WO2007095682A1

WO2007095682A1 - Methods and micro-organisms for modulating the conversion of non-volatile sulfur compounds to volatile thiol compounds

Info

Publication number: WO2007095682A1
Application number: PCT/AU2007/000199
Authority: WO
Inventors: Isak Stephanus Pretorius; Jan Hendrik Swiegers
Original assignee: Australian Wine Research Institute Ltd
Current assignee: Australian Wine Research Institute Ltd
Priority date: 2006-02-24
Filing date: 2007-02-23
Publication date: 2007-08-30
Anticipated expiration: 2008-08-24

Abstract

The present invention relates to a method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound. The method includes either or both of the following steps: (i) exposing the non-volatile sulfur compound to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon- sulfur lyase enzyme capable of converting the non-volatile sulfur compound to volatile thiol compound; and (ii) exposing the non-volatile sulfur compound to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non- volatile sulfur compound to volatile thiol compound.

Description

METHODS AND MICRO-ORGANISMS FOR MODULATING THE CONVERSION OF NON-VOLATILE SULFUR COMPOUNDS TO VOLATILE THIOL

COMPOUNDS

This application claims priority from Australian Provisional Patent Application No. 2006900917 filed on 24 February 2006, the contents of which are to be taken as incorporated herein by this reference.

Field of the Invention

The present invention relates to a method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound.

The present invention also relates to a micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme.

Background of the Invention

The aroma of a product is often one of the most important factors in determining the quality and intrinsic value of the product. For example, small variations in the presence and concentration of volatile aroma compounds can mean the difference between a premium and an average table wine.

In the case of wine, some of the most potent aroma compounds are the volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-l-ol (3MH) and 3-mercaptohexyl acetate (3MHA). The thiol 4MMP has the lowest sensory detection threshold of any volatile thiol, with reported values in water and wine being 0.1 ng I^"1 and 3 ng I^"1, respectively. The concentration found in wine, up to 30 ng I^"1, indicates the importance of these compounds to the aroma of wine.

In wine made from Vitis vinifera var. Sauvignon blanc, volatile thiols are of particular importance to the varietal character as they impart box tree, passionfruit, grapefruit, gooseberry, guava, and at high concentrations, sweaty aromas. 4MMP, 3MH and 3MHA have also been identified in wines made from Vitis vinifera var. Riesling, Colombard, Semillon, Cabernet Sauvignon and Merlot in varying concentrations, and as such can potentially impact on the aroma of wine made from these varieties.

The volatile thiols are almost non-existent in the grape juice and develop during fermentation. For example, 4MMP and 3MH exist in the grapes in the form of aroma- inactive, non-volatile, cysteine bound conjugates and develop during fermentation by conversion of the precursors present in the grape to their volatile form.

Given that small variations in the presence and concentration of volatile aroma compounds can have a significant effect on the quality of products such as wine, there exists a need to be able to modulate the aroma of such products. The present invention relates to a method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound, by exposing the non-volatile sulfur compound to a microorganism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of the Invention

The present invention arises from studies into the overexpression of the tnaA gene in a commercial wine yeast. In model fermentations, the modified yeast results in a significant increase in the amount of 4-mercapto-4-methylpentan-2-one (4MMP) released from the cysteine bound precursor. Sauvignon blanc wine made with the modified yeast shows a significant increase in desirable passionfruit and box tree aromas.

This is in contrast to previous studies which have indicated that although deletion of endogenous genes in S. cerevisiae may lead to a reduction in the release of some volatile thiol compounds from their precursors, over expression of the genes did not result in an increase in the release of the volatile thiol compound. The present invention provides a method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound, the method including either or both of the following steps:

(i) exposing the non-volatile sulfur compound to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and

(ii) exposing the non-volatile sulfur compound to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound.

The present invention also provides a product with an altered release of a volatile thiol compound produced by the above described method.

The present invention also provides a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme.

The present invention also provides a micro-organism including an exogenous nucleic acid encoding a carbon-sulfur lyase or part thereof.

The present invention also provides a method of modulating the aroma of a product including a non-volatile sulfur compound, the method including either or both of the following steps:

(i) exposing the product to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and (ii) exposing the product to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; wherein the aroma of the product is modulated by the conversion of the non-volatile sulfur compound to a volatile thiol compound by the carbon-sulfur lyase.

The present invention also provides a product with altered aroma produced by the above described method.

The present invention also provides a method of modulating the aroma of a wine product including a non-volatile sulfur compound, the method including exposing the product to an isolated enzyme having a carbon-sulfur lyase enzyme activity capable of converting the non-volatile sulfur compound to a volatile thiol compound, wherein the aroma of the product is modulated by the conversion of the non-volatile sulfur compound to a volatile thiol compound by the carbon-sulfur lyase.

The present invention provides a method of fermenting a product including a non- volatile sulfur compound, the method including either or both of the following steps:

(i) exposing the product to a genetically altered sugar-fermenting microorganism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and (ii) exposing the product to an extract derived from a genetically altered sugar-fermenting micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non- volatile sulfur compound to a volatile thiol compound.

The present invention also provides a method of detecting a precursor of a volatile thiol compound in a sample, the method including:

(i) exposing the sample to a carbon-sulfur lyase enzyme capable of converting the precursor to a volatile thiol compