression vectors contai-35 ning the gene coding sequence of the invention, and appropriate transcriptional and translational controls. These methods include in-vitro recombinant techniques. Such techniques are described in Sambrook et al. , 1989. Molecular 21 cloning a laboratory manual, cold spring Harbour press, Plain view, NY and Ausubel FM et al., (1989) Current protocols in molecular biology, John Wiley and Sons, New York, NY.

In order to express a biologically active polypep tide, the nucleotide sequence encoding the polypeptide, or the functional equivalents thereof, fragments of the polypeptide, is inserted into the appropriate expression vector (i.e. a vector that contains the necessary elements 10 for the transcription or translation of the inserted coding sequence). Specific initiation signals may also be required for efficient translation of the polypeptides of the invention. These signals include the ATG initiation codon and adjacent sequences. In cases where the polypeptides, their 15 initiation codons and upstream sequences are inserted into the appropriate expression vector, no additional translatio-nal control systems including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the 20 entire insert. Exogenous transcriptional elements and initation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D et al (1994) Results Prob Cell Differ 20: 125-25 62; Bittner et al. (1987) Methods in Enzymol 153: 516-544) .

In addition a host strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not 30 limited to, acylation, carboxylation, glycosylation, phosphorylation and lipidation. Post translation processing which cleaves a 4prepro' form of the protein may also be important for correct insertion, folding and/or function. Different host cells which have the correct cellular machi-35 nery and characteristic mechanisms for such post-translatio-nal activities maybe chosen to ensure correct modification and processing of the introduced, foreign protein. 22 The nucleotide sequences of the present invention can be engineered in order to alter the coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example mutations may be introduced using techniques which are well known in the state of the art, e.g. site directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon usage, to produce splice variants etc. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular procaroytic or eukaroytic expression host in accordance with the frequency with which particular codons are utilised by the host (Murray E et al. (1989) Nuc Acids Res 17: 477-508). Other reasons for substantially altering the nucleotide sequence(s) of the invention and their derivatives, without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from naturally occurring sequences.

In developing the expression cassette, the various fragments comprising the regulatory regions and open reading frame may be subjected to different processing conditions, such as ligation, restriction enzyme digestion, resection, in-vitro mutagenesis, primer repair, use of linkers and adapters and the like. Thus, nucleotide transitions, trans-versions, insertions, deletions and the like, may be performed on the DNA which is employed in the regulatory regions and/or open reading frame. The expression cassette may be wholly or partially derived from natural sources endogenous to the host cell. Furthermore, the various DNA constructs (DNA sequences, vectors, plasmids, expression cassettes) of the invention are isolated and/or purified, or synthesised and thus are not naturally occurring. The invention further contemplates the use of yet undescribed biological and non biological based expression systems and novel host(s) systems that can be can be utilised to contain and express the gene coding sequence(s) of the invention. 23 'Antisense' or 'partial sense' or other techniques may also be used to reduce the expression of genes in the pathway leading to the production of flavour volatile esters. Full length 'sense' techniques may be designed to 5 increase or reduce the expression of genes in the pathway leading to the production of flavour volatiles. The 'anti-sense' or 'partial sense' molecules may be designed to block translation of mRNA by preventing the transcript from binding to the ribosomes. Ribozymes are enzymatic RNA 10 molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridisation of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead 15 motif ribozyme molecules that can specifically and efficiently catalyse endonucleolytic cleavage of gene sequences of the invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which 2 0 include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide 25 inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridisation with complementary oligonucleotides using ribonuclease protection assays.

'Antisense' molecules and ribozymes of the inven- 3 0 tion may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in-vitro and in-vivo trans-35 cription of DNA sequences of the invention. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 and SP6. Alternatively, antisense cDNA constructs that synthesise PCT/N L99/00737 24 antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the 5 addition of flanking sequences, at the 5' and/or 3' end of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.

A variety of vector/host expression systems can be 10 utilised to contain and express the gene coding sequences of the invention. These include and are not limited to microorganisms such as bacteria (e.g. E coli, B subtilis, Strep-tomyces, Pseudomonads) transformed with recombinant bacteriophage, plasmid or cosmid DNA expression systems, yeast (e.g 15 S. cerevisiae, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Schizosacch. Pombe, Yarrowia) transformed with yeast expression vectors; filamentous fungi (aspergillus nidulans, aspergillus orizae, aspergillus niger) transformed with filamentous fungi expression vec-20 tors, insect cell systems transfected with virus expression vectors (eg baculovirus, adenovirus, herpes or vaccinia viruses); plant cell systems transfected with virus expression vectors (e.g. cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression 25 vectors (e.g Ti or Pbr322 plasmid); or mammalian cell systems (chinese hamster ovary (CHO), baby hamster kidney (BHK), Hybridoma's, including cell lines of mouse, monkey, human and the like.

In the case of plant expression vectors, the 3 0 expression of a sequence (s) of the invention may be driven by a number of previously defined promoters, including inducible and developmentally regulated promoters. The invention further contemplates the use of the individual promoters of the polynucleotide sequence(s) of the present 35 invention for this purpose. In particular an alcohol acyl transferase promoter(s) pertaining to SEQ ID IA and SEQ ID 2A or any promoters particularly responsive to ripening events may be used to drive the tissue specific expression of the target genes. In addition, viral promoters such as the 35S and the 19S promoters of CaMV (Brisson et al. , 1984; Nature, 310: 511 -514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al. (1987); EMBO J 6:307-311). Promoters or enhancers derived from the genomes of plant cells, tissue specific promoters i.e fruit specific promoters, Fbp7 (Columbo et al. 1997; Plant Cell 9; 703-715), 2A11 promoter (Pear et al., 1989, Plant Molecular Biology, 13:639-651), small subunit of 10 Rubisco (Corruzzi et al., 1984; EMBO J 3:16; Broglie et al. , 1984 Science 224:838-843) or timing specific promoters such as ripening specific promoters (the E8 promoter, Diekman and Fisher, 1988, EMBO J, 7:3315-3320) may be used. Suitable terminator sequences include that of the agrobacterium 15 turnefaciens nopaline synthase gene (Nos 3' end), the tobacco ribulose bisphosphate carboxylase small subunit termination region; and other 3' regions known to those skilled in the art. These constructs can be introduced into plant cells by direct DNA transformation, or pathogen mediated transfec-2 0 tion. For reviews of such techniques, see Hobbs S or Murry LE, in McGraw Hill yearbook of Science and technology (1992), Mc Graw Hill NY, PP 191-196 or Weissbach and Weiss-bach (1988) Methods for Plant Molecular Biology, Academic Press, New York, NY, pp421-463, Fillatti et al. , Biotech-25 nology, 5: 726-730. Manipulation of DNA sequences in plant cells may be carried out using the Cre/lox site specific recombination system as outlined in patent application W091-09957.

According to a fourth aspect the invention provides 30 a method for regulating aliphatic and/or aromatic ester formation in fruit, comprising inserting into the genome of a fruit-producing plant one or more copies of one or more DNA sequences of the invention.

The activity of genes according to the present 35 invention involved in the biosynthetic pathway leading to volatile ester formation, hence flavour, may be either increased or reduced depending on the characteristics desired for the modified plant part. The gene sequence may 26 be in the same 'sense' or 'antisense' orientation as the endogenous target gene. Methods well known to those skilled in the art can be used to construct recombinant vectors which will express 'sense' or 'antisense' polynucleotides 5 encoding the gene sequences of the present invention. Such technology is now well known in the art. In the case of the 'plant cell' officiating as 'host cell', the target 'plant cell' may be part of a whole plant or may be an isolated cell or part of a tissue which may be regenerated into a 10 whole plant. The target plant may be selected from any mono-cotyledonous or dicotyledonous plant species. Suitable plants include any fruit bearing plant such as strawberry, citrus (lemon), banana, apple, pear, melon, sweet pepper, peach or mango. Other suitable plant hosts include vegeta-15 ble, ornamental (to include sunflower) and arable crops (to include soybean, sunflower, corn, peanut, maize, wheat, cotton, safflower, and rapeseed). For any particular plant cell, the gene sequences, cDNA, genomic DNA, or synthetic polynucleotide, used in the transformation vector construct, 20 may be derived from the same plant species, or may be derived from other plant species (as there will be sufficient homology to allow modification of related enzyme gene function). The procedure or method for preparing a transfor-mant can be performed according to the conventional techni-25 que used in the fields of molecular biology, biotechnology and genetic engineering.

According to a fifth aspect the invention provides a plant and propagating material thereof which contains in its genome a DNA sequence of the invention or a vector as 30 defined above.

In a preferred embodiment the invention provides a genetically modified strawberry or lemon plant and propagating material derived therefrom which has a genome comprising an expression vector for over-expression or down-35 regulation of an endogenous strawberry or lemon plant gene counterpart of any of the DNA sequences of the invention.

In mammalian cells a number of viral based systems may be utilised. In cases where the adenovirus is used as an 27 expression vector, a gene coding sequence(s) of the invention may be ligated into the adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non essential El or E3 5 region of the viral genome will result in a variable virus capable of expressing the gene sequence(s) in infected host cells (Logan and Shark (1984) Proc Natl Acad. Sci 81:3655-59) In addition transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase 10 expression in mammalian host cells.

Furthermore insect cells such as silkworm cells or larvae themselves may be used as a host. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera 15 frugiperda cells or in Trichoplusia larvae. The gene coding sequence(s) of the invention may be cloned into the nonessential region of the virus, such as the polyhedrin gene, and placed under control of a polyhedrin promoter. Successful insertion of the sequence(s) will render the polyhedrin 2 0 gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S frugiperda cells or Trichoplusia larvae in which the gene sequence or sequences are expressed (Smith et al. (1993) J Virol 46:584; Engelhard et al. (1994) Proc. Natl 25 acad Sci, 91: 3224-7).

The control elements or regulatory sequences of the described systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters and 3' untranslated regions, which interact 30 with host cellular proteins to carry out transcription and translation. Dependent on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, 35 inducible promoters such as the hybrid lacZ promoter of the Bluescript ® phagemid (Strategene, La Jolla) or pSPORT (Gibco BRL) and ptrp-lac hybrids and the like may be used. Other conventionally used promoters are trc and trc promo- WO 00/32789 PCT/NL99/00737 28 ters. The baculovirus polyhedrin promoter may be used for insect cells. As a promoter for filamentous organisms, for example, amalyse, trp C, and the like may be used. As a promoter for yeast for example glyceraldehyde-3-phosphate 5 dehydrogenase may be used. As a promoter for animal cell hosts, viral promoters such as the SV4 0 early promoter, SV40 late promoter and the like may be used. In the yeast Saccha-romyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as the alpha factor, 10 alcohol oxidase and PGH may be used. For review, see Grant et al., 1987 Methods in Enzymology 153: 516-544.

For long term, high yield production of recombinant proteins, stable expression is preferred. For example cell lines which stably express the polypeptides of the 15 invention may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for one to two days in an enriched media before they are swit-2 0 ched to selective media. The purpose of the selectable marker is to confer resistance to selection and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stable transformed cells can be proliferated using tissue culture 25 techniques appropriate to the cell type.

Although the presence/absence of the marker gene expression suggests that the gene of interest is also present, its presence and expression must be confirmed. For example, if the gene sequence (s) of the invention are 30 inserted within a marker gene sequence, recombinant cells can be identified by the absence of gene function. Alternatively a marker gene can be placed in tandem with the gene sequence of interest, under the control of a single promoter. Expression of the marker gene in response to indue-35 tion or selection usually indicates expression of the tandem gene as well.

Any number of selection systems may be used to recover the transformed cell lines. Anti-metabolite, antibi- WO 00/32789 PCT/NL99/00737 29 otic or herbicide resistance can be used as the basis of selection; for example, dhfr which confers resistance to methotrextate (Wiger M. et al. (1980) Proc Natl Acad Sci 77: 3567-70; npt, which confers resistance to the aminoglycosi-5 des neomycin and G-418 (Colbere-Garapin F. et al. (1981) J Mol Biol 150: 1-14) and als and pat, which confer resistance to chlorsulfuron and phosphinotricin acyl transferase, respectively (Murry supra). Additional selectable genes have been transcribed, for example, trpB, which allows cells 10 utilise indole in place of tryptophan, or His D, which allows cells to utilise histinol in place of histidine (Hartman SC and RC Mulligan (1988) Proc Natl Acad Sci 85: 8047-51). Alternatively one could use visible markers such as anthocyanidins, ^-glucuronidase and its substrates, GUS, 15 GFP and variants, and luciferase and its substrate, lucife-rin, which are widely used to identify transformants, but also to quantify the amount of stable protein expression attributable to a specific vector system (Rhodes CA et al. (1995) Methods Mol Biol 55: 121-131.

Host cells transformed with a nucleotide sequen ce (s) of the invention, may be cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. The protein produced by recombinant cells may be secreted or contained intracellularly depending on 25 the sequence and/or the vector used. As will be understood by those skilled in the art, expression vectors containing polynucleotides sequences of the invention, can be designed with signal sequences for direct secretion of the protein product, through a prokaroytic or eukaryotic cell membrane. 30 Other recombinant constructions may join the gene sequences of the invention, to a nucleotide sequence encoding a polypeptide domain, which will facilitate purification of soluble proteins (Kroll DJ et al. (1993). DNA Cell Biol 12: 441-53).

According to a sixth aspect the invention provides a method for producing aromatic and/or aliphatic esters in microorganisms, plant cells or plants comprising inserting into the genome of the microorganism or plant one or more copies of DNA sequences of the invention, and feeding an alcohol and an acyl-CoA to the microorganism or plant (cell) .

Alternatively, host cells which contain the gene 5 sequences or polypeptides thereof according to the invention may be identified by a variety of procedures known to those skilled in the art. These procedures include, but are not limited to DNA-DNA, DNA-RNA hybridisation, amplification using probes (portions or fragments of polynucleotides), 10 protein bioassay or immunoassay techniques which include membrane, solution or chip based technologies for the detection and /or quantification of the nucleic acid or protein.

Nucleic acid amplification based assays involve the 15 use of oligonucleotides or oligomers based on the gene sequences of the invention to detect transforraants containing DNA or RNA encoding the polypeptides. As used herein 'oligonucleotides' or 'oligomers' refer to nucleic acid sequence of at least 10 nucleotides and as many as about 60 20 nucleotides, preferably about 15-30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer. Polynucleotide sequences according to the invention encoding alcohol acyl transferase polypeptides may be used to detect volatile ester (aliphatic and aromatic) 25 formation in fruits. PCR as described in US patent Nos 4,683,195 and 4,965,188, provides additional uses for oligonucleotides based on the alcohol acyl transferase sequence. Such oligomers are chemically synthesised, but they may be generated enzymatically or produced from a 30 recombinant source. Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'-3') and one with antisense (3'-5'), employed under less stringent conditions for detection and /or quantitation of closely related DNA or RNA sequences. For example, polynucleotide 35 sequences encoding the alcohol acyl transferase protein may be used in the hybridisation or PCR assay of plant tissues, more specifically fruit tissues, to detect alcohol acyl transferase protein presence. The form of such quantitative 31 methods may include, Southern or Northern analysis, dot/slot blot or other membrane based technologies; PCR technologies such as DNA Chip, Taqman and ELISA Technologies. All of these technologies are well known in the art and are the 5 basis of many commercially owned diagnostic kits. The polynucleotide sequences according to the present invention herein provide the basis for assays that detect signal transduction events associated with volatile ester production, aliphatic and aromatic, hence flavour formation. 10 A variety of protocols for detecting and measuring expression of the polypeptides of the invention, using either polyclonal or monoclonal antibodies specific for the protein are well known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) 15 and fluorescent activated cell sorting (FACS). These methods are described, among other places, in Hampton R et al. (1990), Serological methods, a laboratory manual, APS Press, St Paul MN) and Maddox DE et al. (1993, J Exp Med 158:1211) .

A variety of labels and conjugation techniques are 20 known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labelled hybridisation or PCR probes for detecting sequences related to polynucleotides as outlined in the first and second aspects of the invention, include oligolabeling, nick 25 translation, end labelling of PCR amplification using a labelled nucleotide. Alternatively, the gene sequence or sequences of the invention, or any portion of it, may be cloned into a vector for the production of the mRNA probe. Such vectors are known in the art, are commercially availa-30 ble, and may be used to synthesise RNA probes in-vitro by addition of an appropriate RNA polymerase, such as T7, T3, or SP6 and labelled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway NJ) , promega (Madisson WI) , and US Biochemical Corp (Cleveland OH) supply commercial 35 kits and protocols for these procedures. Suitable reporter molecules or labels include fluorescent, chemiluminescent, 32 or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.

According to another aspect of the invention, natural, modified or recombinant polynucleotide sequences 5 according to the invention may be ligated to a heterologous sequence to encode a fusion protein. A fusion protein may be engineered with one or more additional domains added to facilitate protein purification (i.e. a cleavage site located between the sequence of the polypeptide and the 10 heterologous protein sequence) so that the polypeptide of the invention may be purified away from the heterologous moiety. The invention further contemplates the creation of fusion proteins, chosen from the group of sequences of the invention, degenerate variants thereof, functional equiva-15 lents thereof, pertaining to 'fruit' of strawberry and lemon, or gene sequences with substantial homology to the sequences of the invention from other 'fruit' tissues concerned with the pathway for the formation of aliphatic or aromatic esters, hence flavour. Such purification facilita-2 0 ting domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobin, and the domain utilised in the FLAGS extension/affinity purification 25 system (Immunex Corp, Seattle, WA). The introduction of a cleavable linker such as factor XA, thrombin or enterokinase (Invitrogen, San Diego CA) between the purification domain and the polypeptide is useful to facilitate purification. One such expression vector provides for the expression of a 30 fusion protein comprising gene sequence(s) which encode polypeptides of the invention, and contains nucleic acid encoding six histidine residues followed by thioredoxin and the enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilised metal ion 35 affinity chromatography as described in Porath et al. , (1992) Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein.

PCT/N L99/00737 33 According to a seventh aspect the invention provides an isolated DNA sequence encoding (a) a polypetide having an amino acid sequence as shown in SEQ ID NO: 3B, or 5 (b) a polypeptide having at least 80% homology with the amino acid sequence SEQ ID NO: 3B, or (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide has aminotransferase activity and is involved 10 in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 3A which encodes a 15 polypeptide having aminotransferase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 3A, or (c) which has at least 70 % homology with any of the sequences of (a) or (b), or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) .

Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO: 3B, or (b) having at least 80% homology with the amino 30 acid sequence SEQ ID NO: 3B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has aminotransferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

According to an eighth aspect the invention provides an isolated DNA sequence encoding 34 (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4B, or (b) a polypeptide having at least 90% homology with the amino acid sequence SEQ ID NO: 4B, or (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide has thiolase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit. 10 In particular the invention provies an isolated DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 4A which encodes a polypeptide having thiolase activity and being involved in the biosynthetic pathway for ali- phatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 4A, or (c) which has at least 7 5 % homology with any of the sequences of (a) or (b), or (d) which is capable of hybridising under strin gent conditions to any of the sequences (a) -(c) .

Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO: 4B, or (b) having at least 90% homology with the amino acid sequence SEQ ID NO: 4B, or (c) being a fragment of polypeptide (a) or (b) , 30 which polypeptide or fragment of polypeptide has thiolase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

According to a ninth aspect the invention provides 35 an isolated DNA sequence encoding (a) a polypetide having an amino acid sequence as shown in SEQ ID NO: 5B, or (b) a polypeptide having at least 90% homology with the amino acid sequence SEQ ID NO: 5B, or (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide 5 has pyruvate decarboxylase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated 10 DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 5A which encodes a polypeptide having pyruvate decarboxylase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 5A, or (c) which has at least 75 % homology with any of the sequences of (a) or (b), or (d) which is capable of hybridising under strin-20 gent conditions to any of the sequences (a) - (c) .

Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence as shown in SEQ 2 5 ID NO: 5B, or (b) having at least 90% homology with the amino acid sequence SEQ ID NO: 5B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has 30 pyruvate decarboxylase activity and is invol ved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

According to a tenth aspect the invention provides an isolated DNA sequence encoding 35 (a) a polypetide having an amino acid sequence as shown in SEQ ID NO: 6B, or (b) a polypeptide having at least 75% homology with the amino acid sequence SEQ ID NO: 6B, or 36 (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for ali-5 phatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 6A which encodes a 10 polypeptide having alcohol dehydrogenase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 6A, or (c) which has at least 65 % homology with any of the sequences of (a) or (b), or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) .

Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO: 6B, or (b) having at least 75% homology with the amino 25 acid sequence SEQ ID NO: 6B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for ali- phatic and/or aromatic ester production in fruit.

According to an eleventh aspect the invention provides an isolated DNA sequence encoding (a) a polypeptide having an amino acid sequence 35 selected from the group consisting of sequences SEQ ID NO: 7B, 8B, 9B and 10B, or (b) a polypeptide having at least 37 i) 55% homology with the amino acid sequence of a 326 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 7B, or ii) 75% homology with the amino acid se quence of a 278 aa fragment from the C terminal end of the coding sequence of Of SEQ ID NO: 8B, or iii) 65% homology with the amino acid se-10 quence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 9B, or iv) 80% homology with the amino acid sequence of a 18 8 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 10B, or (c) a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is 20 involved in the biosynthetic pathway for ali phatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated DNA sequence having a nucleic acid sequence 25 (a) selected from the group consisting of sequen ces SEQ ID NO: 7A, 8A, 9A and 10A, which encodes a polypeptide having alcohol dehydrogenase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic 30 ester production in fruit, or (b) complementary to SEQ ID NO: 7A, 8A, 9A or 10A, or (c) which has at least 55% homology, preferably at least 65% homology, with any of the sequences of (a) or (b), or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) . 38 Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence selected from the group consisting of sequences SEQ ID NO: 7B, 8B, 9B and 10B, or (b) having at least i) 55% homology with the amino acid sequence of a 3 26 aa fragment from the C terminal end of the coding sequence of Of SEQ ID NO: 7B ii) 75% homology with the amino acid sequence of a 278 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 8B, or iii) 65% homology with the amino acid se quence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 9B, or iv) 80% homology with the amino acid se- quence of a 188 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 10B, or (c) being a fragment of polypeptide (a) or (b), which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

According to a twelfth aspect the invention provi- des an isolated DNA sequence encoding (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 11B, or (b) a polypeptide having at least 75% homology with the amino acid sequence of a 181 aa frag- ment from the C terminal end of the coding sequence of SEQ ID NO: 11B, or (c) a fragment of polypeptide (a) or (b), 39 which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in 5 fruit.

In particular the invention provides an isolated DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO: 11A which encodes a polypeptide having alcohol dehydrogenase acti- vity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 11A, or (c) which has at least 4 8 % homology with any of 15 the sequences of (a) or (b), or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) .

Further the invention provides a purified and 20 isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO: 11B, or (b) having at least 75% homology with the amino acid sequence of a 181 aa fragment from the 3' end of SEQ ID NO: 11B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for ali- phatic and/or aromatic ester production in fruit.

According to a thirteenth aspect the invention provides an isolated DNA sequence encoding (a) a polypeptide having an amino acid sequence 35 selected from the group consisting of sequences SEQ ID NO: 12B and 13B, or (b) a polypeptide having at least 40 i) 55% homology with the amino acid sequence of a 176 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 12B, or ii) 3 5% homology with the amino acid se quence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 13B, or (c) a fragment of polypeptide (a) or (b), 10 which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated DNA sequence having a nucleic acid sequence (a) selected from the group consisting of sequences SEQ ID NO: 12A and 13A, which encodes a polypeptide having alcohol dehydrogenase acti- vity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or (b) complementary to SEQ ID NO: 12A or 13A, or (c) which has at least 20% homology, preferably at 25 least 3 0% homology, with any of the sequences of (a) or (b) , or (d) which is capable of hybridising under stringent conditions to any of the sequences (a) -(c) .

Further the invention provides a purified and isolated polypeptide, (a) having an amino acid sequence selected from the group consisting of sequences SEQ ID NO: 12B and 13B, or 35 (b) having at least i) 55% homology with the amino acid sequence of a 176 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 12B, or PCT/N L99/00737 41 ii) 35% homology with the amino acid sequence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 13B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in 10 fruit.

According to a further aspect the invention provi des an isolated DNA sequence encoding (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO:15B, or 15 (b) a polypeptide having at least 41% homology, preferably at least 50% and more preferably at least 8 0% homology, with the amino acid sequence SEQ ID NO:15B, or (c) a fragment of polypeptide (a) or (b) , which 20 polypeptide or fragment of polypeptide has esterase activity and is involved in the biosynthesis pathway for aliphatic and/or aromatic ester production in fruit.

In particular the invention provides an isolated 25 DNA sequence having a nucleic acid sequence (a) as shown in SEQ ID NO:15A which encodes a polypeptide having esterase activity and being involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit, or 30 (b) complementary to SEQ ID NO:15A, or (c) which has at least 53% homology,preferably at least 70% homology, with any of the sequences of (a) or (b).

Further the invention provides a purified and 35 isolated polypeptide, (a) having an amino acid sequence as shown in SEQ ID NO:15B, or (b) having at least 41% homology, preferably at least 50% and more preferably at least 80% homolo- gy, with the amino acid sequence ID NO:15B, or (c) being a fragment of polypeptide (a) or (b) , which polypeptide or fragment of polypeptide has esterase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.

Additonally, the invention provides nucleotide sequences which are suitable as primers for obtaining additional alcohol acyltransferase sequences from various fruit by cloning. These nucleotide sequences (motifs) 15 encode: the amino acid sequence as set forth in SEQ ID NO:14B, or Leu XXX Tyr Pro X X Gly Arg, or Pro Ser Arg Val X X Val Thr X Phe Leu X Lys X 20 Leu lie wherein X refers to any amino acid residue.

According to a fourteenth aspect the invention further provides a method for regulating aliphatic and/or 25 aromatic ester formation in fruit comprising inserting into the genome of a fruit-producing plant one or more copies of one or more DNA sequences as defined above encoding various enzymes involved in the biosynthetic pathway for aliphatic and/or aromatic ester formation in fruit. 30 The levels of protein may be increased for example by the incorporation of additional genes. The additional genes maybe designed to give either the same or different spatial or temporal patterns of expression in the fruit as the target gene.

Further the invention provides a genetically modified strawberry or lemon plant and propagating material derived therefrom which has a genome comprising an expres- PCT/N L99/00737 43 si'on vector for overexpression or downregulation of an endogenous strawberry or lemon plant gene counterpart of any of the DNA sequences as defined above encoding various enzymes involved in the biosynthetic pathway for aliphatic 5 and/or aromatic ester formation in fruit.

Further the invention provides a method for producing aromatic and/or aliphatic esters in microorganisms, plant cells or plants comprising inserting into the genome of the microorganism or plant one or more copies of DNA sequences of this invention encoding a polypeptide having alcohol acyl transferase activity, and one or more copies of DNA sequences of this invention encoding a polypeptide having alcohol dehydrogenase activity, and feeding aldehydes and acyl-CoA to the microorganism or plant (cell).

In a further embodiment the above method further comprises inserting one or more copies of DNA sequences of this invention encoding a polypeptide having pyruvate decarboxylase activity, and feeding alpha-keto acids and acyl-CoA to the microorganism or plant (cell). 2 0 In a still further embodiment the above method further comprises inserting one or more copies of DNA sequences of this invention encoding a polypeptide having aminotransferase activity, and feeding amino acids and acyl-CoA to the microorganism or plant (cell).

In a still further embodiment the above method further comprises inserting one or more copies of DNA sequences of this invention encoding a polypeptide having thiolase activity, and feeding amino acids and fatty acids to the microorganism or plant (cell). 3 0 The DNA sequences of this invention encoding a polypeptide having esterase activity can be disrupted to increase the amount of volatile esters. Molecular strategies to accomplish this are well known in the art.

Strawberry and citrus are among the most popular fruits for natural flavour ingredients because of their flavour, fragrance, aroma and scent. Examples of commercially important aliphatic esters are ethyl acetate (used in artificial fruit and brandy flavours), ethyl butyrate (the 44 primary constituent of apple aromas), isoamyl acetate (the main component of banana aroma) and ethyl butyrate (has a fruity odour, reminiscent of pineapple) . Flavour usage of these esters is among the highest of all natural flavour 5 ingredients. The invention further contemplates the use of the polynucleotide sequences according to the present invention, for the industrial production of 'fruit' flavours which are natural to match the odour fidelity of the natural fruit. As the chemical composition of flavour is quite 10 complex, both the type and ratio of compounds present being the key determinant of flavour quality, this present invention further contemplates the production of novel flavours by the use of the polynucleotide sequences according to the present invention, alone or in combination, to provide novel 15 avenues for natural flavour production in the future.

The invention further contemplates the generation of strawberry and lemon plants which amongst other phenoty-pic modifications may have one of the following characteristics : (a) reduced/enhanced flavour from the reduced/en hanced production of volatile esters, (b) increased disease resistance due to the enhanced production of volatile esters, (c) modified plant-insect interactions, to include 25 increased insect resistance.

In a further aspect, the gene sequences of the present invention are used for the manipulation of flavour in other 'fruits' and or/ industrial processes. The genes/ peptides of the present invention, in particular SEQID 1A 3 0 (strawberry SLE2 7) and SEQ ID IB (lemon CLF26) may be used in the following industrial applications,- with particular reference to the manipulation of fruit derived products, however, not exclusively limited to these: a) in the processed food industry as food additives to enhance the flavour of syrups, ice-creams, frozen desserts, yoghurts, confectionery, and like products, PCT/N L99/00737 45 ' b) flavouring agents for oral medications and vitam ins , c) providing additional flavour/aroma in beverages, including alcoholic beverages, 5 d) enhancing or reducing fruit flavour/aroma/fragran- ce/scent, e) enhancing the flavour/aroma of 'natural products (eg flower scent however, including but not exclusively limited to flower scent), 10 f) enhancing the flavour/aroma of 'synthetic or arti ficial products', g) as an alternative to the industrial synthesis of nature identical flavour/aroma substances, h) for the production of novel combinations of arti-15 ficial flavour substances, i) as antibacterial or as anti-fungal agents, j) fragrance /perfumes in the cosmetics, creams, sun- protectant products, hair conditioners, lengthening agent and fixative in perfumes, suspension aid for 2 0 aluminium salts in anti-perspirants pharmaceuti cals, cleaning products, personal care products and animal care products, k) disinfectant additives, 1) as degreasing solvents for electronics (etc.), m) insect pheromones, n) dye carrier, solvents, insect repellent, miticide, scabicide, plasticiser, deodorants.

An example as way of illustration of the industrial importance of volatile esters (aromatic and aliphatic) 30 is the ester methyl anthranillate. Methyl anthranillate, a methyl ester of o-aminobenzoic acid, apart from being a very important perfumary, aroma, scent ester (it imparts a peculiar aroma known as "foxy" to grapes of the North american species Vitis Labrusca), the same compound is known 35 to serve as a pheromone in various insects. Results of investigations aimed at testing the attractiveness of methyl anthranilate for the the soybean beetle Anomala rufocurea, Motschulsky suggested the potential use of methyl anthrani- 46 late as a lure for mass trapping, as well as for monitoring (Imai et al., Applied Entomology and Zoology, 32: 1, 4 5 -48, 1997). In addition methyl anthrenilate acts as a bird repellent. According to the invention we propose the use of 5 the SEQ1D: 2A for the production of methyl anthranilate, for a number of applied applications.

The present invention also relates to antibodies which specifically bind to the polypeptides as claimed in the invention, compositions comprising substantially puri-10 fied polypeptides, fragments thereof, agonists or alternatively antagonists, and methods of producing polypeptides. In bacterial/prokaroyte systems a number of expression vectors may be selected depending on the used of the polypeptide(s) according to the invention. For example, when large quanti-15 ties of the polypeptide are needed for the induction of the antibody, vectors which drive high levels of expression of fusion proteins that are readily purified are desirable. Such vectors include, but are not limited to the multifunctional E.coli cloning and expression vectors, such as 20 Bluescript® (Stratagene), in which the gene sequence(s) according to the invention, can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of B-galactosidase so that the hybrid protein is produced; pPIN vectors (Van Heekle and Schuster 25 (1989) J Biol chem 264: 503-5509); and the like. pGEX vectors (Promega, Madison WI) may also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general such fusion proteins are soluble and can be easily purified from lysed cells by 30 absorption to gluthionine-agarose beads followed by elution in the presence of free gluthionine. Proteins made in such systems are designed to include heparin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will. 35 In addition to recombinant production the amino acid sequences of the polypeptides, or any parts thereof, may be altered by direct synthesis or by genetic means known by those skilled in the art, and/or combined using chemical 47 methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide. Fragments of the polypeptides according to the invention may be produced by direct peptide synthesis using solid phase techniques (c.f.

Stewert et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San Fancisco, Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 10 431A Peptide Synthesizers (Perkin Elmer, Forster City CA) in accordance with the instructions provided by the manufacturer. Various fragments of each of the polypepides may be chemically synthesised separately and combined using chemical methods to produce full length molecules. 15 The invention further contemplates a diagnostic assay using antibodies raised to each of the polypeptides of the present invention, with or without modification. For example, although not exclusively limited to, alcohol acyl transferase antibodies raised to the polypeptides as outli-2 0 ned in the 6-8th embodiments are useful for the detection of flavour forming esters in various fruits. Diagnostic assays for alcohol acyl transferase include methods utilising the antibody and a label to detect the alcohol acyl transferase protein in various fruit extracts. Frequently the polypepti-25 des and antibodies are labelled by joining them, either covalently or non covalently, with a reporter molecule. A variety of reporter molecules are known and several of them have been previously described (cf. reporter genes).

A variety of protocols for measuring the polypep-30 tide, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Polynucleotide sequence(s) of the invention, SEQ ID 1A strawberry (SLE27), SEQ ID 2A, lemon alcohol acyl transferase proteins (CLF26), or any part thereof, may be used for 35 diagnostic purposes, to detect and quantify gene expression in various tissues (eg leaves, stems and in particular fruits (climacteric and non climacteric)), hence volatile ester (aliphatic and aromatic) formation, hence flavour at 48 various stages of plant maturation. The diagnostic assay is useful to distinguish between absence, presence and excess expression of the alcohol acyl transferase proteins and to monitor expression levels during development. 5 The following examples are offered by way of illustration and not by limitation.

Example 1: Method for the detection of volatiles The composition of the headspace of the strawberries was analysed using Solid Phase Micro-extraction for trapping and 10 concentrating the emitted volatiles. The fibres used were 100 fivn Polydimethyl siloxane (PDMS) supplied by Supelco. Analysis was performed on a Fisons GC 8000 system, coupled to the Fisons MD800 mass spectrometer. The gas chromatograph was temperature programmed, with a plateau at 8 0 °C for 2 15 min., followed by a ramp of 8°C/min to 250 °C for 5 min. The injector port was operated at 250°C for thermal desorp-tion during 60 sec. of the SPME fibers, using the deactivated standard 0.7 ml split/splitless injection port liner with zero split. The column used was a HP-5 with a length of 20 50 m and internal diameter of 0.32 mm and a film thickness of 1.03 lira, and operated with helium at a pressure of 3 8kPa. Eluted components were identified by their mass spectrum, using the Fisons Masslab software and the NIST library. The identity of the components was verified where possible by 25 using known substances as reference (most of them obtained from Aldrich).

Headspace analyses were performed both on the plant and in the laboratory using detached fruits. The fruits were put onto new clean aluminium foil and covered with a glass 30 funnel closed with aluminium foil. The SPME fiber was inserted in the funnel through the foil closing, and the fiber was exposed to the air in the funnel during 3 0 min. Figure 2 shows a typical chromatogram obtained after head-space measurement of red ripe fruit of strawberry grown in 3 5 the greenhouse. The importance of esters to the overall «' WO 00/32789 PCT/NL99/00737 49 volatile emission from the ripe strawberry fruit can be clearly observed.

Example 2: Construction of a strawberry red fruit stage and lemon young fruit peel cDNA library, mass excision and 5 random sequencing 2.1 Messenger RNA isolation and cDNA libraries construction Total RNA was isolated from strawberry fruit red stage of development using the method described by Manning K. [Analytical Biochemistry 195, 45-50 (1991)] . The cultivar used was 10 Fragaria X ananassa Duch. cv. Elsanta. Plant material for the isolation of RNA from lemon (citrus Limon cv. Meyer) was the green peel of a small fruit (1.2 x 2 cm) of the cultivar. Isolation of RNA from lemon was done using the same method as described for strawberry. The cDNA libraries for 15 both strawberry and lemon where produced as a custom service by (Stratagene) in the lambda zap vector. Messenger RNA was isolated from total RNA using the polyA+ isolation kit (Pharmacia). 2.2 Mass excision and random sequencing The ExAssist™/SOLR™ system (Stratagene) was used for mass excision of the pBluescript SK(-) phagemid. The excision was done according to the manufacturer's instructions using 20 x 103 pfu from the non amplified library for each excision. High quality plasmid DNA from randomly picked colonies was 25 extracted using the QIAGEN BioROBOT 9600. Colonies were grown overnight in 3ml Luria Broth medium (10g/l tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) supplemented with 100 mg/litre Ampicillin, centrifuged at 3000 RPM for 10 min. and the pellet was used directly for plasmid DNA isolation by 30 the robot. Each DNA isolation round consisted of 96 cultures .

Insert size was estimated by agarose gel electrophoresis after restriction enzyme digestion of the pBluescript (SK-) vector with EcoRI and Xhol. Inserts with length 50 above 500 bp were used for sequencing. Plasmid DNA from the selected samples were used for polymerase chain reaction (PCR) sequencing reactions using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit and the MJ Research 5 PTC-200 DNA Engine™ thermal cycler. The T3 and T7 universal primers were used for sequencing from the 5' and 3' ends respectively. PCR program was according to the Dye Terminator manufacture's protocol (ABI PRISM). The ABI 373, 370A and 310 sequencers (Applied Bio-systems) were used. Sequen-10 ces were edited manually to remove vector and non reliable sequences and submitted to the Blast homology search program using BlastX and BlastN (Altschul et al. J. Mol. Biol. 215, 403 - 410, 1990) provided by the National Centre for Bio-technological Information on the world wide web. The straw-15 berry and lemon sequencing data received from the Blast search was transferred using a self designed macro program to a database made using the software Access (Microsoft). Sequences with a BlastX score above 80 were considered as having a significant homology (Pearson, Curr. Opinion. 20 Struct. Biol, 1, 321 - 326, 1991) and they were used for data interpretation in the Access and Excel programmes (Microsoft). The cDNA clones: SLE2 7 (SEQ ID NO: 1A), CLF26 (SEQ ID NO: 2A), SLF96 (SEQ ID NO: 3A), SLG150 (SEQ ID NO: 4A) , SLH51 (SEQ ID NO: 5A) , SLB39 (SEQ ID NO: 6A) , SLF193 25 (SEQ ID NO: 7A), SLF122 (SEQ ID NO: 8A), SLD194 (SEQ ID NO: 9A), SLF17 (SEQ ID NO: 10A), SLF138 (SEQ ID NO: 11A), SLG16 (SEQ ID NO: 12A), SLG144 (SEQ ID NO:13A) were selected for further analysis.

Example 3: Characterisation of selected cDNAs 3.1 Sequencing the entire length cDNAs Plasmid DNA from the selected cDNAs was used for PCR sequencing reactions using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit and the MJ Research PTC-200 DNA Engine™ thermal cycler. Each individual selected cDNA 35 was sequenced completely on both strands. DNA and protein 51 alignment *contigs' were performed using the GeneWorks software {IntelliGenetics, Oxford). 3.2. RNA isolation from different tissues and organs of 5 strawberry and lemon.

RNA isolation from both strawberry and lemon tissues was done as described for the preparation of total RNA for the construction of the cDNA libraries [Manning K. Analytical Biochemistry 195, 45-50 (1991)]. For both species green 10 house grown plant material was used. From strawberry the different tissues/organs used for RNA isolations were; leaf, root, petiole, flower, green fruit, white fruit, turning fruit, red fruit, red fruit without seeds, seeds and overripe fruit. For lemon the different tissues/organs used were; 15 root, leaf, flower bud, albedo, green fruit peel (1.2 x 2 cm), green fruit peel (2x3 cm), green fruit peel (3.5 x 6 cm), green fruit peel (6x8 cm), yellow fruit peel (2 weeks fruit detached). 3.3 Expression analysis using Northern blots Ten /ig of each RNA sample was denatured by glyoxal (1.5M) prior to electrophoresis on a 1.4% agarose gel in 15mM sodium phosphate buffer pH = 6.5 (without Ethidium bromide). Equal loading of RNA was verified by visualising equal amount of RNA from each sample on a 1% TAE buffer [lx : 25 0.04M Tris-acetate / 0.001M ethylenediaminetetra-acetate (EDTA)] agarose gel prior to loading. Blotting was done using Hybond N+ membrane (Amersham) in 25 mM Sodium phosphate buffer pH = 6.5 overnight.

After fixation (2h 800c) blots were hybridised as 30 described by Angenent et al. 1992 (Plant Cell 4, 983-993). The hybridisation probes were made by random labelling oligonucleotide priming (Feinberg and Vogelstein, Anal. Biochem. 137, 266-267, 1984) of the entire cDNAs. The hybridised membranes were autoradiographed at -7 0°C with 35 intensifying screens. In Figure 3 the expression pattern of PCT/N L99/00737 52 strawberry alcohol acyl transferase (SLE2 7, SEQ ID NO: 1A) is demonstrated by a Northern blot loaded with total RNA extracted from 10 different plant tissues and/or organs from strawberry. The expression pattern of strawberry alcohol 5 acyl transferase is in perfect match with the accumulation of volatile esters in the strawberry plant. The gene SLE2 7, (SEQ ID NO: 1A) is expressed at it lowest level in the white fruit, gradually increasing as fruit develops reaching its highest levels in the turning stage and the red stage when 10 the volatile ester compounds can be detected at their maximum. In other strawberry plant tissues/organs which do not produce those volatile fruity esters the expression of the gene can not be detected even when the film is exposed to a longer period after hybridisation. 15 The expression of the lemon alcohol acyl transfe rase (CLF26 SEQ ID NO:2A) seems to be correlated with the accumulation of volatile monoterpene compounds in the lemon tree tissues/organs (Figure 4) . These compounds can be converted by this enzyme to their ester derivatives as a 20 result of alcohol acyl transferase enzyme activity. The gene is more highly expressed in the flower bud. Analysis by Gas chromatograph (GCMS) shows high concentrations of terpene compounds produced in the flower bud such as limonene. Expression analysis of montoterpene cyclases (genes directly 25 responsible for the formation of monoterpenes) isolated from lemon thus shows a similar pattern of expression to the lemon alcohol acyl transferase (CLF26, SEQ ID NO:2A). Expression of the gene was also strongly detected in the early stages of fruit development and in the flower bud 30 whilst no expression could be detected in the root and albedo tissues. This data provides evidence for a link between the presence of terpene compounds mainly monoterpenes and the lemon alcohol acyl transferase (CLF26, SEQ ID NO:2A).

The expression of other three genes part of the metabolic pathway described in this invention for the formation of volatile ester compounds, in particular aliphatic esters in fruits is shown in Figure 5. Thiolase (SLG150, 53 SEQ ID NO: 4A) alcohol dehydrogenase (SLB3 9, SEQ ID NO: 6A) and pyruvate decarboxylase (SLH51, SEQ ID NO: 5A) gene expression was monitored at different organs or tissues of the strawberry plant. The data shown provides evidence that 5 thiolase (SLG150, SEQ ID NO: 4A) , alcohol dehydrogenase (SLB3 9, SEQ ID NO: 6A) and pyruvate decarboxylase (SLH51, SEQ ID NO: 5A) are involved in ripening processes such as the production of strawberry flavour and aroma compounds. Clones SLF193 (SEQ ID NO: 7A) , SLF122 (SEQ ID NO: 8A) , 10 SLD194 (SEQ ID NO: 9A), SLF17 (SEQ ID NO: 10A), SLF138 (SEQ ID NO: 11A), SLG16 (SEQ ID NO: 12A), SLG144 (SEQ ID NO:13A) and SLF96 (SEQ ID NO: 3A) were also tested for the expression and they were all appearing to have upregulated expression in the ripening fruit (data not shown). 3.4 Co-ordinate expression of genes in the biosynthetic pathway leading to aromatic and aliphatic ester formation The co-ordinate expression pattern of genes of the biosynthetic pathway leading to aromatic and aliphatic ester formation was simultaneously monitored using arrays of 20 cDNAs [Ermolaeva et al. , 1998, Nature Genetics, Vol, (20), 19-23]. The method allows one to simultaneous quantitatively and statistically analyse the expression profiles of genes pertaining to a particular metabolic pathway, such as pathway leading to aliphatic and aromatic ester formation, 25 flavour, fragrance, aroma and scent. The experimental and hybridisation conditions followed were according to Schena et al., (PNAS, 93: 10614 - 10619) . The expression levels derived from four images were statistically analysed with an analysis of variance modelling the factors dye, origin, and 30 spot levels on a logarithmic scale giving the significant ratios of expression between the spots of origin (eg. green versus red). As way of illustration the expression profiles Of SEQ ID 1A, SEQ ID 3A, SEQ ID 5A, SEQ ID 11A, SEQ ID 12A, analysed in the green versus red and turning versus red 35 stages of fruit development are outlined in Figure 10. The co-ordinated expression pattern of the genes during straw 54 berry fruit development is in perfect match with the accumulation of volatile ester compounds aromatic and aliphatic during strawberry fruit development. In addition alcohol acyl transferase enzyme activity was detected by Perez et al., (J. Agric. Food Chem. 1996, 44, 3286-3290) to be elevated dramatically between the white and the turning stages of strawberry fruit development. This correlates well with a dramatic increase in expression of the genes of the biosynthetic pathway as shown in Figure 10. A supporting detail is the fact that volatile ester compounds can be detected in their maximum when the fruit is completely red and this is probably a result of gene activation at earlier stages between the white and turning stage of development. 3.5 DNA isolation from strawberry leaf tissue.

Genomic DNA was isolated from green house grown strawberry plants (cv. Elsanta) . Young folded leaves kept 2 days in water in the dark were used for the isolation. 1 gram of leaf material was ground in liquid nitrogen and immediately added to 7 ml DNA extraction buffer (100 mM Tris-Cl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 0.2% B- mercaptoethanol) . After incubation for 30 min. at 600C one volume of chloroform:iso-amyl alcohol (24:1) was added to the tubes. The samples were then centrifuged for 10 min. at 4000 rpm and the aqueous phases were transferred to 4.5 ml isopropanol and incubated for 30 min. at -20°C. Nucleic acids were spun down at 4000 rpm for 12 min. washed with 70% ethanol and air dried. Pellets were dissolved in 3 ml TE and 0.25 volumes of NaCl (5M) and 0.1 volume absolute ethanol were added. Samples were centrifuged for 10 min at 4000 rpm and supernatants were transferred to 4.5 ml isopropanol for precipitation. Pellets were washed with 70% ethanol dried and dissolved in 0.5 ml TE buffer (lOmM Tris-Cl, ImM EDTA pH = 8.0). RNA was selectively precipitated by the addition of 0.6 volumes of 8M lithium chloride. The RNA was pelleted by centrifugation 10 min. 4000 rpm and the DNA was precipitated again with the addition of 0.6 volume isopropanol. Pellets were washed with 55 70 % ethanol, dried and dissolved in 0.5 ml sterile water. The amount of DNA was determined by comparing to known amounts of phage lambda DNA. 3.6 Southern blot analysis for strawberry alcohol acyl transferase (SLE27, SEQ ID N0:1A) Aliquots of 2 fig strawberry genomic DNA were digested for 16h with the restriction enzymes: Hindlll, Xbal, Xhol and EcoRI and separated on a 0.7% TAE (0.04M Tris-acetate pH = 8.0 and ImM EDTA pH = 8.0) agarose gel. The DNA was then denatured in 0.4M NaOH for 3 0 min. and then transferred to a Hybond N+ membrane in 0.4M NaOH. Blot hybridisation and probe preparation was done as described previously for Northern blots. After hybridisation the blot was washed first under low stringency conditions (52°C with 2 times half an hour 2xSSC/0.1% SDS) and after observation of the results by autoradiography of the film they were washed further under stringent conditions (65°C with 2 times half an hour 0.1xSSC/0.1% SDS) and autoradiographed again.

In order to investigate whether the different esters formed in the fruit are a result of an activity of one single enzyme or a multigene family, a Southern blot was performed using strawberry genomic DNA and the full length SLE27 cDNA (SEQ ID N0:1A) which served as a probe (Figure 6) . Since the cultivar used for the investigation is an octaploid the interpretation of the results is more difficult than that for a diploid plant. However, the results obtained suggest the possible existence of another homolog of strawberry alcohol acyl transferase. This additional copy might arise from one of the parental lines with a similar function to the one isolated, or may represent another homolog with different function or substrate specificity to SLE27 (SEQ ID NO: 1A). 3.7 Alcohol acyltransferase sequences from other fruit. 56 Additional alcohol acyltransferase sequences from melon, strawberry vesca, banana, apple, lemon and mango were obtained using degenerate primers synthesised to the motif sequence ID number 14B using methods well known in the art such as polymerase chain reaction (PCR) and reverse genetic approaches such as RT-PCR. Such techniques are described in Sambrook et al., 1989. Molecular cloning a laboratory manual, cold spring Harbour press, Plain view, NY and Ausubel FM et al., (1989) Current protocols in molecular biology, John Wiley and Sons, New York, NY. The esterase gene was cloned using PCR techniques using the following degenerate primer : 5'-GG (T/A) TGGGG I (T/G) CTA(T/C) TCTTGC-3' ) and reverse genetic approaches such as RT-PCR. Such techniques are described in Sambrook et al., 198 9. Molecular cloning a laboratory manual, cold spring Harbour press, Plain view, NY and Ausubel FM et al., (1989) Current protocols in molecular biology, John Wiley and Sons, New York, NY.

Example 4 Strawberry alcohol acyl transferase (SLE27, SEQ ID NO:IB) protein analysis 4.1 Cloning to the expression vector pRSET B The E.coli expression vector pRSETB (Invitrogen), was used for expression studies of strawberry alcohol acyl transferase (SLE2 7, SEQ ID NO: 1A). This pRSETB vector contains the T7 promoter which can be induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and therefore by inserting the desired gene downstream of this promoter, the gene can be expressed in E. coli. In addition, DNA inserts were positioned downstream and in frame with a sequence that encodes an N-terminal fusion peptide. This sequence includes (in 5' to 3' order from the N-terminal to C-terminal), an ATG translation initiation codon, a series of six Histidine residues that function as a metal binding domain in the translated 57 protein, the Anti-Xpress epitope, and the enterokinase cleavage recognition sequence.

The original pRSETB was primarily used for the insertion of the gene encoding the Green Fluorescent Protein 5 (GFP). The GFP gene was fused to the pRSETB vector using the BamHI and Hindlll restriction sites located at the multiple cloning site (MCS) as can be seen in Figure 7. This construct for the expression of GFP served as control for the experiments together with the empty pRSETB vector. 10 Cloning the GFP gene to the pRSETB vector inserted an additional Sail restriction site at the 3' of the GFP gene and together with the BamHI site located at the 5' of the GFP gene served as sites for the cloning of the strawberry alcohol acyl transferase gene (SLE2 7, SEQ ID N0:1A). 15 The BamHI and Sail sites were introduced to the 5' and 3' respectively of the strawberry alcohol acyl transferase coding sequence by the use of PCR. The 4 52 amino acid open reading frame of the SLE27 (SEQ ID N0:1A) clone was amplified with the pfu DNA polymerase (Stratagene) and primers 20 AAP165 (5'-CGGATCCGGAGAAAATTGAGGTCAG) and AAP166 (5'-CGTCGA-CCATTGCACGAGCCACATAATC) according to the manufacturers instructions. The PCR product was cloned into PCR-script vector (Stratagene) , cut out with BamHI and Sail and further inserted (as a translation fusion) into the corresponding 25 restriction sites in the pRSETB vector. 4.2 Bacterial expression and partial purification using the His tag columns.

The pRSET B vector harbouring the strawberry alcohol acyl transferase (SLE27, SEQ ID NO: 1A) was used to transform E. 30 coli strain BL21 Gold DE3 (Stratagene) as described by the manufacturer. For bacterial expression typically 1 ml of overnight liquid culture grown at 3 7°C in Luria Broth (LB) medium (10g/l tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) supplemented with 100 mg/liter Ampicillin was diluted 35 50 times in the same medium and grown until the OD600 reached 0.4 (usually at 37°C except when different temperatures 58 for growth during induction were examined). At this stage Isopropylthio-beta-D-galactoside (IPTG) was added to a final concentration of ImM in order to induce expression . After one and a half hour of induction the cells were harvested by centrifugation at 4000 x g for 15 min. Pellet and a sample from the supernatant were kept for SDS gel analysis. The cells were further processed as described by the Ni-NTA Spin Columns manufacturers (QIAGEN) for protein purification under native conditions. 4.3. Recombinant protein detection using Western blot The supernatant (S) , pellet (P) and elute from the Ni-NTA columns (E) samples were diluted with 2x SDS-sample buffer [(standard Laemmli, pH 6.8 Tris /glycine/SDS/glycerol + 50mM Dithiothreitol(DTT)]. The samples were heated for 5-10 min at 95°C, followed by a short spin of 30 seconds, full speed. From the samples 10/zl was loaded on 10% SDS-gel (mini-gel, 1mm, 7x9cm). The gels were run with standard buffer (25mM Tris 192mM, glycine 0.1% SDS, pH 8.3) at 100-200V, 50-30mA, ca 3hr with 16°C water cooling. After complete run, gels were used for Coomassie Brilliant Blue (CBB) stain or Western blot. For CBB staining, the gel was fixed during lhour in 40% Methanol, 10% Acetic Acid in water. Then incubated overnight with CBB staining solution (0.05% CBB-250 in 40% Methanol, 10% Acetic acid in water) and destained by repeated washing in 25% Methanol, 10% Acetic acid in water. Western blot was onto Polyvinyl di fluoride (PVDF), (Immobilon P, Amersham) pre-wetted with Methanol. Transfer buffer was Towbin buffer plus 0.1% SDS (25mM Tris, 192mM Glycine, 20% 0.1% SDS, pH 8.3). Transfer was submarine (Novex blot unit, plate electrodes, 3cm distance) at 200mA overnight plus 2hr. at 4 00mA, cooled in ice-water with cooling block. Transfer was checked by using pre-stained markers as a reference.

After transfer the blot was washed in Tris buffer saline TBS (20mM Tris-HCl pH7.5 / 150mM NaCl)- Tween20 (0.02%), blocked with 2% fat free milk in TBS-Tween20, then 59 washed with TBS-Tween 20. Then first Antibody was anti-Xpress (Invitrogen) 1/1000 in 2% BSA TBS-Tween for 3hr. incubate at room temprature, then 4x washed with TBS-Tween 20. Second Antibody is GAR-AP (Boehringer) 1/3000 in 2% BSA 5 TBS-Tw (TBS + 0.05% Tween - 20) for l-2hr. incubation at room temprature, then 4x washed with TBS-Tween 20. Final wash was in water, then in Alkaline phosphatase-buffer (lOOmM Tris pH9.2, ImM MgCl2) . The staining reaction was with nitroblue tetrazolium/5-bromo-4-chloro-3-indolphospha-10 te/alkaline phosphatase-buffer (45/xl nitroblue tetrazolium 30mg/ml in 70% dimethylformamide, 33/zl 5-bromo-4-chloro-3-indol phosphate 15mg/ml in dimethylformamide in 10ml of alkaline phosphatase buffer). The staining reaction was stopped by rinsing with water. Western blotting was used in 15 order to determine the amount of the recombinant SLE2 7 (SEQ ID NO: IB) protein in the elute after the Ni-NTA column purification compared to the pellet and supernatant before loading on the column. Figure 9 shows a western analysis of the pellet supernatant and elute after partial purification 2 0 on the Ni-NTA column. 4 samples were loaded from each pellet, supernatant and elute originating from 4 different growth and induction of the E. coli cells. A commercial antibody recognising an epitope peptide fused to the N-terminus of the SLE27 recombinant protein showed relatively 25 high expression of SLE27 (SEQ ID NO: IB) in the elute from the Ni-NTA column. The molecular weight of the recombinant SLE27 (SEQ ID NO:IB) protein (SAAT) recombinant protein was 54 kDa. The enzyme exhibited a broad pH range (pH optimum around 8.3) . As a control for the experiment served the 30 Green Fluorescent Protein (GFP) partially purified in the same method as done for SLE27 (SEQ ID NO: IB) . No band was detectable at the level of the SLE27 (SEQ ID NO:IB) recombinant protein in the GFP lane. 4.4 Method for establishing alcohol acyl transferase activity of the strawberry SLE27 (SEQ ID NO:IB)-encoded protein. 60 The alcohol acyl transferase activity of the strawberry cDNA SLE2 7 (SEQ ID N0:1A) encoded protein was established by analysing the volatile esters produced from acyl-CoA and different alcohols. In a 1.8 ml glass vial with a small magnetic stirring bar, 325 fil of buffer (50 mM Tris/HCl pH 8.0, containing 1 mM dithiothreitol) , 25 jil of protein extract and 25 (al of acyl-CoA (4 mM in buffer) were subsequently pipetted. The enzyme reaction was started by the addition of 25 ^1 of alcohol solution (160 mM in buffer). The vial was quickly closed with a snap cap and placed in a waterbath (at 35°C) with a stirring device. After 15 min enzyme reaction with continuous stirring, the vial was opened, 0.29 g of solid CaCl2 was added (final concentration 5M) and the vial was quickly closed again with an open top crimp cap. The high CaCl2 concentration was used to stop further enzymatic reactions and to drive the volatiles into the headspace. After addition of CaCl2, the mixture was incubated again at 35°C while stirring. The volatile components released into the headspace were trapped using solid phase micro-extraction (SPME; Supelco Inc., Bellefonte, PA, USA) , exposing the fused silica fiber coated with 100 lm polydimethylsiloxane (PDMS; Supelco Inc.) to the headspace for 15 min. The presence of esters in the alcohol stock solutions and the spontaneous formation of esters were checked by adding 25 |al of buffer instead of protein extract into the reaction vial.

The volatiles trapped by SPME were separated and identified by gas chromatography (GC 8000 Series; Inters-cience B.V., Breda, The Netherlands) coupled to mass spectrometry (MD-800, Fisons Instruments, Interscience) as described by Verhoeven et al. (1997) [Chromatographia Vol. 46, No 1,2]. Compounds were thermally desorbed (2500C) from the SPME-fiber for 1 min. Separation of compounds was on a capillary HP-5 column (50 m x 0.32 mm, film thickness 1.05 £tm; Hewlett Packard) with He (3 7 kPa) as a carrier gas. The oven temperature was programmed at 2 min at 80°C, then an increase of 8°C min"1 to 250°C, and finally 5 min at 250°C. 61 Mass spectra of eluting compounds were generated at 70eV each second. The MD 800 Masslab software (Interscience) was used to record mass spectra and to calculate peak areas. Volatile compounds were identified by screening the NIST 5 library for comparable mass spectra and by comparison with authentic reference compounds. 4.5.Substrates used by the strawberry alcohol acyl transfe-rase-encoded enzyme (SLE 27: SEQ ID NO: IB) The histidine-released protein elute from E. coli expressing 10 the strawberry alcohol acyl transferase gene (SLE27, SEQ ID NO:1A) was incubated with both acetyl-CoA and 1-butanol. The chromatograms obtained by SPME of volatiles released into the headspace indicated a clear peak of acetic acid 1- butyl ester (Figure 8B) . This ester was not detected when the 15 protein (Figure 8C) , alcohol substrate (Figure 8D) and acetyl CoA were absent (Figure 8E). Moreover, when the SLE27 (SEQ ID NO:IB)-encoded protein elute in the assay was replaced by an equal amount of histidine-released elute from lysate of E. coli expressing only the histidine-tag (i.e. 20 the same vector without the SLE27 (SEQ ID NO.-1A gene), the ester could not be detected either (Figure 8G). To investigate which substrates can be used by the SLE2 7 (SEQ ID NO:IB)-encoded protein to produce volatile esters with acetyl-CoA, the alcohol species in the assay was varied. 25 Table 1 shows all substrates tested and the species that can be used by the SLE27-encoded enzyme (SEQ ID NO: IB) . Extensive in vitro enzyme assays using both GC-MS and radio-GC revealed that a comprehensive array of acyl-CoAs and alcohols (short and long chain with even and uneven carbon 3 0 number, aromatic, cyclic and aliphatic, branched and unbran-ched, saturated and unsaturated) can serve as substrates for the SLE27-encoded enzyme (SEQ ID NO:IB) enzyme in vivo (Table 1 & 2).

The alcohol acyltransferase activity of the the SLE27-35 encoded enzyme (SEQ ID NO:IB: SAAT) encoded protein was established in two ways: (1) For the activity with different WO 00/32789 PCT/NL99/00737 62 alcohols and acetyl-CoA, [i4C] -acetyl-CoA was used as substrate and the formation of (radio-labeled) products analyzed using radio-gas chromatography and quantified using liquid-scintillation counting. Radio-GLC was performed 5 essentially as described previously [Bouwmeester, H.J. (1999) Plant Physiol. 121: 173-180 (1999)] Temperature programming was as follows: 70 °C for 10 min, 10 min"1 to 270 °C and a final time of 10 min. For quantification of the enzyme activity, 1.4 /xg SAAT protein was diluted to 100 /x 1 10 with 50 mM Tris-buffer (pH 8.3, to which [14C]-acetyl-CoA (0.1 mM at 0.1 Ci.mol"1 in routine assays) and alcohol (20 mM in routine assays, 2 /xl of hexane stock.) were added. After incubation for 30 min at 30 °C, the assays were cooled on ice for 15 min and 7 00 /x 1 of hexane were added. Assays were 15 vortexed, centrifuged and a 600-/xl portion of the hexane phase was removed for liquid scintillation counting. The ratio of hexane soluble radioactivity (esters) to the total radioactivity added as acetyl-CoA was used to calculate product formation. (2) For the utility of different acyl-20 CoAs, assays containing 325 /xl of buffer (50 mM Tris/HCl pH 8.0, containing 1 mM dithiothreitol) , 25 /xl of protein elute and 25 /xl of acyl-CoA (4 mM in buffer) were stirred in a glass vial. The enzyme reaction was started by the addition of 25 /xl of alcohol (160 mM in buffer) . After 15-min incuba-25 tion with continuous stirring (35 °C) , solid CaCl2 was added (final concentration 5 M). Volatiles released into the vial headspace (at 35 °C with stirring) were subsequently trapped for 15 min by exposing a fused silica fiber coated with 100 /xm polydimethylsiloxane (PDMS) to the headspace for 30 min. 30 The SPME-trapped volatiles were analyzed by GC-MS, as described previously [Verhoeven et al. , (1997). Chromato-graphia 46, 63-66 ] .

Table 1. Substrate specificity of the SLE27 (SAAT), SEQ ID NO: IB recombinant enzyme towards different types of alco-35 hols. Comparison of esterification activity with different alcohols (20 mM) , and using [14C] acetyl-CoA (0.1 mM) as acyl donor. Activity ( mean + SD, n = 2) is expressed as nmol 63 product formed per hour per fj.g enzyme. a- the ester product was not reported to occur in commercial strawberry varieties (data from Honkanen and Hirvi. 1990). b no detectable activity, n.d. - not described. Ester properties are from 5 Arctander, (1969).

Table 1 Alcohol Carbon no.

Activity Ester formed Ester properties methanol C1:0 0.43 ± 0.10 methyl acetate ethereal, sweet ethanol C2:0 0.41 ± 0.07 ethyl acetate pineapple, ethereal 1 -propanol C3:0 2.60 + 0.26 1 -propyl acetate powerful celery odor 2-propanol C3:0 1.30 ± 0.15 2-propyl acetate ethereal, sweet, banana, fruity 1-butanol C4:0 2.29 ± 0.25 1 -butyl acetate fruity, diffusive 2-butanol C4:0 3.11 + 0.02 2-butyl acetate n.d. 3-methyl-! -butanol C5:0 3.68 ± 0.25 3-methyl-l-butyl acetate fruity, banana, sweet (Isoamylalcohol) (isoamyl acetate) 1-hexanol C6:0 8.44 + 0.37 1-hexyl acetate apple, cherry, pear, floral c/s-2-hexen-l -ol C6:l 6.05 + 0.55 cis-2-hexenyl acetate" n.d. c/s-3-hexen-l-ol C6:l 4.06 ± 0.14 ris-3-hexenyl acetate green, sharp-fruity lrorts-2-hexen-l -ol C6:l 9.20 ± 0.65 rranj-2-hexenyl acetate fresh-green, sweet, fruity rrans-3-hexen-1 -ol C6:l 1.25 + 0.07 /ra/is-3-hexenyl acetate" n.d. 1-heptanol C7:0 14.89 ± 4.12 heptyl acetate* sweet, apricot like 1-octanol C8:0 16.36 ± 2.69 1-octyl acetate fruity, floral, jasmine, herbaceous 1-nonanol C9:0 14.00 + 0.11 1-nonyl acetate* floral, fruity (mushroom-gardenia) 1-decanol C10:0 7.79 + 0.10 1 -decyl acetate floral, orange-rose furfurylalcohol C5:0 0.72 + 0.06 furfuryl acetate" fruity, banana benzylalcohol C7:0 0.68 + 0.04 benzyl acetate sweet, floral, fruity, fresh 2-phenylethylalcohol C8:0 1.58 ± 0.12 2-phenylethyl acetate floral, rose, honey like undertone, sweet, fruity linalool C10:0 b linaiyl acetate" floral-woody odor with faint citrus note Tabel 2.

Acceptance of different types of acyl-CoAs as co-substrates by the SLE27 (SAAT), SEQ ID NO: IB recombinant enzyme. 10 Esters formed after 15 min reaction of SAAT protein with the substrates indicated were trapped using headspace-SPME and GC-MS. a- the ester product was not reported to occur in commercial strawberries [data from Honkanen and Hirvi. 1990 .Dev. Food Sci. Amsterdam : Elsevier Scientific Publications, 15 125-193.]. n.d.- not described. Ester properties are from Arctander, S. Perfume and Flavor Chemicals (Steffen Arctander' s Publications, Las Vegas, NV, (1969).

PCT/N L99/00737 Tabel 2: AcylCoAs (Carbon no.) Alcohol Ester formed Ester properties n-propionyl CoA (C3:0) 1-butanol 1-butyl propanoate* ethereal, banana n-butyryl CoA (C4:0) 1-propanol 1-propyl butyrate sharp, pungent, rancid, sweaty, sickening n-butyryl CoA (C4:0) 1-butanol 1-butyl butyrate fruity, pineapple 2-butyryl CoA (C4:0) 1-butanol 1-butyl- 2 -butyrate" fruity, apple, banana and pineapple n-crotonoyl CoA (C4:l) 1-butanol 1-butyl crotoate* n.d. n-hexanoyl CoA (C6:0) 1-propanol 1-propyl hexanoate" wine-like, cheese n-decanoyl CoA (C10:0) 1-butanol 1-butyl decanoate" Brandy (Whisky- Cognac) -like odor benzoyl CoA (C7:0) 1-butanol 1-butyl benzoate" mild floral-balsamic odor Radio-GC analysis after incubations of the SLE27 (SAAT), SEQ ID NO: IB recombinant enzyme with [14C] -acetyl-CoA and either octanol or hexanol showed the production of 14C-labeled octylacetate and 14C-labeled hexylacetate, respectively (Figure 11). The kinetic properties of SAAT were determined using [14C] -acetyl-CoA and five different alcohols. The apparent Km and Vmax for acetyl-CoA were 104.2 /iM and 44.5 nmol product .h"1./ig1 protein, respectively. The apparent values of Km (in mM) and Vmax (in nmol product . h"1. fig1 protein) for octanol were 5.7 and 26.0, for hexanol 8.9 and 6.1, for butanol 4 6.1 and 4.2, for trans-2-hexenol 16.8 and 10.5 and for cis-2-hexenol 17.9 and 6.0, respectively. Activity was also detected with aromatic (benzyl- and phenylethyl-) and cyclic (furfuryl-) alcohols though activities were low compared to 1-octanol (4 to 10%). In contrast, no activity could be detected with the terpene alcohol linalool.

Example 5 Verification of alcohol acyl transferase activity of the proteins from melon, strawberry vesca, banana, apple, mango and lemon.

The alcohol acyl transferase activity of the proteins having the sequences SEQ ID NO:16B to 23B, derived from strawberry vesca, banana, apple, mango, lemon (3x) and melon, respectively, was established in a similar way as described for PCT/N L99/00737 strawberry protein encoded by SEQ ID N0:1A in Example 4. From an overnight culture of E.coli, 0.75 ml was transferred into a 2 ml glass crimp vial, 0.3 g CaCl2 was added and a small magnetic stirrer was put into the vial. The vial was closed with a crimp cap, and incubated during 15 min at 50 deg. celsius. The headspace of the vial was sampled with a 100 micro PDMS solid phase microextraction (SPME) device, supplied by Supelco, during this period. The SPME fibre was transferred into the injection port of a Fisons G8600 gaschromatograph, and desorbed at 250 deg. Celsius during 10 seconds. Bottom split was closed during this period of 10 seconds, and reopened afterwards. A HP-5 column was used with 50m length, 0.32 mm ID and 1.05 micron film thickness. The oven was programmed for 2 min. at 80 deg. Celsius, ramp of 8 deg Celsius /min. to 250 deg. Celsius, for 5 min. at a head pressure of 47 kPa. A MD 800 mass spectrometer was used for the detection and identification of volatile esters, and the resulting mass spectra from M/z 35 to 400 were compared with the NIST database for identification. Where possible, retention times were compared to that of authentic reference compounds, obtained from Sigma or Aldrich.

The chromatograms obtained are shown in Fig. 12-18, of which Fig. 12 shows the results of using SEQ ID N0:1A for comparison. The data obtained by the chromatograms are summarized in the following tables 3 to 9, respectively.

Table 3.

Shows ester formation (A) E. coli cell cultures expressing the strawberry alcohol acyl transferase (SLE2 7, SEQ ID NO: 1A) . (B) E. coli cell cultures expressing the empty vector cassette. Cultures supplemented with 1-butanol (Fig. 12) .

Peak number Retention time Ester formed 1 6 . 797 butanoic acid, isopropyl ester 2 7 .027 acetic acid, butyl ester 3 9.027 propionic acid, butyl ester 4 . 048 ester, not identified 11. 028 butanoic acid, butyl ester 8 .368 hexanoic acid, butyl ester 7 .068 acetic acid, benzyl ester 6 13.248 pentanoic acid, butyl ester 9 17.278 butanoic acid, phenylmethyl ester Table 4.

Shows ester formation (A) E. coli cell cultures expressing the melon alcohol acyl transferase (MAY5, SEQ ID NO:23A)) . (B) E. coli cell cultures expressing the empty vector 15 cassette. No alcohol supplements (Fig. 13).

Peak number Retention time Kster Formed 1 6.267 acetic acid, isobutyl ester 2 7 . 047 acetic acid, butyl ester 3 8 .557 unknown ester 4 9.037 propionic acid, butyl ester 9.167 acetic acid, pentyl ester 6 .048 unknown ester 7 .438 acetic acid, 2-ethyl butyl ester 8 11.038 butanoic acid, butyl ester 9 11.408 acetic acid, hexyl ester .078 acetic acid, benzyl ester 11 .378 hexanoic acid, butyl ester 12 17.048 acetic acid, phenyl ethyl ester 13 17.088 formic acic, unknown ester 14 17.298 butanoic acid, phenyl ethyl ester Table 5.

Shows ester formation (A) E. coli cell cultures expressing the apple alcohol acyl transferase (MAY3, SEQ ID NO:18A).

(B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with isoamylalcohol, n-butanol, cis-2-hexen-l-ol and isopropanol(Fig. 14).

Peak number Retention time Ester formed 1 2 11.038 17.089 butanoic acid, butyl ester formic acid, unknown ester Table 6.

Shows ester formation (A) E. coli cell cultures expressing the citrus alcohol acyl transferase (CLF2 6: SEQ ID NO:2A). 10 (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with 1-butanol (Fig. 15) .

Peak number Retention time Ester formed 1 7 . 047 acetic acid, butyl ester 2 .086 propanoic acid, 2 methyl, butyl ester Table 7.

Shows ester formation (A) E. coli cell cultures expressing the banana alcohol acyl transferase (MAY2, SEQ ID NO:17A). (B) E. coli cell cultures expressing the empty vector 2 0 cassette. No alcohol supplements (Fig. 16).

Peak number Retention time Ester formed 1 17 . 089 formic acid, unknown ester Table 8.

Shows ester formation (A) E. coli cell cultures expressing 25 the apple alcohol acyl transferase (MAY3, SEQ ID NO:18A). (B) E. coli cell cultures expressing the empty vector cassette. Both cultures supplemented with isoamylalcohol, n-butanol, cis-2-hexen-l-ol and isopropanol (Fig. 17).

Peak number Retention time Ester formed 1 2 11.268 15.268 acetic acid, 1,2-hexenyl ester butanoic acid, 1, 2-hexenyl ester Table 9.

Shows ester formation (A) E. coli cell cultures expressing the strawberry vesca alcohol acyl transferase (SUN1, SEQ ID NO:16A (B) E. coli cell cultures expressing the empty vector 10 cassette. Both cultures supplemented with isoamylalcohol, n-butanol, cis-2-hexen-l-ol and isopropanol (Fig. 18).

Peak number Retention time Ester formed 1 6.977 acetic acid, butyl ester 2 8.287 acetic acid, iso-amyl ester 3 11.278 acetic acid, 2 hexenyl ester Sequence listing INFORMATION FOR SEQ ID NO: 1A (SLE27) WO 00/32789 PCT/NL99/00737 69 "SEQUENCE CHARACTERISTICS: LENGTH 1632 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 1A ACCT ACTTT GCC AAAAT GGAG AAAATTGAGGT C AGT AT AAATTCC AAAC A-C ACC ATC AAACC ATC AACTT CCT CT AC ACC ACTT C AGCCTT AC AAGCTT ACC-CTCCTGGACCAGCTCACTCCTCCGGCGTATGTCCCCATCGTGTTCTTCTACC-CC ATT ACT GACC AT GACTTC AAT CTTCCTC AAACCCTAGCT GACTT AAGA-C AAGCCCTTT CGGAGACT CTC ACTTTGT ACTAT C C ACT CTCT GGAAGGGT-C AAAAAC AACCT AT AC AT CG AT GATTTT GAAGAAGGT GTCCC AT ACCTT-GAGGCTCGAGTGAATTGTGAC AT GACT GATTTT CT AAGGCTTCGG AAAAT CGAGTGCCTTAATGAGTTT GTT CC AAT AAAACC ATTT AGT AT GGAAGC A-ATATCTGATGAGCGTTACCCCTTGCTTGGAGTTCAAGTCAACGTTTTCGAT-TCTGGAATAGCAATCGGTGTCTCCGTCTCTCACAAGCTCATCGATGGAG-G AACGGC AGACT GTTTT CT C AAGT CCT GGGGTGCT GTTTTTCGAGGGT-GTCGTGAAAATATCATACATCCTAGTCTCTCTGAAGCAGCATTGCTTTTCC-CACCGAGAGATGACTTGCCTGAAAAGTATGTCGATCAGATGGAAGCGTTAT-GGTTT GCCGG AAAAAAAGTT GCT AC AAGGAGATTT GT ATTT GGT GT GAAAG-CC AT ATCTTC AATTC AAGAT GAAGCGAAGAGCGAGTCCGT GCCC AAGCC AT -CACGAGTTCATGCCGTCACTGGTTTTCTCTGGAAACATCTAATCGCTGCTTC-TCGGGCACT AAC ATC AGGT ACT ACTTCAACAAG ACTTT CT AT AGCGGCC- WO 00/32789 PCT/NL99/00737 70 C AGGC AGTGAACTT AAGAAC ACGGAT GAAC AT GG AG AC AGT GTT GGAT A-AT GCCACT GGAAACTT GTTCT GGT GGGC AC AGGCC AT ACTAGAGCT AAGT-C AT AC AAC ACC AG AG AT C AGT GAT CTT AA.GCT GT GT GACTT GGTT AACTT G-CTC AAT GGATCT GTC AAAC AAT GT AACGGT GATT ACTTT GAGACTTTC AAG-GGTAAAGAGGGAT AT GGAAGAAT GT GC G AGT ATCT AGATTTT C AGAGGAC-TATGAGTTCTATGGAACCAGCACCGGATATTTATTTATTCTCGAGCTGGAC-TAATTTTTT C AACCC ACTT GATTTT GGAT GGGGGAGGAC ATC AT GGATT G-GAGTT GC AGGAAAAATT GAAT CT GC AAGTT GC AAGTT CAT AAT ATT AGTT C-C AAC AC AAT GCGGTT CT GGAATT GAAGCGT GGGT G AAT CT AGAAGAAG A-GAAAAT GGCT ATGCT AGAAC AAGAT CCCC ATTTT CTAGCGTT AGC ATCT C-C AAAGACCTT AATTT AAAGAT ATT GATT AAGAAAGATT AT GT GGCT CGT G-C AATGTTTCGATTTT GC AGT GAAT AAGGTTT AAATT AGTT C ACC AGCC AAT -CAATAAAATGCAAGT AT GAT AGACTTTGT CTACGT AT GTT ATCCGAAT GT-GTTTCCATATGCTTGTAACCAATATAGCTCTTTATTGTAACAAATGCTCTAT-T AAGCTTCT AGCT AT AAAGTT ATTT ATCT ATT AAAAAT AAAACT AT GG AAGT-TTT ACC AAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: IB (SLE27) SEQUENCE CHARACTERISTICS: LENGTH: 452 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL ACYL TRANSFERASE WO 00/32789 PCT/NL99/00737 71_ •SEQUENCE DISCRIPTION FOR SEQ ID NO: IB MEKIEVSINSKHTIKPSTSSTPLQPYKLTLLDQLTPPAYVPIVFFYPITDHDFNL- PQTLADLRQALSETLTLYYPLSGRVKNNLYIDDFEEGVPYLEARVNCDMTDFL- RLRKIECLNEFVPIKPFSMEAISDERYPLLGVQVNVFDSGIAIGVSVSHKLIDGG- TADCFLKSWGAWRGCRENIMPSLSEAALLFPPRDDLPEKYVDQMEALWFAG- KKVATRRFVFGVKAISSIQDEAKSESVPKPSRVHAVTGFLWKHLIAASRALTS- GTTSTRLSIAAQAVNLRTRMNMETVLDNATGNLFWWAQAILELSHTTPEIS- DLKLCDLVNLLNGSVKQCNGDYFETFKGKEGYGRMCEYLDFQRTMSS- MEP APDIYLFS SWTNFFNPLDFGWGRT SWIGV AGKIES ASCKFIILVPTQCGS- GIEAWVNLEEEKMAMLEQDPHFLALASPKTLI 72 INFORMATION FOR SEQ ED NO: 2A (CLF26) SEQUENCE CHARACTERISTICS: LENGTH: 1613 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: CITRUS LIMON ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 2A CTT ATTT AAAAGTT CAT C AAC AAATT GTT CT ACC ACTT ACC ATTT CT C ATAG-CTCTGCAAGTTCGGATTTGACTCTTTCTCTTTTCCTCATTCCGGCCGGTGTT-GATAGTTAC ATTTTGGC AC AAT G AAAATTC ACGTT AAGGAGTC AAC AATT A-TACGCCCTGCTCAAGAAACACCCAAGCATCGCCTACAAATATCCGACCTA-GACAT GATTGTGCC ATCC AATT ACGTTCCC AGT GTGT ATTT CT ATCGGCG-GT CC AGTGACTGC ACCG ATTTTTTT G AAGTT GGTTT GCT G AAGAAGGCT CT-G AGCGAAGTT CTTGTGCCGTTTT AC CC C GTT GCCGGAAGGTT GC AG AAG-G AT GAAAAT CGC AAAATT GAG ATT CT ATGT AACGGAGAGGG AGTTTT GTT-T CT GGAGGCCGAAAC AAGTT GT GGT ATT GAT GATTTCGGT G ACTT CT C A-C AAGGCTCGAAACT CCT GACGCTT GTTCC AACT GTT GGT GAT AC AAAGGA-TATATCCTCCCATCCACTCTTGATGGCACAGGTAACTTATTTCAAATGTG-GAGGCGTTT GT GTT GGAACT AG AGT GAATC AT AC ACT GGT AG AT GG AGCTT-CAGCGTACCATATCATCAACTCATGGGCGGAGACGACGCGTGGCGTTCC-TATTAGC ACTCAACC ATTCT AT GAT CGGACC AT ACT GAGT GTT GGGGTTC-CAACTTCTCCCAAATTCCATCACATTGAATATGACCCGCCTCCTTCCAT- WO 00/32789 PCT/NL99/00737 73 GAACGCTCCTCCTACCCAAAATCCTGAAATCATTTCTACCGCAATCCTTAAC-CT ATC ACTT GATC AAATCC AC ACCCT C AAAGAGAAATCT AAG AC AGATC AT -GAACCC AACGT C AAGT AT AGT AGGAT GGCGATCCT AGC AGC AC AT ATCT GG-CGT AGC AT GT GT AAAGCGCGCGGATTATCTGAT GATC AAGTT AGC AAGTT A-CACTTTCCTACAGACGGACGACAGAGATTGAATCCACCACTCCCGCCTGGA-TATTTT GGAAATGT AATTTTC ACC ACGTCGTT GACGGCTT CAT CGGGT GA-T ATCCT AAGT GAACC ATT GAAT CAT ACT GTT GAAAG AATT C AAAAAGC AT -T AAAGCGGAT GGACGAT GAGTATTTGAAATC AGC ACTT GCTT ACCT AAAG-C AACAGCCTGATTT AAAT GCT CT ACGGAAAGG AGG CC AC ATTT AC AAGTGC-CCT AACCT C AAT ATCGT C AATTT GGC AAAT AT GCC AAT GT AT GTT GCG AATT -TT GGAT GGGGCC AGC CGAT ATTT GCGAGGAT CGTT AAC AC AT ATT AT GAAG-GGAT AGC AC AT ATTT ATCC AAGTCCGAGC AAT GAT GGGACCTT GTC AGT G-GTT AT AAACTCGGT AGCC GAT C AC AT GC AGCTGTT C AAG AAGTT CTTT -TACGAGATCTTTGATTAAGGTATGAAAGACCTAGGTATTTTATATTTTCTA-GAAAT GT C ACTTTTTTTTTTTTTTTTTTTT GGGGGCGC AAAT GTT GT CTT ACTT GGAATTTT AT AT ATTTT AATCC AT GTTTTT AT GG AAGGC AGT GGTGTTG-CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 2B (CLF26) SEQUENCE CHARACTERISTICS: LENGTH: 434 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear WO 00/32789 PCT/NL99/00737 74 OTHER INFORMATION: CITRUS LIMON ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 2B MKIHVKESTIIRPAQETPKHRLQISDLDMIVPSNYVPSVYFYRRSSDCTDF-FEVGLLKKALSEVLVPFYPVAGRLQKDENRKIEILCNGEGVLFLEAETSCGIDD-F GDFSQGSKLLTLVPT V GDTKDIS SHPLLMAQ VT YFKCGGV C V GTRVNHTL-VDGAS AYHIIN S WAETTRGVPIST QPF YDRTILS V GVPT SPKFHHEEYDPPP SM-NAPPT QNPEIISTA3LNLSLDQIHTLKEKSKTDHEPNVKY SRMAILAAHIWRSM-CKARGLSDDQVSKLHFPTDGRQRLNPPLPPGYFGNVIFTTSLTASSGDILSEPLN-HTVERIQKALKRMDDEYLKSALAYLKQQPDLNALRKGGHIYKCPNLNIVN-LANMPMYVANFGWGQPIFARIVNTYYEGIAHIYPSPSNDGTLSWINSVADHM-QLFKKFF YEIFD. 75 INFORMATION FOR SEQ ID NO: 3A (SLF96) SEQUENCE CHARACTERISTICS: LENGTH: 1586 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY AMINOTRANSFERASE SEQUENCES DISCRETION FOR SEQ ID NO:3A AAACCGTCGGCGTCTGTAAATGCGTCGCCGCTCCGGAGAAGACAGAGTA-CAAGACTCAGGTGAATCGCAATGCCAACATGGCCAAGCTTCAAGCCGGT-TAT CTTTTTCC AGAGATT GCGAGGAGGAGGAAT GCGC ACTT GC AGAAGC AC-CCT GAT GCGAAG AT AATTCC ACTT GG AATT GGT GAT ACT AC C G AGCC AAT -T CC AGAAT AT AT AACCT CT GC AAT GGCAAAGAG AGC ACTT GC C AT GTCC AC-CCT AGAGGGTT AC AGT GGTT AT GGACCTGAAC AAGGT GAAAAGCC ACT GA-GAGTTGCAATTGCTAAAACGTTTTATGGCGACCTTGGCATAGAGGAAGAT-GACATATTTGTTTCTGATGGGGCAAAATGTGACATATCCCGCCTTCAGGT-TCTTTTT GGGGCGGAT AAAAC AAT AGC AGTGC AAG ATCC AT CGT AT CCGGC-TTAT GT AG ACT C AAGT GTT ATT AT GGGCC AG AC AGG AC AGT AT C AG AAAT C -TGTTCAGAAGTTTGGAAACATCGAGTACATGAGGTGTACTCCCGATAATG-GATTTTTTCCT GAT CT GT CCT CT ACT AAGCGAAC AGAT AT CAT ATTTTT CT -GTTC ACC AAAC AAT CCT ACT GGTT CT GCT GC AAC AAGGGAG C AACT G AC A-C AACTT GT AAAGTTTGCC AAGGAT AAT GGTT C AATC AT AGT CT AT GATTCT- WO 00/32789 PCT/NL99/00737 76 GC AT AT GCC AT GT AT AT GT C AGAT GAT AATCC ACGCTCC AT CTTT GAAATCC-CT GGAGCT AAAGAT GTT GC ACTT G AGAC AT CAT C ATTT AGT AAGT ATGC CG-GATT C ACT GGAGTTCGTTT GGGGT GGACT GT GGTTCC AAAGC AGTT GC AG-T ATTC AGAT GGTTTTC AAGTT GCC AAGGATTTC AACCGC ATT GTTT GT ACTT -GCTT C AAT GGT GC ATCC ACT ATT AT CC AAGCT GGT GGT CT GGCTT GCCTT-C AACC AAAGGGT GTT AAGGCT AT GC ACGGT GTGAT AAATTT CT AC AAA-GAAAAT ACT AAGAT CAT AAT GGAGACGTTT AACT CTCTT GGCTTT AACGT G-T AT GG AGGGAC AAACGCT C CAT AT GT GT GGGTCC ACTTCCCT GG AC AAAGC -T CCTGGGAT GT GTTT GCT GAGATCCTTGAGAAGACT CAT GTGGT AACC A-C ACCT GGAAGT GGCTTT GGACCT GGT GGT GAAGGTTT CAT C AGGGT AAGT G-CCTTT GGAC AC AGGAAAAATAT ATT AGAAGC AT GT AAAAGATT C AAGC AAT -T AT AC AAGT GAGG ACT GCGGAT CT GAATT GT AGACC AGTTT CT ACTGC AT G-CT AGTT G AAC CT ATTT GC CTCCCATTT C CGTT CT AT GCT AAAT ATTTT AG-C ACGTTCC AATTCCGT ATT C AGTTT GTCGGCTTT AGTTT AT GAATT AT GGA-GATTTTAGCT ATT GT AAAAAT GATTCGAT C AGCCTT GTTTTC ATGT GTT A-C ACTT AATT GTT GT AAC ATTT GT GAGGAT CAGAAGCTTT GATTCT GTTT GC~ T AG AAT AGT AT AATTTT ACCT AAAT AAAGT GGTT GAT CTTTCTT GGC CTG-CAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 3B (SLF96) SEQUENCE CHARACTERISTICS: LENGTH: 397 TYPE: Peptide STRANDNESS: Single WO 00/32789 PCT/NL99/00737 77 TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY AMINOTRANSFERASE SEQUENCES DISCRETION FOR SEQ ID NO:3B MAKLQAGYLFPEIARRRNAHLQKHPDAKIIPLGIGDTTEPIPEYITSAMAKRA-LAMSTLEGYSGY GPEQGEKPLRVAIAKTFY GDLGIEEDDIF V SDGAKCDISR-LQ VLF GADKTIAV QDP S YP A YVD S S VTMGQT GQ Y QKS VQKF GNIE YMRCTPD-NGFFPDLSSTKRTDHFFCSPNNPTGSAATREQLTQLVKFAKDNGSIIVYDSAYA-MYMSDDNPRSIFEIPGAKD VALET S SF SKY AGFT GVRLGWTVVPKQLQ Y SDG-FQVAKDFNRIVCTCFNGASTIIQAGGLACLQPKGVKAMHGVINFYKENTKI-IMETFNSLGFNVYGGTNAPYVWVHFPGQSSWDVFAEILEKTHWTTPGSGFGP-GGEGFIRVS AFGHRKNELE ACKRFKQLYK 78 INFORMATION FOR SEQ ID NO: 4A (SLG150) SEQUENCE CHARACTERISTICS: LENGTH: 1775 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY THIOLASE SEQUENCE DISCRETION FOR SEQ ID NO: 4A CGCTCCTTT GATTT CCTTGTTTC AATT AT C AAGAGT ATGGAGAAAGCGAT -C AAC AGGC AGAAGGTT CTCCTCGACC AT CTCCGACCTT CTT CTT CTTC-CGACGACT CTTCT CTCTCCGCGTCGGT AT GTGCGGCT GGGGAT AGCGCT G-CGT AT GCT AGGAAT CAT GT CTTT GGGGACGAT GT C GTC ATCGTT GC AGCTT-TT CGC ACTCC ACT CT GC AAGGCT AAGCGT GGCGGCTT C AAGT AT ACTT AT G-CTGATGATCTCCTCGCACCTGTCCTCAAGGCCGTGGTTGAGAAAACC AATC-TCAATCCCAAGGAAGTCGGGGATATTGTTGTCGGTACCGTCTTGGCCCCAG-GATCTCAGAGAGCTAGCGAATGCAGGATGGCTGCTTTCTATGCTGGCTTCC-CT GAG ACT GTGCCGGTT AGAACT GT G AAC AG AC AAT GTTCGT CT GGC CTC-C AAGC AGTT GCT GAT GTTGCTGCT GCC ATT AGAGC AG GGTTTT AT GAT ATT -GGCATTGGTGCTGGTTTGGAATCCATGACTGCAAACCCAATGGCATGG-G AAGGGG AT GTT AAT CCT AAAGT AAAG AT CTTT G AAC AAGC C C AGA ATT GC-CTT CTT CCT ATGGGAGTC ACCTC AGAAAAT GTT GCTC ATCGTTTT GGT GTTT -CAAGACAGGAGCAAGATCAGGCTGCAGTTGACTCTCATAGAAAGGCAGCT-GCTGCT GCT GCT GCTGGT AG ATTT AAAG AT GAAAT C ATC C CTGT GGC AAC- WO 00/32789 PCT/NL99/00737 79 C AAG ATT GTT GAT CC AAAAT CT GGT GAT GAGAAACCT GTT AC AATCTCT -GTT GATGAT GGGATTCGAAAC AC AAC ATT GGCGGACCT AGC AAAGCT -GAAGCCT GT GTTT AAGAAAGAT GGGACC ACC ACT GCT GGT AATTCT AGT -C AAGTT AGT GAT GGT GCT GG AGCT GTTCTCTT GAT GAAG AGAAGT GTTGC-CGACC AAAAAGGATT GCCG ATT CTT GGT GT ATT C AGGAATTTT GTT GCT -GTT GGT GTGGAT CCT GCC ATC AT GGGT GTT GGCCC AGCT GCT GC AATT C-CAGTT GC AGTT AAGGC AGCT GGTTT AGAGCTT GAT GAT ATT GACCTTTTT -GAGAT AAAT GAGGCTTTT GC ATCCC AATTT GT GT ATT GCCGT AAC AAGCT G-GGACTT GAT CC AGAAAAAAT C AAT GTT AACGGAGGT GC AAT GGCC ATCGGC-C ATCC ACTT GGT GC AAC AGGT GCCCGGT GT GTT GCC ACT CTTTTGCAT GAGAT G AAGCGT CGT GGT AAAGACT GCCGCT AT GGAGT GATCTC AAT GT G-C ATAGGC AC AGGGAT GGGT GC AGCC GCT GTTTTT GAAAGAGGAGAC C G-GACCGAT GAACT CT GC AAT GCTC GC AAGGTT GAAT C ACTC AACTT CTT ATC-C AAGGATGTTCGGT AGT AG AG AAT GGTT AGT GAC AGG AGCT ATT CC AAT C A-AT AAT GTTTGGT GGAGTCT GAAAATC ATAGT AAAGC ACT GG AAT AACGTT G-CT AAGTTTTTCGTT GGGT ACT ACCTT GTTT ATT GGG AT GG AAT AC AC AT G-TAGTTGGTTTGTTCTCCCAGACCTCCCACTTGTTGGCATATTCATTTTTGTC-C AACCT AAAAAGTT CC ATTTT ATAGGACTTC AT CT C AAT AAC ATT GGGTTT G-CGCC ACT AAAGC AGT GCCT AAAACTGT AATT GGGT AATTTT GGT AT ACCT -GTTT GCT ACTTTT CTTTT CT AAGTT AATC AAGC CCT GC CC AC CTC AT A-T AAAAAAAAAAAAAAAA AAAAAAA INFORMATION FOR SEQ ID NO: 4B (SLG150) SEQUENCE CHARACTERISTICS: WO 00/32789 PCT/NL99/00737 80 LENGTH: 458 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY THIOLASE SEQUENCE DISCRETION FOR SEQ ID NO: 4B MEKAINRQKVLLDHLRP S S S SDD S SLS AS VC AAGD S AA Y ARNHVF GDD WI-VAAFRTPLCKAKRGGFK YTY ADDLL AP VLKAV VEKTNLNPKE V GDI V V GT V -LAPGSQRASECRMAAFYAGFPETVPVRTVNRQCSSGLQAVADVAAAIRAGFY-DIGIGAGLESMT ANPMAWEGD VNPKVKIFEQ AQNCLLPMGVT SENVAHRFGV -SRQEQDQAAVDSHRKAAAAAAAGRFKDEIIPVATKIVDPKSGDEKPVTISVD-DGIRNTTLADLAKLKPVFKKDGTTTAGNSSQVSDGAGAVLLMKRSVADQKGL-PILGVFRNFVAVGVDPAIMGVGPAAAIPVAVKAAGLELDDIDLFEINEA-F ASQF VY CRNKLGLDPEKINVN GGAMAIGHPLG AT G ARC V ATLLHEMKRRGK-DCRY GVISMCIGT GMG AAAVFERGDRTDELCNARKVESLNFLSKD VR WO 00/32789 PCT/NL99/00737 81 INFORMATION FOR SEQ ID NO: 5A (SLH51) SEQUENCE CHARACTERISTICS: LENGTH: 2141 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY PYRUVATE DECARBOXYLASE SEQUENCE DISCRETION FOR SEQ ID NO: 5A ATTTTCACTCAGAGTCTCAATCTTTCATCACAAAAATTCCCATTTGATCA-CAAAAAAGTTTCAACCTTTAAACCTCCATGGACACCAAGATTGGCTC-CATCGACGTCTGCAAAACCGAGAACCACGACGTCGGTTGTTTACCAAACAG-CGCCACCT CC ACCGTT C AAAACT CAGTCCCTT CC ACCTCCCTC AGCT CCGC-CGACGCCACCCTCGGCCGCCACCTGGCACGCCGCCTCGTTCAAATCGGCGT-CACCGACGTCTTCACCGTCCCCGGCGACTTCAACTTGACCCTTCTTGACCAC-CTCATCGCCGAGCCCGGCCTCACCAACATTGGCTGCTGCAACGAGCT-CAACGCCGGGTACGCCGCCGACGGCTACGCGCGGTCGCGTGGCGTCGGCG-CGTGCGT GGT GACTTTC ACT GTT GGT GGACT GAGT GTGCT GAACGCGATCG-CCGGCGC GT AT AGT GAG AATTT GCCGGT GATTT GT ATT GTT GGT GGGCC-CAACTCT AAC GATT ATGGGACT AACCGGATT CTTC ACC AT ACT ATT GGGTT -GCCGGACTTC AGTC AAGAGCTCCGGT GCTTT C AGACCGT GACTTGCTTT -C AGGCTGT GGTGAAT AATCT GGAGGAT GC AC AT GAG AT GATT GAT ACT GC A-ATTT CGACT GCGTT GAAAG AAAGC AAGCCT GT GT AT ATC AGC ATT GGCT G-C AACTT GGCT GGGATTCCT C ATCCT ACTTTC AGCCGT G AACCT GTT C C ATTT- WO 00/32789 PCT/NL99/00737 82 ;TC ATT GT CTCC AAAATT GAGC AAT AAGT GGGGATT AGAGGCT GC AGT G-GAGGCT GCTGC AG AGTT CTTGAAC AAGGC AGT GAAGCC AGTT AT GGT GGG-CGGGCCC AAACT GCGCT CT GC ACAT GCT GGT GAT GCCTTT GTT GAACT GGC-T GATGCTT CT GGATTTGCT CT GGCT GT GAT GCC ATC AGC AAAGGGGC AAGT -GCC AGAGC ACCACCCCC ATTTC ATCGGAACGT ACT GGGGT GCT GT GAGC AC-TGCCTTTTGT GCT GAG ATT GT GGAGT CT GC AGAT GC AT ACTT GTTT GCT G-GGCCGATTTT CAAT GACT AC AGCT C AGTT GGGT ACTCGCT CCTT CT CAA-GAAAGAGAAGGCGATC ATT GT GC AGCC AGATCGT GT GACGAT AGGGAAT -GGCCCT AC ATTT GGTT GT GTT CTC AT G AAGG ATTT C CTCTT AGGC CT AG-C AAAGAAGCT GAAGC AT AAC AAC ACT GCT CAT GAGAACT ACCGC AGGATCT -TTGTGCCTGATGGCCACCCTCTGAAGGCTGCACCCAAAGAACCTTTGAGG-GTTAATGTTCTGTTCAAACACATTCAGAATATGCTGTCAGCTGAAACCGCT-GT GATT GCT GAGAC AGGGGACT CAT GGTTT AACT GT C AGAAGCT G AAATT G-CC ACCCGGCT GCGGGT AT GAGTTCC AAAT GC AAT AT GGAT C AATT GGTT G-GT C AGTT GG AGC AACT CTTGGGT AT GCT C AGGCT GT ACCT GAG AAGC G AGT -GATTT CTTTC ATT GGT GAT GGGAGCTTCC AGGT GACT GCT C AAG AT GTGTC-C ACAAT GATTCGAAATGGAC AGAGAACC ATT ATTTTCCT GAT AAAC AAT G-GTGGAT AC ACCATT GAAGT GGAAATCC ATGAT GGACC AT AC AAT GT GAT -C AAG AACT GGAACT AC ACT GG ACT GGTT GAT GC AAT C C AC AAT GGG-GAAGGC AAGT GCTGGAC AACC AAGGT GCGTT GCG AAGAGGAGCT GATT -GAAGC AAT AG AGACT GC AAAT GGACCC AAGAAGGAT AGCTT CTGCTT C ATT -GAGGTGATT GTTC AC AAGG AT GAT ACC AGC AAAGAGTTGCTT GAGT GGGG-GTCTAGGGTTTCTGCTGCCAACAGCCGCCCACCTAATCCTCAGTAAAACTC-TCCTGTGTCATATGAAGGCCTTCATTCACATTCACAGATTTAGATCAAGC- WO 00/32789 PCT/NL99/00737 83 C AAGCT CTT GT GC AAATTTTCCTT AT GTTTTTCCT GT C AACT GGAGAAT GGT-GTCTGTCAAGTTTTTTTTACACTACAGTGATTTCTGGTTTGTCTGTATATT-T C CTT CT GAAT ATT AGT ATCTTCT GATTTTT C AATT G ATC AAATT CTGT-GATCCT AAAT GGTTT GT GGAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 5B (SLH51) SEQUENCE CHARACTERISTICS: LENGTH: 605 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY PYRUVATE DECARBOXYLASE SEQUENCE DISCRETION FOR SEQ ID NO: 5B MDTKIGSIDVCKTENHDVGCLPNSATSTVQNSVPSTSLSSADATLGRHLARRL-VQIGVTD VFTVPGDFNLTLLDHLIAEPGLTNIGCCNELNAGYAADGY ARSR-GV GAC WTFT V GGLS VLNAIAGAY SENLP VICIV GGPN SND Y GTNRILHHTI-GLPDFSQELRCFQTVTCFQAWNNLEDAHEMIDTAISTALKESKPVYISIGCN-L AGIPHPTF SREP VPF SLSPKLSNK W GLEAA VE AAAEFLNK A VKP VM V GGPKL-RSAHAGDAFVELADASGFALAVMPSAKGQVPEHHPHFIGTYWGAVSTAF-CAEIVESADAYLFAGPIFNDYSSVGYSLLLKKEKAIIVQPDRVTIGNGPTFGCV-LMKDFLLGLAKKLKHNNT AHENYRRIF VPDGHPLKAAPKEPLRVNVLFK-HIQNMLSAETAVIAETGDSWFNCQKLKLPPGCGYEFQMQYGSIGWSVGATL-GYAQAVPEKRVISFIGDGSFQVTAQDVSTMIRNGQRTIIFLINNGGYTEEVEIH- WO 00/32789 PCT/NL99/00737 84 DGPYNYIKNWNYT GLVD AIHNGEGKCWTTKVRCEEELIEAIETANGPKKDSF-CFIEVIVHKDDTSKELLEWGSRVSAANSRPPNPQ WO 00/32789 PCT/NL99/00737 85 INFORMATION FOR SEQ ID NO: 6A (SLB39) SEQUENCE CHARACTERISTICS: LENGTH: 1415 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 6A T AATCT AGCTT CT GC ACC AAAACT AT C AGAT AATT AAG AAT CT GCC AC AG A-GAAAATGGTGATGTCTATCGAGCAGGAACACCCCAAGAAGGCATCTGGAT-GGGCTGCAAGAGATTCATCTGGTGTTCTCTCTCCCTTCAGTTTCTCCAGAAG-GGAAACCGGAGAGAAAGACGTGACGTTC AAAGT GAT GT ACT GT GGGATTT -GCCATTCGGACCTTCACATGGTCAAGAATGAATGGGGCTTCTCTACCTATC-CT CT GGTTCC AGGGC AT GAG ATT GTT GGT GAAGT G ACGG AAGT AGGAAG-C AAT GT AC AAAAATTC AAAGTT GGAGAC AG AGTCGGT GTT GGAT GC ATT GT -GGGATCTT GCCGATCTTGT GAAAATTGT ACCGACC ACCTT GAGAACT ACT G-CCCCAAACAGATACTCACTTACGGTGCCAAGTACTACGACGGAACCACCAC-CT AT GGCGGTT ACT CT GAC ATT AT GGT GGCCGAT GAAC ACTT CAT AGT ACG-CATCCCAGACAACTTGCCTCTTGATGGTGCTGCGCCGCTCCTATGTGCCGG-GATTACAACCTACAGCCCCCTGAGATATTTCGGACTTGACAAGCCCGGCAT-GCATGTAGGTGTGGTCGGCCTAGGCGGTTTAGGCCACGTCGCCGTGAAGT-TT GCC AAGGCT AT GGG AGT G AAGGTT AC AGT GATT AGT AC ATCC CCT AA- WO 00/32789 PCT/NL99/00737 86 GAAAGAGGAGGAAGCTCGT AAAC ACCT AGGAGCT GACTCGTTTTT GGTT AG-CCGTGACCAAGATCAAATGCAGGCTGCCATTGGTACCATGGATGGGAT-C ATTGACACGGTTT CT GCACAACATCCTCT CCTGCCTTTGATTGGTTT GTT-GAAGT CT CAT GGAAAGCTTGTT AT GGTT GGT GC ACC AGAGAAGCCT CTT-GAACT GCC AGTTTTTCCTTT ACT CAT GGGAAGAAAGAT GGT AGCT GGT AG-CGGCATTGGGGGTATGAAGGAGACACAAGAGATGATAGATTTTGCAGC-CAAGCACAACATTACAGCAGACATCGAAGTCATACCAATCGACTACTTG-T AAC ACT GCT AT GGAGCGT CT AGT C AAAGC AGAT GT C AG AT ACCGTTTT GT -CAT CGAC ATT GG AAAC AC ACT G AAGGCT AGCT CTT AAATT CT GC AAT C C A-GACT GGAT C AAT GAAGAAAC AAGAAC AG AAACGG AGACT G ATTT AGT GT -CATACTCGGTGTTGGTTTTCCTTGTAGCATTTTTTGTTGTCTGCTACATGA-AT AAT GAT C AC AT GAACAACT GCCTT CT GT GATGATTT GAT AAT AAAAG A-ATAC AT GAAC AAT GAT ACT GCCTT CTTTT GT AAT GTTTTTT ACT AT AT AAT -C ATTT C AAATT ATTTTGCT A- TATCTCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 6B (SLB39) SEQUENCE CHARACTERISTICS: LENGTH: 333 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 6B WO 00/32789 PCT/NL99/00737 87 MVMSffiQEHPKKASGWAARDSSGVLSPFSFSRRETGEKDVTFKVMYCGICHS- DLHMVKNEWGFSTYPLVPGHEIVGEVTEVGSNVQKFKVGDRVGVGCIVGSCR- S CEN CTDHLENY CPKQDLT Y GAKYYDGTTT Y GGY SDIMV ADEHFIVRJPDNL- PLDGAAPLLCAGITTYSPLRYFGLDKPGMHVGWGLGGLGHVA\7KFAKAM- GVK VT VISTSPKKEEE ARKHLG AD SFL VSRDQD QMQ AAIGTMD GIIDT V S AQH- PLLPLIGLLKSHGKLVMVGAPEKPLELPVFPLLMGRKMVAGSGIGGM- KETQEMIDFAAKHNITADIEVIPIDYL WO 00/32789 PCT/NL99/00737 88 INFORMATION FOR SEQ ID NO: 7A (SLF193) SEQUENCE CHARACTERISTICS: LENGTH: 1227 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 7A GGAAAC AGGAGC AAC GGACGT AAGATT C AAAGT GTT GT ACT GT GGAGT AT-GCC ATTCGGAC AT AC ACAT GGCC AAAAAT GATT GGGGGACTT CT ACCT AT C-CT ATT GT ACCT GGGC AT GAACTT GTT GGT GT AGT AAC AGAAGT AGGAT G-C AAAGT AAAGAAATT C AAAAGTT GGAGAC AAGGT CGGT GTT GGTT GC AT G-GT CGACT C AGACC AACTT GCG AAAATTGTAT CC ATC ACCT AGAAAATT ACT -GTCCGAAT CT GAT AC AAACCT ACGGTTCT AAAT ACT ACGACGGAACC AT GA-C AT ACGGAGGTT ACT CGAAC AAC AT GGT GACT GAT GAGC ACTT C ATTGT-TCGGATCCCGGACAACTTACCTCTTGATGGCGCTGCTCCGCTTCTATCTGC-CGGGATT AC AACTT AC AGCCC AT GGAGAT ATT AT GGACTT GAC AAACCCGG-TATGCATCTTGGTGTTGAATGGCCTAGGCGGTTTAGGTCACGTCCGCCGT-TAAATTTGCCAGGGCTTTGGGGCTCAAGGTTACAGTCATTAGTACCTCCCC-TAATTAAAGAAGGAGGCAGCTATGGAACATCTCCCGCGCTGATGCATTCCC-TGCTTAGAACTGACCAAGATCAGATGGAGGCTGCCATGAGCACAATGGAT-GGT ATC ATT GAC AC AGTTCCT GC AGTTCGACCT CT AGAGCCTTT G ATTT -C ATT GTT GAAGACT AAT GGAAAAGTT GTT ACCGTTGGT AT AGC AGT GC AGC- WO 00/32789 PCT/NL99/00737 89 C ACTCGAT CTCCC AGTTTT CCCTTT GATAAT AGGAAGGAAGAT GGT AGCT G-GTAGT GC C ATT GGAGGT AT GAAAGAGAC GCAAGAGAT GATT GATTTT GCT G-CT GAAC AT AAC AT AAC AGCT GAC AT C GAGGT C AT C C CG ATT GATT ACCT-GAAC ACCGC AAT GGAACGCGTT GTC AAAAAAGAT GTC AGGTTTCGATTT GT-CATCGACGTTGAGAACACATTGTAAGTCCGCCTAAGTTTTTCATTCAATTCT-GTT AAT AAG ACT AT GC ATT AAT AT AT GACT GACT CTCC AT AGGAT GG AGT-T AT C AGT CTT C AAATTT CT AG AC AT ATTTT GT GATC AAAT AAAT GGAAT GGCTTT GTTTTCCTTTTCC ACT AAGATT AGATTT C AGTT GT ATT GTTTTT AAAGA-G ATT GAT GTTTTT ATT AATT GT AAC AGT GTT ATC AGT CT AATC AT -TAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 7B (SLF193) SEQUENCE CHARACTERISTICS: LENGTH: 326 TYPE: partial peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 7B ETGATDVRFKVLYCGVCHSDIHMAKNDWGTSTYPIVPGHELVGVVTEVG-CKVKKFKSWRQGRCWLHGRLRPTCENCIHHLENYCPNLIQTYGSKYYDGTM-TYGGYSNNMVTDEHFIVRIPDNLPLDGAAPLLCAGITTYSPWRYYGLDKPGM-HLGVEWPRRFRSRPPLNLPGL W GSRLQ SL VPPLDCEG GS Y GT S P ALMH SLLRT - WO 00/32789 PCT/NL99/00737 90 D QD QME AAMS TMDGIIDT VP A VRPLEPLISLLKTN GKWTV GIA V QPLDLP VF -PLIIGRKMV AGS AIGGMKET QEMTDF AAEHNIT ADIE VIPID YLNT AMERVVKK-DVRFRFVTDVENTL INFORMATION FOR SEQ ID NO: 8A (SLF122) SEQUENCE CHARACTERISTICS: LENGTH: i063 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 8A GCAAAGTGCAAAAATTTAAAGTTGGAGACAAAGTTGGTGTTGGGTGCTTGG-T AGGCT CAT GC AAAACTT GCG AC AGCT GT GCT AACGATTT GGAGAACT ACT -GCCCCAAACAGATACAGACTTACGGCGCCAAGTACCTTGACGGAACAAC-C AC AT ACGGCGGTT ACT CT GAC AT CAT GGT GGC GGAT GAGGC CTTTGT A-ATCCGTATTCCGGACAACCTGCCTCTTGAGGGTGCTGCTCCTCTCCTATGTG-CCGGAATCACAACTTACAGTCCCCTGAGGTATTTCGGACTTGACAAACC-CGGCATGCATGTCGGGGTGGTTGGCCTTGGCGGTTTAGGCCATGTCGCGGT-GAAGTTT GCC AAGGCTTTGGGGGTT AAT GTC AC AGT GAT C AGT ACCT CCGC-T AAT AAG AAAG AT G AAGCT ATT AAAC AC CTTGGTGCT GATT CTTTCTT GGT-CAGTCGTGACCAAGATCAGATGCAGGCTGCCATGGGAACATTGGACGGTAT-CATCGACACAGTTTCCGCAGTCCACCCCCTCCCACCTTTGATTAGTTTATT-GAAGGCT AAT GG AAAGCTT GTT AT GGTT GGAGC ACC AGAGAAGCC ACTT -GAGCT ACC AGTTTTTT CTTT AAT AAT GGGAAGGAAG ACTTT AGCCGGT AG- WO 00/32789 PCT/NL99/00737 91 T AATATCGGAGGT AT C AAGGAGAC AC AAGAGAT GAT AGATTT GGC AGC-CAAACACAACATAACGGCCGACATCGAGATTATCCCCATCGACTATTTGAA-C ACT GCT AT GGAGCGT CTTGCT AAAGGGGAT GTT AGAT ACCGTTTT GT -C ATCGAC AT C GGAAAC AC ATT GAAGCCGGCC ATTT AAATTT GC ATTT C G AT-C AGAAACT GAAT C AAGC GAGGT CGAGAGGCCT ACGT AAC AAT GC AAAC AT-GTGCTAGCTTGTTCTTGGAGTAGTCTTTAGCTTTTCTCTGATGTATTCCATC-T GTTTTGTTC AT GTCCC ATCTT ATT ATGAGAAAAAT GT GGGT ACCGT GGA-T ATT GAAT AAAT GAAGAGCT ACT GGAACGAT GGTTT C A-CAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 8B (SLFI22) SEQUENCE CHARACTERISTICS: LENGTH: 278 TYPE: partial peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 8B KVQKFKVGDKVGVGCLVGSCKTCDSCANDLENYCPKQIQTYGAKYLDGTT-TY GGY SDIMV ADE AF VIRIPDNLPLEGAAPLLC AGITT YSPLRYF GLDKPGM-HVGWGLGGLGHVAVKFAKALGVNVTVISTSANKKDEAIKHLGADSFLVSRD-QDQMQAAMGTLDGIIDTVSAVHPLPPLISLLKANGKLVMVGAPEKPLELPVF- WO 00/32789 PCT/NL99/00737 92 SLIMGRKTLAGSNIGGKETQEMIDLAAKHNITADIEIIPIDYLNTAMERLAKGD-VRYRFVTDIGNTLKPAI INFORMATION FOR SEQ ID NO: 9A (SLD194) SEQUENCE CHARACTERISTICS: LENGTH: 1228 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 9A GCAAGAGATTCATCTGGTGTCCTCTCTCCCTTCAATTTCTCCAGAAGG-GAAACCGGAGAGAAAGACGTT AT GTT C AAAGT GTT GT ACT GT GGAATTT GC-C ATT CGGACCTT C AC AT GGT C AAGAAT GAAT GGGGCTTCTCT ACCT ATCCT-TT GGTCCCGGGGC AT GAGATT GTT GGT GAAGTT ACGGAAGTAGGGAG-C AAAGT AC AAAAATTT AAAGTT GGAGAC AG AGT C GGTGTT GGAT GCGTTGT-GGGAT CTT GC CGAT CTT GT GAAAATT GT ACCGAC C ACCTT GAG AACT ACTG-CCCCAAACAGATACTCACTTACGGTGCCAAGTACTACGACGGAACCACCAC-CT AT GGCGGTT ACT CTGAC ATT AT GGT GGCCGACGAAC ACTT CAT AGT ACG-CATCCCAGACAACTTGCCTCTTGATGGCGCTGCGCCGCTCCTATGTGCCGG-GATTACAACCTACAGCCCCCTGAGATATTTCGGACTTGACAAGCCCGGCAT-GCATGTAGGTGTGGTCGGCCTAGGCGGTTTAGGCCACGTCGCCGTGAAGT-TTGCC AAGGCT AT GGG AGT GAAGGTT AC AGT GATC AGT ACGTCCCCT AA- WO 00/32789 PCT/NL99/00737 93 G AAAGAGG AGG AAGCT CTT AAAC ACCT AGG AGCT GACT CGTTTTTCGTT AG-CCGT GAC CAAGATC AAAT GC AGGCT GCC ATT GGT ACC AT GGAT GGGAT -C ATT GAC AC AGTTT CT GC AC AAC ATCCTCTCCTGCCTTT GATT GGTTT GTT -GAAGTCTCATGGAAAGCTTGTTATGGTTGGTGCACCAGAGAAGCCTCTT-G AACTTCC AGTTTTT CCTTT ACT CAT GGGAAGAAAGAT GGGT AGCT GG-TAACCGGCATTTGGGGGTATGAAGGAGACACAAGAGATGATAGATTTTGC-T GCC AGGC AC AAC AT AAC AGC AG AC AT CGAAGT CAT AC AATCGACT ACT-T AAAC ACT GCT AT GGAGCGTTTAGTC AAAGC AGAT GT C AGAT ACCGTTTT -GT C ATCGAC ATT GGAAAC AC ACT G AAGGCT AGC ACTT AAATT CT GC AAT C-C AGACTGT AT C AAT GAAGAAAC AAGAAC AG AAACT GAG ATT GATTT GGTGT-CATACTCCGCCTATGGTTTTCCTTACAGCATTTTTTGTTGTTTGCTACATGA-AT AACGATC AC AT GAACT GT GAT GATTT GAT AAT AAAAGAAT AC AT AAA-CAAAAAAAACAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 9B (SLD194) SEQUENCE CHARACTERISTICS: LENGTH: 283 TYPE: partial Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 9B ARDSSGVLSPFNFSRRET GEKD VMFKVL Y CGICHSDLHMVKNE W GFST YPL-VPGHEIVGEVTEVGSKVQKFKVGDRVGVGCWGSCRSCENCTDHLENYCP- WO 00/32789 PCT/NL99/00737 94 KQILTYGAKYTDGTTTYGGYSD1MVADEHFIVRIPDNLPLDGAAPLLCAGIT-T Y SPLRYF GLDKPGMHV G WGLGGLGHV AVKFAKAMGVKVT VISTSPK-KEEEALKHLGADSFFVSRDQDQMQAAIGTMDGIIDTVSAQHPLLPLIGLLKSH-GKL VMV GAPEKPLELP VFPLLMGRKMGS W INFORMATION FOR SEQ ID NO: 10A (SLF17) SEQUENCE CHARACTERISTICS: LENGTH: 852 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 1 OA GTCCCCTGAGGTATTTCGGACTTGACAAACCCGGCATGCATGTCGGGGTG-GTTGGCCTTGGCGGTTT AGGCC AT GTCGCGGT GAAGTTT GCC AAGGCTTT G-GGGGTT GAGGT C AC AGT GAT C AGT AC CTCCGCT AAT AAGAAAG AT GAAGC-TATTAAACACCTTGGTGCTGATTCTTTCTTGGTCAGTCGTGACCAAGATCA-GATGCAGGCTGCCATGGGAACATTGGACGGTATCATCGACACAGTTTCTG-C AGT CC ACCCCCT CCC ACCTTT GATT AGTTT ATT GAAGGCTAAT GGAAAGC-TTGTT ATGGTT GGAGC ACC AGAGAAGCC ACTT G AG CTACC AGTTTTTT CTT -TAATAATGGGAAGG AAG ACTTT AGCCGGT AGT AAT ATCGGAGGT ATC AAG-GAGACACAAGAGATGATAGATTTGGCAGCTAAACACAACATAACGGCCGA-CATCGAGGTCATCCCCATCGATTATTTGAACACTGCAATGGAGCGTCTTGC- WO 00/32789 PCT/NL99/00737 95 TAAAGGGGATGTT AGATACCGGTTT GTC ATCGACATCGG AAAC ACATT -GAAGCCGGCC ACTT AAATTT GC ATTTCGATC AGAAACT GAATC AAGC GAT -GTCGAGAGGCCTACGT AAC AAT GT AAAC AT GT GCTAGCTT GTT CTT GT AG-TAGTCTTTAGCATTTCTCTGATGTACTCCTTCTGTTTTGTTCATGTTCCATCT-T AT AAT AAG ATT CTT ATT AT GAAAAAAAT AT GGT ACCGT GGAT ATT GAAT AAAT G AAG AACT ACT GGAAC AAT GGTTT C AC AAATT ATTT GT GGT GC-TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 1 OB (SLF17) SEQUENCE CHARACTERISTICS: LENGTH: 188 TYPE: partial peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 10B PLRYF GLDKPGMHV GW GLGGLGHV AVKF AKALGVE VT VI ST S ANKKDE AIK-HLG AD SFL V SRDQDQMQ AAMGTLD GIIDT VS AVHPLPPLISLLKAN GKLVM-V GAPEKPLELP VF SLIMGRKTL AGSNIGGIKET QEMIDL AAKHNIT A-DEEVIPIDYLNTAMERLAKGDVRYRFVIDIGNTLKPAT INFORMATION FOR SEQ ID NO: 11A (SLF138) SEQUENCE CHARACTERISTICS: WO 00/32789 PCT/NL99/00737 96 LENGTH: 663 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 11A AGTTTGGTCTTGATGTGGGTGGATTAAGGGGAGGGAT ATT GGGACTT G-GAGGT GTTGGAC AC AT GGGGGT GAAGAT AGC AAAGGCT AT GGGAC AC-CAT ATC ACCGT GAT AAGCTCTT CT GAT AAGAAGAAAAAAGAGGCCTTGGAG-CATATTGGTGCTGATGAGTACTTGGTGAGCTCTGATGCCACCCAAATGCAA-GAGGCTATGGACTCACTGGATTACATTATTGACACCATTCCAGTGTTCCAC-CCTCTTGAGC CTT ACCT CTCTTT GTT GAAGCTT GAT GGG AAGTT GAT CTT-GATGGGTGTTAT CAAC ACCCC ATTGC AATTTGT CT CT CC ATT GGTC AT GCTT -GGGG AGGAAG AC GAT C ACCGGGAGCTTTGTGGGGAGC ATGAAGG AGATG-GAGGAGAT GCT CG AGTT CT GC AAAG AG AAAG AGCT GAAACG AT GATT-GAAGT GGTG AAGAT GGACT AC AT C AACGAAGCTTT C GAAAGGTT GGAGAA-GAACGACGTTAGGT AC AGGTTCGTT GT GGATT GTT GCCGGC AGC AAT CTT -GAT C AAT AAG AAAG AAAG AAGGC AT CAT CG AGT GTT GTCCT ATTTT-T ATCGAGT ACT CT GT CTCATCTTAT CTT AAAC AAT AT AAAT AAAC AAA-GAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 1 IB (SLF138) SEQUENCE CHARACTERISTICS: LENGTH: 181 WO 00/32789 PCT/N L99/00737 97 TYPE: partial Peptide STRANDNESS: Single TOPOLOGY. Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 1 IB F GLD V GGLRGGILGLGGV GHMGVKIAKAMGHHITVIS S SDKKKKE ALEHI-GADEYLVSSDATQMQEAMDSLDYIIDTIPVFHPLEPYLSLLKLDGKLILM-GVINTPLQFVSPLVMLGEEDDHRELCGEHEGDGGDARVLQRERAETMIEVVK-MDYINEAFERLEKNDVRYRFVVDCCRQQS 98 INFORMATION FOR SEQ ID NO: 12A (SLG16) SEQUENCE CHARACTERISTICS: LENGTH: 694 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 12A GT GC ATT GCT AT GCCT AT G AAGGC AAG AT GC AAGAAC AT CT GC AATT AT GT -GAAGACGAGTTT AAAAAGAT AAT GAAG AT AAATTT CAT GT CTGC AT GGTT -TCT GGTAAATGCCGTT GGC AGAAGAATGCGAGATC AT AAAT C AGGAGGTT C-CATCATATTGTTGACCTCGATTGTTGGAGCTGAAAGAGGGCTTTATACAG-GAGCT GTT GCCT AT GGT GCAT GTT CGGC AGC ACTGC AGC AGTT AGT AAG-GTCGTCGGCATT GGAGATTGGAAAAT ACC AGATC AGGGTT AAT GC AATCG-C ACGT GGTTT GC ATTT GGAAGAT GAGTTTCCT AAGT CT GT GGG AAT AG AGA-GAGC AAAGAAGCT GGT GAAT GAT GC AGTT C CGCT GG AG AGAT GGCTT GAT -GTT AAAAAT GAT GT GGCTT C AAGT GT C AT AT ATTT GGTC AGT GAT GGTT-CAAGGTACATGACGGGC AC AACT AT ATTT GTTGAT GGGGC AC AGTCTC-TCGTGAGGCCTCGAATGCGTTCTTATATGTGATTCTTGCTCCTATTATATCC-TCCT AGCC ATT ATT AGCT ACTT AGGTTT GTT CAT ACTTC AT AGGT GAACT -C ATT AGCT ATT CTT AC ATTT GTTCCTT AT GAAT AAAGAAGTC AAGATT -CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA WO 00/32789 PCT/NL99/00737 99 INFORMATION FOR SEQ ID NO: 12B (SLG 16) SEQUENCE CHARACTERISTICS: LENGTH: 176 TYPE: partial Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 12B VHCY AYTiGKMQEHLQLCEDEFKKIMKINFMS AWFLVNAV GRRMRDHKSGG-SHLLTSIVGAERGLYTGAVAYGACSAALQQLVRSSALEIGKYQIRVNAIARGL-HLEDEFPKS VGIERAKKLVND AVPLERWLD VKND V AS S VTYLVSDGSRYMT-GTTIFVDGAQSLVRPRMRSYM 100 INFORMATION FOR SEQ ID NO: 13 A (SLG144) SEQUENCE CHARACTERISTICS: LENGTH: 1010 TYPE: partial cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 13 A GGAAACT ACC AT C AATTTT GGGT CT AAGAAGATT GC AGTT GTT ACTGGAGC-C AAC AAAGGGATT GGACTT GAGATT AGC AAGC AATT AGCT GCT AAAG-GAGTT GGGGT GGT ATTAAC AGC AAGAG AT GT GAAGAGAGG AAC AGAAGCT-GCTGAAAATCTT AAGGCTT CT GGGTTCTCT GAT GT GGT ATTTC AT C AGCT A-GATGT AAC AGAGCCGACT ACT ATTGGTT CTTT GGC AAACTTT CTT GAAACG-C AATTT GGAAAGCTT GAC AT ATT GGTT AAC AAT GC AGGAGT C GTT GGAT CT-GTAT ACCTC AC AGCCGACT AT GATCC AGT GC AAAC AT ACGAGAC AGCG AG-GGATT GTTT GAAAAC AAACT ATT AT GGGCT C AAGC AAGTC AC AG AAGC ACTT GTT CC GCT GCTTC AAAAATCT GAAGCT GC AAGG AT AGTC AAT GTCTCTTC-CGGATT AGG AC AGCT AAG AAAT ATTGG AAAT GAGAAGGCC AAGAAGGAGC-T AGGAGAT GC AGAT AACCT C AAC G AGG AG AAAGT GGAC AAGCT AGTT GAG-GAATTT CT GGAGGAT GT G AAAC AGGATT C GAT AG A AT C C AAAGGCT GGCC -TCTAAGTATATCTGCCTACATTGTCTCAAAAGCAGCTCTGAATGCTTATA-CAAGACTCTTGGCAAAGAAGTATCCCCATATTGCCATAAACGCAGTTGGTC-CAGGTTATACCAAAACAGACCTCAATAATAATTCCGGGATTCTCACAGTT- WO 00/32789 PCT/NL99/00737 101 GAAGAAGCT GC AGT AGGTCCT GT GAGGCTGGCTTT GAT AGCCGAAACT A-GAATTT CCGGCCTCTTCTTC AAC AGAAAT GAAGAGTCGACCTTT GATT AG-GT C AACGT GATCCCTGAT GAACT GGACT ATTTT AGATTTT C AG AAT GTGC-TT GATTTT GTT GAAGT ATTT AT GGGATTT GT ATGT AT ACTTT GAT GT AT -C ATT GT ATT AAT AGAGC AC AT GTT GT GAT -CAAAAAAAAAAAAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 13B (SLG144) SEQUENCE CHARACTERISTICS: LENGTH : 283 TYPE: partial Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY ALCOHOL DEHYDROGENASE SEQUENCE DISCRETION FOR SEQ ID NO: 13B ETTINF GSKKI AWT GANKGIGLEISKQL AAKGV GVVLT ARD VKRGTE AAENL- KASGFSDWFHQLDVTEPTTIGSLANFLETQFGKLDILVNNAGVVGSVYL- TADYDPVQTYETARDCLKTNYYGLKQVTEALVPLLQKSEAARIVNVSSGL- GQLRNIGNEKAKKELGDADNLNEEKVDKLVEEFLEDVKQDSIESKGWPLSI- S AYIV SKAALN AYTRLL AKK YPHIAIN AV GP GYTKTDLNNN S GILT VEE AAV G- PVRLALIAETRISGLFFNRNEESTFD WO 00/32789 PCT/NL99/00737 102 INFORMATION FOR SEQ ID NO: 14B (Motif 1) SEQUENCE CHARACTERISTICS: ' LENGTH: 12 TYPE: amino acid STRANDNESS: Single TOPOLOGY: Linear HYPOTHETICAL SEQUENCE: NO SEQUENCE DISCRETION FOR SEQ ID NO: 14B Trp Thr Asn Phe Phe Asn Pro Leu Asp Phe Gly Trp WTNF FN PL DF GW WO 00/32789 PCT/NL99/00737 103 INFORMATION FOR SEQ ID NO: 15A (MAY 1; SEST) SEQUENCE CHARACTERISTICS: LENGTH: 877 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: MANGO ESTERASE SEQUENCE DISCRETION FOR SEQ ID NO: 15A ATGAGGCCACAAATAGTGTTATTCGGAGATTCAATAACGGAGCAATCTTT CGGATCAGGTGGTTGGGGTTCTTCTCTTGCTGACACTTACTCTCGCAAGGC TGAT GT ATT AGTT CGTGGCT AT GGTGGCT AC AAT ACT AGATGGGC ATTGTT CTTGTT AT GTC ACATTTT CCCTCT GC AC AAT AAAAT ACCT CC AGCCGTCAC CACAATTTTCTTTGGGGCTAATGATGCAGCCCTTCTTGGGAGAACGAGTG AAAGGCAGCATGTTCCCGTGGAAGAATACAAGAACAATCTCAGAAAAAT GGTTCAGCATTTGAAGGAAGTCTCCCCCACGATGCTAGTTGTGCTTATTAC TCCACCACCAATTGATGAGGAAGGGCGTAAAGCATATGCACGATCCGTTT ATGGTGAGAAAGCTATGAAAGAGCCTGAGAGGACAAATGAAATGGCTGG AGTTTATGCTAGACATTGTGTTGAACTGGCAAAAGATCTTCCTGCCATTGA TCTGTGGTCCAAGATGCAGGAAACAGAAGGTTGGCAGAAAAAATTCCTCA GT GATGGGTT GC ACCTT AAGT C AG AAGGC AAT GC AGT GGTT C ACC AAGAA GTTGTGAGAGTTCTAAAAGAAGCATGGTTTTCTCCTGAACAAATGCCATAT GATTTTCCTCACCAATCAGTAATTGATGGAAAACACCCTGAGAAAGCTTT CCAACTGCAATGCCCTGCTGAATTCTAGTCAAGACAGGCTTGGAAATTTG TT CTCT CTTTC AATTTTT CT ATTT G ATG AAAAG ATTTGG ACT GCTTTTTCCT 104 AGTCATGCCAAATGAAACAGTGTTAGCCTTTTGCCTATTTTATCAGATGCT GATATGCGCTCTGTGTCGAC INFORMATION FOR SEQ ID NO: 15B (MAY1;SEST) SEQUENCE CHARACTERISTICS: LENGTH: 243 TYPE: peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: MANGO ESTERASE SEQUENCE DISCRETION FOR SEQ ID NO: 15B MRPQIVLFGDSITEQSFGSGGWGSSLADTYSRKADVLVRGYGGYNTRWALFL LCHIFPLHNKIPPAVTTIFFGANDAALLGRTSERQHVPVEEYKNNLRKMVQHL KEVSPTMLVVLITPPPIDEEGRKAYARSVYGEKAMKEPERTNEMAGVYARHC VELAKDLPAIDLWSKMQETEGWQKKFLSDGLHLKSEGNAVVHQEWRVLKE AWFSPEQMPYDFPHQSVIDGKHPEKAFQLQCPAEF WO 00/32789 PCT/NL99/00737 105 INFORMATION FOR SEQ ID NO: 16A (SUN1;VAAT) SEQUENCE CHARACTERISTICS: LENGTH: i488 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY VESCA ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 16A ATGG AG AAAATT G AGGT C AGT AT AATTTCCAAAC AC ACC ATC AAACC ATC AACTTCCTCTTCACCACTTCAGCCTTACAAGCTTACCCTGCTCGACCAGCT CACTCCTCCATCGTATGTCCCCATGGTATTCTTCTACCCCATTACTGGCCCT GCAGTCTTCAATCTTCAAACCCTAGCTGACTTAAGACATGCCCTTTCCGAG ACTCTCACTTTGTACTATCCACTCTCTGGAAGGGTCAAAAACAACCTATAC ATCGATGATTTTGAAGAGGGTGTCCCATACCTTGAGGCTCGAGTGAACTG TGACATGAATGATTTTCTAAGGCTTCCGAAAATCGAGTGCCTAAATGAGTT T GTTCC AAT AAAACC ATTT AGT AT GG AAGC AAT AT CTG AT G AGCGTT ACC CTTTGCTCGGAGTTCAAGTTAACATTTTCAACTCCGGAATAGCAATCGGGG TCTCCGTCTCTCACAAGCTCATCGATGGAAGAACTTCAGACTGTTTTCTCA AGTCGTGGTGTGCTGTTTTTCGTGGTTCTCGTGACAAAATCATACATCCTA ATCTCTCTCAAGCAGCATTGCTTTTCCCACCAAGAGATGACTTGCCTGAAA AGTATGCCCGTCAGATGGAAGGGTTATGGTTTGTCGGAAAAAAAGTTGCT ACAAGGAGATTTGTATTTGGTGCGAAAGCCATATCTGTAATTCAAGATGA AGCAAAGAGCGAGTCCGTGCCCAAGCCATCACGAGTTCAGGCTGTCACTA GTTTTCTCTGGAAACATCTAATCGCTACTTCTCGGGCACTAACATCAGGTA WO 00/32789 PCT/NL99/00737 106 CT ACTTCAAC AAGACTTTCT AT AGC AACCC AGGT AGTGAAC AT AAGATCA CGGAGG AAC ATGG AG AC AGT GT GGG AT AAT GCC ATT GGAAACTTGATATG GTTCGCTCCGGCC AT ACTAGAGCT AAGTC AT ACAAC ACT AG AGATCAGTG ATCTTAAGCTGTGTGACTTGGTTAACTTGCTCAATGGATCTGTCAAACAAT GTAACGGTGATTACTTTGAGACTTTCATGGGTAAAGAGGGATATGGAAGC AT GT GCGAGT ATCT AG ATTTT C AGAGGACT AT GAGTTCT ATGGAACC AGC ACCAGAGATTTATTTATTCACGAGCTGGACTAATTTTTTCAACCAACTTGA TTTTGGATGGGGGAGGACATCATGGATTGGAGTTGCAGGAAAAATTGAAT CTGCATTTTGCAATCTCACAACATTAGTTCCAACACCATGCGATACTGGAA TTGAAGCGTGGGTGAATCTAGAAGAAGAAAAAATGGCTATGCTAGAACA AGATCCCCAGTTTCTAGCACTAGCATCTCCAAAGACGCTAATTTCAAGAT ATTGATT AAGGAAGATT AT GCGGCTCGT GCAATGTTTCC ATTTT GTTGTGA TTAAGGCTTAAATTAGTTCACCAGCCAATCAATAAGATGCAAGTATGATA GACTCGGTCTACGTATGTTATCCG INFORMATION FOR SEQ ID NO: 16B (SUN1; VAAT) SEQUENCE CHARACTERISTICS: LENGTH: 455 TYPE: peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: STRAWBERRY VESCA ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 16B WO 00/32789 PCT/NL99/00737 107 MEKIEVSIISKHTIKPSTSSSPLQPYKLTLLDQLTPPSYVPMVFFYPITGPAVFNL QTLADLRHALSETLTLYYPLSGRVKNNLYIDDFEEGVPYLEARVNCDMNDFL RLPKIECLNEFVPIKPF SMEAISDERYPLLGV QVNIFN SGIAIGV S V SHKLIDGRT SDCFLKSW C AVFRGSRDKIIHPNLS Q AALLFPPRDDLPEK Y ARQMEGLWFV G KKVATRRFVFGAKAISVIQDEAKSESVPKPSRVQAVTSFLWKHLIATSRALTS GTTSTRLSIATQVVNIRSRRNMETVWDNAIGNLIWFAPAILELSHTTLEISDLK LCDLVNLLNGSVKQCNGDYFETFMGKEGYGSMCEYLDFQRTMSSMEPAPEI YLFTSWTNFFNQLDFGWGRTSWIGVAGKIESAFCNLTTLVPTPCDTGIEAWV NLEEEKMAMLEQDPQFLALASPKTLISRY INFORMATION FOR SEQ ID NO: 17A (MAY2; BAAT) SEQUENCE CHARACTERISTICS: LENGTH: 1291 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: BANANA ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 17A ATGAGCTTCGCTGTGACCAGAACAAGCCGGTCTTTGGTCACTCCATGCGG GGTCACGCCGACGGGCTCGCTCGGCCTCTCCGCCATCGACCGGGTGCCCG GCCTCAGGC ATAT GGTGCGGTCGCT ACACGT GTTCAGGC AAGGCCGGGAG CCGGCCAGGATCATCAGGGAAGCACTGTCGAAGGCGCTGGTGAAGTACTA CCCCTTCGCGGGGCGGTTCGTGGACGATCCCGAGGGCGGCGGCGAGGTTC GTGTCGCTTGCACTGGCGAGGGCGCTTGGTTCGTCGAGGCCAAGGCGGAC TGCAGCTTGGAGGACGTGAAGTACCTCGATCTCCCGCTCATGATCCCTGA WO 00/32789 PCT/NL99/00737 108 GGACGCGCTCCTGCCCAAGCCCTGCCCGGGACTGAACCCCCTCGACCTCC.

CTCTCATGCTGCAGGTGACAGAGTTCGTGGGCGGCGGATTCGTGGTCGGC CTCATCTCCGTCCATACCATCGCCGACGGCCTCGGCGTCGTCCAGTTCATC AACGCCGTCGCCGAGATCGCCCGTGGCCTGCCGAAGCCCACCGTGGAGCC TGCATGGTCCCGGGAGGTCATACCCAACCCACCTAAGCTGCCTCCCGGTG GCCCGCCCGTGTTCCCCTCCTTCAAGCTGCTCCACGCCACCGTCGACCTAT CCCCTGACCACATCGATCACGTCAAGTCCCGACACTTGGAGCTCACCGGC CAGCGCTGCTCTACCTTCGACGTCGCCATCGCCAACCTGTGGCAGTCCCGC ACGCGCGCCATCAACCTGGACCCAGGCGTCGACGTGCACGTGTGCTTCTT CGCCAACACTCGCCACCTGTTGCGCCAGGTCGTCCTCCTGCCCCCCGAGG ATGGCTACTACGGCAACTGCTTCTACCCGGTGACCGCCACCGCCCCAAGC GGCAGGATCGCATCGGCCGAGCTCATCGATGTCGTCAGCATCATCAGGGA CGCCAAGTCGAGGCTGCCGGGCGAGTTCGCCAAGTGGGCTGCCGGGGATT TCAAGGACGACCCTTACGAGCTCAGCTTCACGTACAACTCGCTGTTCGTGT CGGACTGGACCCGGCTCGGCTTCCTCGACGTCGACTACGGCTGGGGCAAG CCCCTCCACGTTATACCGTTCGCGTACTTGGACATCATGGCGGTCGGCATC ATCGGGGCGCCGCCGGCGCCGCAAAAGGGGACTCGGGTGATGGCGCAGT GCGTCGAGAAGGAGCACATGCAGGCGTTCCTGGAAGAGATGAAAGGCTT CGCTT AAACC AGCAGCAGT GT AGT ACTT GTC AGT ATCC WO 00/32789 PCT/NL99/00737 109 INFORMATION FOR SEQ ID NO: 17B (MAY2; BAAT) SEQUENCE CHARACTERISTICS: LENGTH: 419 TYPE: peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: BANANA ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 17B MSFAVTRTSRSLVTPCGVTPTGSLGLSA1DRVPGLRHMVRSLHVFRQGREPAR IIREALSKALVKYYPF AGRF VDDPEGGGEVR V ACT GEGAWFVE AKADCSLED VKYLDLPLMIPEDALLPKPCPGLNPLDLPLMLQVTEFVGGGFVVGLISVHTIA DGLGVVQFINAVAEIARGLPKPTVEPAWSREVIPNPPKLPPGGPPVFPSFKLLH ATVDLSPDHIDHVKSRHLELTGQRCSTFDVAIANLWQSRTRAINLDPGVDVH V CFF ANTRHLLRQVVLLPPEDGYY GN CF YP VT ATAPSGRIAS AELID VV SURD AKSRLPGEFAKWAAGDFKDDPYELSFTYNSLFVSDWTRLGFLDVDYGWGKP LHVIPFAYLDIMAVGIIGAPPAPQKGTRVMAQCVEKEHMQAFLEEMKGFA INFORMATION FOR SEQ ID NO: 18A (MAY3; AAAT) SEQUENCE CHARACTERISTICS: LENGTH: 1485 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: APPLE ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 18A WO 00/32789 PCT/NL99/00737 ATGTCATTCTCAGTACTTCAGGTGAAACGATTGCAACCGGAACTTATAACT CCGGCAAAGTCAACGCCTCAAGAAACAAAGTTTCTCTCAGATATTGACGA CCAAGAAAGCTTGAGAGTTCAGATTCCAATCATAATGTGTTACAAAGACA ACCCTT C ACTT AATAAAAATCGT AATCCCGTT AAGGC AATT AGGG AAGCC TT AAGT AG AGC ATT AGT GT ATT ACTACCCCTT AGCTGGAAGGCTT AGGG A AGGGCCTAATAGAAAGCTCGTGGTCGATTGCAATGGTGAAGGTATCTTGT TCGTTGAGGCTTCTGCTGATGTCACACTTGAGCAACTAGGAGACAAAATT CTACCCCCTTGTCCACTTTTAGAGGAGTTCTTATATAATTTTCCAGGCTCTG ATGGAATTATTGATTGTCCTTTGCTGCTGATTCAGGTGACCTGTCTTACAT GTGGAGGTTTC AT ACTTGC ATT GCGCCT AAACC AC ACAATGT GT GATGCA GCTGGATTGCTCTTGTTCCTGACCGCCATCGCGGAGATGGCAAGAGGCGC ACATGCACCATCTATTCTACCAGTGTGGGAGAGAGAGCTCTTGTTCGCTCG AGATCCACCAAGAATTACATGTGCTCGTCATGAATATGAAGACGTGATTG GTCATTCTGATGGCTCATACGCATCCAGTAACCAGTCAAACATGGTTCAA CGATCTTTCTACTTTGGTGCCAAGGAGATGAGAGTCCTTCGAAAACAGAT TCCACCCCACCTAATTTCCACTTGCTCCACATTTGACTTGATCACAGCTTG TTTGTGGAAATGTCGCACTCTTGCACTTAACATTAATCCAAAAGAGGCTGT TCGAGTTTCATGCATTGTCAATGCACGAGGAAAGCACAACAATGTACGTC TTCCCTTGGGATACTATGGCAATGCATTTGCATTTCCAGCTGCAATTTCGA AGGCTGAACCTCT ATGC AAAAAT CC ACT GGGAT AT GCTTTGG AGTT GGT G AAGAAGGCT AAAGCTACC AT GA ATGAAGAAT ACTT AAG AT C AGT GGC AG ATCTTTTGGTACTAAGAGGGCGACCTCAATATTCATCGACAGGAAGTTATT T AAT AGTTTCTG AT AAT ACGCGT GT AGGTTTT GG AG AT GT C AATTTT GGAT GGGGACAGCCGGTATTTGCTGGACCCGTCAAGGCCTTGGATTTGATTAGC TTCTACGTTCAACACAAAAAC AAC ACAG AGG AT GGAAT ATT GGTACC AAT WO 00/32789 PCT/NL99/00737 GTGTTTGCCATCCTCGGCCATGGAGAGATTTCAGCAGGAACTAGAGAGGA TT ACT C AGG AACCT AAGG AGG AT AT AT GT AAC AACCTT AGAT C AACTAGT C AAT GAT GT AAGT GTT AAACGT AAT GC ACTTT CTGT AAT GT AG AGTTGTGT CTCTTGGAACTT ATCNC AAGAGTT AT AGCTGTT ATCCAAAGGTCT GAATGT T ATTAAAAAAT AGCC AAT AAT AAG INFORMATION FOR SEQ ID NO: 18B (MAY3; AAAT) SEQUENCE CHARACTERISTICS: LENGTH: 454 TYPE: peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: APPLE ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 18B MSFSVLQVKRLQPELITPAKSTPQETKFLSDIDDQESLRVQIPIIMCYKDNPSLN KNRNPVKAIREALSRALVYYYPLAGRLREGPNRKLVVDCNGEGILFVEASAD VTLEQLGDKILPPCPLLEEFLYNFPGSDGIIDCPLLLIQVTCLTCGGFILALRLN HTMCDAAGLLLFLTAIAEMARGAHAPSILPVWERELLFARDPPRITCARHEYE DVIGHSDGSYASSNQSNMVQRSFYFGAKEMRVLRKQIPPHLISTCSTFDLITAC L WKCRTLALNINPKE AVRVSCIVNARGKHNNVRLPLGY Y GNAF AFP AAISKA EPLCKNPLGYALELVKKAKATMNEEYLRSVADLLVLRGRPQYSSTGSYLIVS DNTRVGFGDVNFGWGQPVFAGPVKALDLISFYVQHKNNTEDGILVPMCLPSS AMERFQQELERITQEPKEDICNNLRSTSQ *- WO 00/32789 PCT/NL99/00737 112 INFORMATION FOR SEQ ID NO: 19A (MAY4; MAAT) SEQUENCE CHARACTERISTICS: LENGTH: 1380 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: MANGO ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 19A ATGAT AATC ACGGT GAAGG AGTCG ACG AT GGT CCCGCCGTCGGCGG AG AC GCCGAGGATATCTCTGTGGAACTCCAACGCCGATCTGGTGGTTCCCCGATT TCATACTCCCAGCGTTTACTTCTACCGGCCCACCGGGGCCATAAACTTCTT TGATGGTAAGTTGCTCAAGGAGGCTCTCGGCAAGGCTCTGGTGCCGTTCT ACCCAATGGCGGGGCGGTTAAAGCGTGACGAAGATGGAAGGATTGAGAT CGATTGTAATGCTGAAGGCGTCTTGTTTGTTGAGGCCGAAACTCCCTCTGT TATTGATGATTTTGGTGACTTTGCGCCCACTTTAGAGCTCAAGCAGCTCAT TCCGACAGTGGATTACTCCGGCGGGATCTCTACGTATCCCCTATTGGCGTT ACAGGTTACTCACTTCAAATGTGGTGGAGTTTCACTTGGTGTAGGTATGCA ACACCATGCGGCAGATGGATTTTCTGGTCTTCACTTTGTAAACACATGGTC AGACATTGCTCGTGGTCTTGATGTTAACATCACCCTGTTCATTGACCGGAC TCTGCTCAGAGCACAGGATCCCCCTCAGCCTACTTTCCCACACACATGGA ATACCAGGCCGCCTCCTTCCCTGAAAACTCCTCCACCAGCAGTTTCTGAGC CTACTGCTGTCTCCATTTTTAAGTTGACGCGGGACCAGCTCAACATCCTCA AAGCCAAGGCCAAAG A AGATGGT AAC ACT AT C AACT AT AGCTC AT ATG AG ATGCTGGCGGGTCATGTCTGGAGATCTGCATGCAAGGCACGCGGCTTATC T GATGATC AAGAG ACT AAATT GT AC ATAGCAACT G ACGG ACGT GCT AGAT 113 T AATCCCCCC ACTTCC ACCT GGTT ACTTT GGG AAT GT GAT ATTT AC AGCC A CACCAATGGCAGTAGCAGGTGATCTCCAGTCAAAGCCTATATGGTATGCT GCTGGCCAGATTCATGATGCCTTGGTTCGAATGGACAACGACTATTTAAG GTCAGCCCTCGATTACCTAGAGCTTCAGCCTGATTTATCAGCATTAGTTCG TGGT GCCC ATAC ATTT AGGT GTCC AAAT CT CGGGATT ACT AGTT GGGTTAG ACT GCCAAT AC AT GAT GC AGATTTTGGTTGGGGTCC ACCC AC ATTT AT GGG GCCTGGTGGGATT GC AT ATGAAGGCTT AT C ATTT GT ATT GCC AAGCCCT AC AAATGATGGGAGCTTATCAGTTGCCATCTCTCTACAATCTGAACACATGA AACTGTTTCAGAAGTTCTTTT ATGAT ATTTAA INFORMATION FOR SEQ ID NO: 19B (MAY4; MAAT) SEQUENCE CHARACTERISTICS: LENGTH: 431 TYPE: peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: MANGO ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 19B MIITVKESTMVPPSAETPRISLWNSNADLVVPRFHTPSVYFYRPTGAINFFDGK LLKEALGKALVPFYPMAGRLKRDEDGRIEIDCNAEGVLFVEAETPSVIDDFGD FAPTLELKQLIPTVDYSGGISTYPLLALQVTHFKCGGVSLGVGMQHHAADGF SGLHFVNTWSDIARGLDVNITLFIDRTLLRAQDPPQPTFPHTWNTRPPPSLKTP PPAVSEPTAVSIFKLTRDQLNILKAKAKEDGNTINYSSYEMLAGHVWRSACK ARGLSDDQETKLYIATDGRARLIPPLPPGYFGNVIFTATPMAVAGDLQSKPIW Y AAGQIHD ALVRMDNDYLRS ALD YLELQPDLS ALVRG AHTFRCPNLGITS W WO 00/32789 ,,„ PCT/NL99/00737 114 VRLPIHDADFGWGPPTFMGPGGIAYEGLSFVLPSPTNDGSLSVAISLQSEHMK LFQKFFYDI INFORMATION FOR SEQ ID NO: 20A (CLE75; LAAT2) SEQUENCE CHARACTERISTICS: LENGTH: 1436 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 20A ATCCACACTAATAATTCTTTCATATGCTCGGGGATGGATCTCCAAATCACC TGCACCGAAATCATCAAGCCTTCTTCGCCGACGCCTCAACACCAAAGTAC CTATAAACTTTCAATTATTGATCAATTAACTCCTAATGTTTACTTTTCCATC ATTCTCTTGTATTCAAAAGCTGGTGAAAGTACCGCCAAAACTTCAGATCA CCTCAAAGAATCTCTTTCAAATACATTAACCCACTACTATCCTTTAGCTGG GCAACTCAAATATGATCAACTTATTGTTGATTGTAACGACCAAGGTGTCCC GTTCATCGAAGCACACGTCACCAACGACATGCGTCAGCTTCTTAAAATAC C AAAT ATT G ATGTTCTCGAAC AACT CCT ACC ATTC AAACCGC AT G AGGGTT TTGATTCTGATCGTTCCAACCTAACCGTTCAGGTCAATTACTTTGGTTGTG AAGGAATGGCGATTGGTCTGTGCTTCAGACACAAAGTTATTGATGCAACA ACGGCTGCATTCTTTGTTAAGAACTGGGGTGTAATTGCTCGTGGTGCTGGA GAAATTAAGGACGTGATCATTGATCATGCTTCCCTGTTTCCCGCAAGAGAT TTATCGTGCTTAACAAAGAGTGTTGACGAAGAGTTTTTGAAGCCAGAGTC TGAAACAAAGCGCTTTGTGTTTGATGGTGCCACTATAGCTTCTTTACAAGA WO 00/32789 PCT/NL99/00737 115 AACGTTTGCAAGTTTTGAACGACGTCCAACACGCTTTGAGGTTGTGTCAGC AGTTATTTT GGGT GCTTT GAT AACT GC AACG AG AGA AT CTG AT GAT GAGTC TAACGTTCCTGAACGTTTGGACACGATAATTTCAGTGAATCTACGGCAGA GAATGAATCCACCATTCCCGGAGCATTGCATGGGGAATATAATATCCGGG GGATT AGT GT ATT GGCC ACTGGAGAAAAAAGTTG ATT ACGGGT GTTT AGC AAAAGAGATTCATGAATCAATAAAGAAAGTGGACGATCAATTTGCGAGG AAGTTCTATGGGGACGCAGAGTTCTTGAACCTGCCGAGGCTTGCGGGTGC TGAGGATGTGAAGAAGCGGGAGTTTTGGGTTACTAGTTGGTGCAAAACTC CGCTGTATGAAGCTGATTTCGGGTGGGGGAATCCTAAGTGGGCAGGCAAC TCC ATGAGGCTT AAT C AG ATT ACT GTTTT CTTT GAC AGT AGT G ATGGT GAG GGAGTTGAAGCTTGGGTGGGGTTGCCCAGAAAAGACATGGCTCGATTTGA AAAAGATTCTGGCATCCTTGCTTACACTTCCCCTAATCCAAGCATATTTTG AGGGTTTATTTATTTTTTATTGCACTGTTTGTTATTTGTACTGGCTTGCTGG GAACATATTCTGGCAAATTTCGCTGATGCAAGTATCATTCTCCATAAAAAT GTCAAAAAAAAAAAAAAAAA INFORMATION FOR SEQ ID NO: 20B (CLE75; LAAT2) SEQUENCE CHARACTERISTICS: LENGTH: 426 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 20B 116 MDLQITCTEIIKPSSPTPQHQSTYKLSIIDQLTPNVYFSIILLYSKAGESTAKTSD HLKESLSNTLTHYYPLAGQLKYDQLIVDCNDQGVPFIEAHVTNDMRQLLKIP NIDVLEQLLPFKPHEGFDSDRSNLT V QVNYF GCEGMAIGLCFRHKVID ATT AA FFVKNWGVIARGAGEIKDVIIDHASLFPARDLSCLTKSVDEEFLKPESETKRFV FDGATIASLQETFASFERRPTRFEVVSAVILGALITATRESDDESNVPERLDTIIS VNLRQRMNPPFPEHCMGNIIS GGLVYWPLEKKVD YGCLAKEIHESIKKVDDQ FARKFYGDAEFLNLPRLAGAEDVKKREFWVTSWCKTPLYEADFGWGNPKW AGNSMRLNQITVFFDSSDGEGVEAWVGLPRKDMARFEKDSGILAYTSPNPSIF INFORMATION FOR SEQ ID NO: 21A (CLF35; LAAT3) SEQUENCE CHARACTERISTICS: LENGTH: 1648 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 21A GCTAGGCTGGCTTTCATTTAGCTTCCATCTCTTTCTCTCTGTCAATAACTCA TGGCTGCAATTGAAAACAGAGTAACACTAAAGAAGCATGAGGTTACCAA AGTCACCCCTTTCGTCAACCCCAACTCAAAGACGACGTCGTTTACTCTCGA TCTCACCTATTTCGACTTTTTCTGGTTCAAGAATCCTCCTGTGGAACGCCTC TTCTTCT AT GAG ATGACT G ACTTG ACGT GGGATTT ATT C AACT C GGAGATC CTCCCAAAGCTGAAGCACTCCCTTTCCTTCACTCTCCTTCATTACCTCCCTC TTGCTGGTCACATCATGTGGCCGCTGGATGCCGCAAAGCCTGCCGTCTACT ACTTTCCCG ACC AAAACG ACGGCGTTT C ATT C GC AGTTGCT GAGTGGTCTT WO 00/32789 117 PCT/NL99/00737 CCGAGTGCCACGCAGGCTTCCATCACCTCTCCGGCAACGGAATCCGCCAA GCAGTTGAATTTCATCCTCTTGTGCCCCAGTTGTCGCTTACGGACGATAAA GCTGAGGTAATTGCCATCCAAATAACACTGTTTCCGAATCAAGGCTTCTCA ATTGGTGTTTCATCTCACCATGCAATTCTTGATGGAAAAACTTCGACCTTG TTCCTGAAATCTT GGGCTT ATTT GT GC AAAC AATT ACAATT AT GCCATCAC CCTTGTTTGTCACCTGAACTAACCCCTCTTCTCGACCGGACTGTCATCAAA GATCCGACAGGTCAGGACATGCTGCAA.CTGAATAAGTGGGTTGTCGGGTC GGATAATTCGGATCCCCAGAAGATACGGAGCTTGAAGGTTTTACCATTCT TAGACTCTGAGTCTCTGAACAAATTGGTCCGAGCCACATTTGAGTTGACG CGTGAAGATATTACGAAACTCAGGCACAAGGTTAATCATCAGTTATCAAA ATC AT C AAAAT C AAAGC AAGTTCGTTT ATC AACTTTT GTGCTT AC ATT AGC TTATGTGTTTGTTTGCATGGCTAAAGCTAAATTAGCCAAAGCCAAAACTGA AGCTGAAGCTGCAGCAGGTAATGATGAAATTAAAAATATTATTGTGGGAT TCACTGCGGATTATAGGAGCCGTTTGGATCCTCCAATTCCACTTAATTATT TTGGTAACTGCAATGGGAGACATTGTGAGACTGCAAAAGCAAGTGATTTC GTTCAAGAAAATGGGGTTGCTTTTGTTGCAGAGATGTTAAGTGATATGGTC AAAGGGATCGAT GCGGAT GC C ATTG AAGCC AAT GATGAT AAGGTTTC AGA AATATTGGAAATTCTGAAAGAAGGAGCAATGATTTTTTCTGTGGCTGGCTC GACCCAATTTGATGTTTACGGGTCGGATTTCGGGTGGGGGAGGCCCAAGA AGGTGGAGATTGTGTCAATAGATAGGACACAAGCCATCTCTTTGGCAGAG AGAAGAGATGGAGGAGGCGGCGTTGAGGTTGGAGTTGTTTTAGAGAAGC AACAAATGGAGGTTTTTGAATCTGTATTTGCTGATGGACTGAAAAATGAT CTTGTTTAATT AATGATGT ATCATCTAAATTTCTC AATAT ATT ATTGGTC AT ATTC AAAAG A AAT AAATT ATT GCGG ATTTTTGT G ACC ACC AAAT AAAAT A CTCTTTTTTGAAAAAAAAAAAAAAAAAA WO 00/32789 n8 PCT/NL99/00737 INFORMATION FOR SEQ ID NO: 21B (CLF35; LAAT3) SEQUENCE CHARACTERISTICS: LENGTH: 491 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 21B MAAIENRVTLKKHEVTKVTPFVNPNSKTTSFTLDLTYFDFFWFKNPPVERLFF YEMTDLTWDLFNSEILPKLKHSLSFTLLHYLPLAGHIMWPLDAAKPAVYYFP DQNDGVSFAVAEWSSECHAGFHHLSGNGIRQAVEFHPLVPQLSLTDDKAEVI AIQITLFPNQGFSIGVSSHHAILDGKTSTLFLKSWAYLCKQLQLCHHPCLSPEL TPLLDRTVIKDPTGQDMLQLNKWVVGSDNSDPQKIRSLKVLPFLDSESLNKL VRATFELTREDITI^LRHKVNHQLSKSSKSKQVRLSTFVLTLAYVFVCMAKAK LAKAKTEAEAAAGNDEIKNIIV GFT ADYRSRLDPPIPLNYFGNCNGRHCETAK ASDFVQENGVAFVAEMLSDMVKGIDADAIEANDDKVSEILEILKEGAMIFSV AGSTQFDVY GSDFGWGRPKKVEIV SIDRTQAISLAERRDGGGGVEV GVVLEK QQMEVFESVFADGLKNDLV WO 00/32789 PCT/NL99/00737 INFORMATION FOR SEQ ID NO: 22A (CLB82; LAAT4) SEQUENCE CHARACTERISTICS: LENGTH: 1520 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 22A AACATGGC AGC AAGCTCACT GC ATGGC AAAGAAGCTACAGTT AT ATATCC TTCTGAGCCAACCCCATCTACGGTTTTGTCTCTCTCAGCTCTTGATTCTCAG CTTTTCTTGCGTTTCACTATTGAGTATCTCTTGGTCTATAGACCTCGCCCTG GTTTGGACCCACTTGCTACCGTGGCTCGTGTCAAGTCCGCACTCGCCAAAG CCTTGGTTCCTTACTATCCCCTCGCGGGTCGGGTCAGAGCTAAACAAGAC GGGTCGGGCTTATTGGAAGTCGTGTGTCTAGGCCAAGGCGCTGTGTTCAT CGAAGCCGTCGACCGTGAAAGTACGATCACCGATTTTGAGAGTGCTCCCA GGTATGTTACTCAGTGGAGGAAACTGCTGTCGTTATACGTGGCGGATGTTC TCAAAGGGGCCCCACCTCTTGTCGTTCAGCTGACTTGGCTTAGAGATGGA GCCGCAGCGCTCGGTATTGGCTTTAACCATTGTGTTTGCGATGGTATCGGC AGCGCCGAGTTCCTCAACTTGTTTACTGAGTTATGTACGAGCCGTCATAAC GAACTGGGTGGTGGCCATTCTCTGCCGAAACCCGTTTGGGATCGCCACCT AATGAACTCCTCCTCATCACGTCAACAGCATGCAGATACACGTGCCAGCT CAGTGAGTCACCTGGAATTCAACAGAGTGGCTGATCTTTGTGGTTTTGTTT CTCGTTTTTCCAACGAAAGGCTTGTTCCCACTTCAATAACGTTCGATAAAC GACGCTTAAACGAGCTGCGGAAGCTGGCTCTGTCCACGAGTCGACCCAGT GAGCTGGCTTACACGTCATTTGAAGTTCTTTCAGCTCATGTGTGGAGAAGC WO 00/32789 PCT/NL99/00737 120 TGGGCTAGGTCGTTGAATCTTCCGTCGAATCAAATCTTGAAGCTTCTATTT AGCATCAATGTACGTAACCGTGTCAAGCCGAGTCTCCCCAGTGGCTATTA TGGCGAT GC ATTT GT ATT AGGCT GTGCTC AAACGAGGGTT AAAGATTTGA CAGAGAAGGACTTAGGGCATGCAGCAATGTTGGTTAAAAAGGCGAAAGA GAGAGTTGATAGTGAGTATGTGAAGTCGGTCATCGACTCAGTGAGTCACA CGAGAGCGTGTCCCGACTCAGTCGGGGTGTTGATAGTGTCGCAGTGGTCA AGGCTAGGGTTAGAGAGAGTTGACTTTGGGATGGGGAGGCCGACTCAAGT GGGTCCCATTTGCTGCGACAGGTATTGCCTGTTTCTACCGGTTTTCAATCA GACGGACGCTGTTAAGGTGATGGTGGCGGTCCCCACAAGTGCAGTTGACA AGTATGAGCATCTCGCGAAGGGCTTATGCTGGTGAGGACCACACCGCATG ATGACCCCACCATGTAATACGTTGACTTATAAACTCAGTTTGACTTTTAAC TTTTTTAACAAGTGATGGAATTTCAGTGATTGACTCATCACTTTGATCCTG ATCCAATAAATAATTGAATTGAGTTCAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA INFORMATION FOR SEQ ID NO: 22B (CLB82; LAAT4) SEQUENCE CHARACTERISTICS: LENGTH: 447 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 22B MAASSLHGKEATVIYPSEPTPSTVLSLSALDSQLFLRFTIEYLLVYRPRPGLDPL ATVARVKSALAKALVPYYPLAGRVRAKQDGSGLLEVVCLGQGAVFIEAVDR 121 ESTITDFESAPRYVTQWRKLLSLYVADVLKGAPPLWQLTWLRDGAAALGIG FNHCVCDGIGSAEFLNLFTELCTSRHNELGGGHSLPKPVWDRHLMNSSSSRQ QHADTRASSVSHLEFNRVADLCGFVSRFSNERLVPTSITFDKRRLNELRKLAL STSRPSELAYTSFEVLSAHVWRSWARSLNLPSNQILKLLFSINVRNRVKPSLPS GYYGDAFVLGCAQTRVKDLTEKDLGHAAMLVKKAKERVDSEYVKSVIDSV SHTRACPDSVGVLIVSQWSRLGLERVDFGMGRPTQVGPICCDRYCLFLPVFN QTDAVKVMVAVPTSAVDKYEHLAKGLCW INFORMATION FOR SEQ ID NO: 23A (MAY5; MEAAT) SEQUENCE CHARACTERISTICS: LENGTH: 1468 TYPE: cDNA STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: HONEY DEW MELON ALCOHOL ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 23A ATGACTTCTCTTTTCACGTACGAAAATGCCAACCAGAATTGATTGCACCAG CAAATCCTACACCCTATGAATTTAAACAACTTTCTGATGTGGATGATCAAC AAAGCTTAAGGCTTCAATTGCCATTCGTAAATATCTATCCCCATAATCCAA GTTTGGAGGGAAGAGAT CC AGT G AAGGT AAT AA AGG AAGC AATTGG AAA GGCGTT GGTGTTCT ACT AT C CTTT AGC AGGA AG ATTG AGAGAAGGGCCAG GTAGAAAGCTTTTTGTTG AATGT AC AGGT G AAGGAAT CTT GTTT ATT GAAG CGGATGCAGATGTGAGCTTAGAAGAATTTTGGGATACTCTTCCATATTCAC 122 .. TTTC AAGC ATGC AG AAC AAT ATT AT AC AT AACGCTTT AAATT CTGATG AAG TCCTCAATTCTCCATTATTGCTCATTCAGGTGACACGACTCAAGTGTGGAG GTTTC ATTTTTGGT CTTT GTTTC AATC AT ACT ATGGCAGAT GGTTTTGGT AT TGTCCAATTCATGAAGGCTACAGCGGAGATAGCTCGTGGAGCTTTTGCTC CATCTATTTTACCAGTATGGCAAAGAGCTCTCTTAACCGCAAGAGACCCTC CCAGAATCACTTTTCGCCACTATGAATACGACCAAGTAGTCGACATGAAG AGCGGCCTCATTCCAGTCAATAGCAAGATCGATCAATTATTCTTCTTTAGC CAACTTCAAATCTCCACCCTTCGCCAAACTTTGCCAGCCCACCTTCACGAT TGCCCTTCCTTCGAGGTCCTCACTGCCTATGTTTGGCGCCTCCGTACCATA GCCCTTC AATTT AAGCC AGAGG AGGAAGTGCGGTTT CTTT GCGT AAT GAA TCTACGCTCGAAGATCGACATACCATTAGGGTATTATGGTAATGCGGTAG TTGTTCCTGCAGTAATCACCACCGCTGCGAAGCTTTGTGGGAACCCACTTG GTTATGCTGTAGACTTGATTAGGAAGGCCAAGGCTAAGGCAACGATGGAG TACATAAAGTCTACGGTGGATCTTATGGTGATTAAAGGACGACCCTATTTC ACTGTAGTTGGATCATTTATGATGTCAGACCTAACGAGAATTGGGGTTGA AAACGTGGACTTTGGATGGGGAAAGGCCATTTTTGGAGGACCTACAACCA CAGGGGCCAGAATTACACGAGGTTTGGTAAGCTTTTGTGTACCTTTCATGA AT AGAAAT GGAGAAAAGGGAACT GCGTT AAGTCT AT GCTTGCCTCCTCCA GCCATGGAAAGATTTAGGGCAAATGTTCATGCCTCGTTGCAAGTGAAACA AGTGGTTGATGCAGTTGATAGCCATATGCAAACTATTCAATCTGCTTCGAA ATAAATAATATTGTTGAAGGTGGGTCTGAGTTGACTCGACCATATCGATG CATGCAAGCTTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTG CTGT INFORMATION FOR SEQ ID NO: 23B (MAY5; MEAAT) WO 00/32789 PCT/NL99/00737 123 SEQUENCE CHARACTERISTICS: LENGTH: 456 TYPE: Peptide STRANDNESS: Single TOPOLOGY: Linear OTHER INFORMATION: LEMON ACYL TRANSFERASE SEQUENCE DISCRETION FOR SEQ ID NO: 23B MDFSFHVRKCQPELIAPANPTPYEFKQLSDVDDQQSLRLQLPFVNIYPHNPSL EGRDPVKVIKEAIGKALVFYYPLAGRLREGPGRKLFVECTGEGILFIEADADV SLEEFWDTLPYSLSSMQNNIIHNALNSDEVLNSPLLLIQVTRLKCGGFIFGLCF NHTM ADGF GIV QFMKAT AEI ARG AF APSILP V W QRALLT ARDPPRITFRHYE Y DQWDMKSGLIPVNSKIDQLFFFSQLQISTLRQTLPAHLHDCPSFEVLTAYVW RLRTIALQFKPEEEVRFLCVMNLRSKIDIPLGYYGNAVWPAVITTAAKLCGN PLGYAVDLIRKAKAKATMEYIKSTVDLMVIKGRPYFTWGSFMMSDLTRIGV ENVDFGWGKAIFGGPTTTGARITRGLVSFCVPFMNRNGEKGTALSLCLPPPA MERFRANVHASLQVKQWDAVDSHMQTIQSASK 124

Claims (48)

Claims
1. An isolated nucleic acid sequence encoding a protein comprising the amino acid sequences LxxxYPxxGR, wherein x constitutes any amino acid sequence, and said protein having alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
2. An isolated nucleic acid sequence encoding a protein comprising the amino acid sequence LxxxYPxxGR and either of WTNFFNPLDFGW or a 70% homologue of this peptide and PSRVxxVTxFLxKxLI, wherein x constitutes any amino acid sequence, and said protein having alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
3. An isolated nucleic acid sequence encoding a protein which has at least 70% homology w ith a protein or fragment thereof encoded by a nucleic acid sequence according to claim 1 or claim 2.
4. An isolated nucleic acid sequence encoding a protein which has at least 80% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 3B, said protein or a fragment thereof has aminotransferase activity and is involved in the biosynthetic pathway for aliphatic and /or aromatic ester production in fruit.
5. An isolated nucleic acid sequence encoding a protein which has at least 90% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 4B, said protein or a fragment thereof has thiolase activity and is iNTnilSCrUAL PROPERTY Grr;CE OF N.Z. 125 involved in the biosynthetic pathway for ahphatic and/or aromatic ester production in fruit.
6. An isolated nucleic acid sequence encoding a protein which has at least 90% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 5B, said protein or a fragment thereof has pyruvate decarboxylase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
7. An isolated nucleic acid sequence encoding a protein which has at least 75% homology with a protein having an amino acid sequence as shown in SEQ ID NO: 6B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
8. An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 7B, 8B, 9B and 10B, or which has at least; i) 70% homology with the amino acid sequence of a 326 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 7B, or ii) 75% homology with the amino acid sequence of a 278 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 8B, or iii) 70% homology with the amino acid sequence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 9B, or 126 iv) 80% homology with the amino acid sequence of a 188 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 10B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
9. An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 11B, or which has at least 75% homology with the amino acid sequence of a 181 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 11B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
10. An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 12B and 13B, or which has at least; 70% homology with the amino acid sequence of a 176 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 12B, or 70% homology with the amino acid sequence of a 284 aa fragment from the C terminal end of the coding sequence of SEQ ID NO: 13B, said protein or a fragment thereof has alcohol dehydrogenase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
11. An isolated nucleic acid sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 15B, or which has at least 80% homology 127 with the amino acid sequence SEQ ID NO: 15B, said protein or a fragment thereof has esterase activity and is involved in the biosynthetic pathway for ahphatic and/or aromatic ester production in fruit.
12. 12. A purified and isolated protein encoded by a nucleic acid according to any of claims 1-3, said protein having alcohol acyl transferase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
13. 13. A purified and isolated protein encoded by a nucleic acid according to claim 4, said protein having aminotransferase activity and is involved in the biosynthetic pathway for aliphatic and /or aromatic ester production in fruit.
14. 14. A purified and isolated protein encoded by a nucleic acid according to claim 5, said protein having thiolase activity and is involved in the biosynthetic pathway for ahphatic and/or aromatic ester production in fruit.
15. 15. A purified and isolated protein encoded by a nucleic acid according to claim 6, said protein having pyruvate decarboxylase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit.
16. 16. A purified and isolated protein encoded by a nucleic acid according to any of claims 7-10, said protein having alcohol dehydrogenase activity and is involved in the biosynthetic pathway for ahphatic and/or aromatic ester production in fruit.
17. 17. A purified and isolated protein encoded by a nucleic acid according to claim 11, said protein having esterase activity and is involved in the biosynthetic pathway for aliphatic and/or aromatic ester production in fruit. 128
18. 18. A vector comprising a nucleic acid according to any of claims 1-11.
19. 19. A vector according to claim 18, wherein said nucleic acid according to any of claims 1-11 is aligned for anti-sense expression.
20. 20. A host transformed or transfected with a vector according to claim 18 or claim 19.
21. 21. A host according to claim 20, wherein said host is a fruit bearing plant.
22. 22. A genetically modified microorganism, plant cell or plant and propagating material thereof having within its genome a vector according to claim 18 or claim 19.
23. 23. A method for producing or enhancing the production of volatile esters in a microorganism, plant cell or plant comprising i) inserting into the genome of said microorganism, plant cell or plant at least one copy of a nucleic acid sequence according to any of claims 1-11 or a vector according to claim 18. ii) culturing said micro-organism or plant (cell) under conditions optimal for gene expression. iii) optionally feeding alcohols and an acyl-CoA and/or aldehydes and acyl-CoA and/or alpha-keto acids and acyl-CoA and/or amino acids and acyl-CoA and/or amino acids and fatty acids to said microorganism or plant (cell). iv) optionally isolating/capturing the product. | i^TZLLHCTLi/.L F.cC^irfiT I Gri:iCE Cr Is!.-. 1 ;. r •:-» Of0*;3 """ 'V i J LwCJ;I;1;129;
24. 24. A method for regulating aromatic and/or aliphatic esters ester formation in fruit, comprising inserting into the genome of a fruit-producing plant one or more copies of one or more nucleic acid sequences as defined in any of claims 1-11, or a vector according to claim 18.;
25. 25. A method according to claim 23 or 24, wherein said plant is strawberry,;citrus (lemon), banana, apple, pear, melon, tomato, sweet pepper, peach or mango.;
26. 26. A method for suppressing the production of volatile esters in a microorganism, plant cell or plant capable of producing volatile esters, comprising i) inserting into the genome of said microorganism, plant cell or plant, a vector according to claim 19 or ii) a nucleic acid according to any of claims 1-11 aligned for sense expression.;iii) culturing said micro-organism or plant (cell) under conditions optimal for gene expression.;
27. 27. A method according to claim 26, wherein said plant is strawberry, citrus, (lemon), banana, apple, pear, melon, tomato, sweet pepper, peach or mango.;
28. 28. A method for producing a flavor and/or antimicrobial compound comprising;;i) transforming or transfecting a suitable host with a vector according to claim 18,;ii)culturing said host under conditions suitable for the expression of said nucleic acid,;iii) optionally isolating the formed product.;razors n rr r* 130
29. 29. A method for producing a flavor and/or antimicrobial compound in a cell-free lysate expression system comprising expressing at least one nucleic acid according to any of claims 1-11 in the presence of a suitable substrate and optionally isolating the formed product, wherein said free lysate system contains all the components necessary for expression of said nucleic acid and processing of the expressed product.
30. 30. A flavor and/or antimicrobial compound comprising a protein according to any of claims 12-17.
31. 31. Use of a purified protein according to any of claims 12-17, for the production an antibody to inhibit the synthesis of a compound according to claim 30.
32. 32. An antibody to a protein according to any of claims 12-17.
33. 33. Use of a compound according to claim 30; (i) as an anti-microbial agent (ii) as a pest controlling agent. (iii) in the processed food industry as a food additive to enhance the flavor of syrups, ice-creams, frozen desserts, yogurts, confectionery and like products and/or as an anti-fungal agent. (iv) as a flavoring agent for oral medications and vitamins and/or as an anti-fungal agent. (v) for providing additional flavor/aroma in beverages, including alcoholic beverages. (vi) for enhancing or reducing plant flavor/aroma/fragrance/scent. (vii) for enhancing the flavor/aroma of natural products and/or synthetic and/or artificial products. ! iNiaiEau; L ?;;crzii7Y GrrJCE C? N.Z. - h AUG :: :s ; EJ¥r;n 131 (viii) for the industrial synthesis of nature identical flavor/aroma substances and/or artificial flavor/aroma substances. (ix) for the biological control of the interaction between plants and insects and/or the interaction between plants and micro-organisms. (x) for providing flavor/aroma in cosmetics, creams, sun-protectant products, hair conditioners, cleaning products, personal care products and health care products. (xi) as a disinfectant additive.
34. 34. Use of a nucleic acid according to anyone of claims 1-11 as a molecular marker or diagnostic tool.
35. 35. A composition comprising a flavor and/or antimicrobial compound according to claim 30.
36. 36. A composition according to claim 35 which is a pharmaceutical.
37. 37. A composition according to claim 35 which is a neutraceutical.
38. 38. Use of a composition according to claim 35 for augmenting or enhancing the aroma and/or taste of food or non food products and/or protection of food or non food products against fungal contamination and/or pest infestation.
39. 39. Use of a composition according to claim 35 for the biological control of pests.
40. 40. Use of a composition according to claim 35 for the protection of stored products. 132
41. 41. A diagnostic kit for screening fruit for the presence of volatile aliphatic and/or aromatic ester compounds comprising; i) one or more purified proteins as claimed in any of claims 12-17, or ii) one or more nucleic acid sequences as claimed in any of claims 1-11, or iii) one or more antibodies as defined in claim 32.
42. 42. A method for screening fruit i) for fruit quality trait attributes, such as flavor, fragrance, aroma, scent, texture, shape, or ii) to distinguish between cultivars/varieties of fruits based on their volatile ester profiles, or iii) to monitor harvest time of fruit(s) for quality trait attributes, or iv) to monitor post-harvest fruit(s) for quality trait attributes, or v) to monitor shelf-life of fruits, or vi) to monitor timing for application of pesticides based on volatile ester profiles, or vii) to monitor fruit resistance capacity based on their volatile ester profiles, comprising detecting the presence of; i) one or more purified proteins as claimed in any of claims 12-17, or ii) one or more nucleic acid sequences as claimed in any of claims 1-11, or iii) one or more antibodies as defined in claim 32. 133 at routine time points using the diagnostic kit of claim 41, and using this as an indicator of the type or amount of volatile esters being released from the fruit at a given time point.
43. 43. An isolated nucleic acid as claimed in any one of claims 1-11, substantially as hereinbefore described with reference to any example thereof.
44. 44. A purified isolated protein encoded by a nucleic acid according to claim 43.
45. 45. A vector comprising a nucleic acid according to claim 43.
46. 46. A host transformed or transfected with a vector according to claim 45.
47. 47. A method as claimed in any one of claims 23-29 wherein the nucleic acid or vector comprises a nucleic acid of claim 43.
48. 48. An antibody to a protein according to claim 44. 3 I OCT 2003 .RECEIVED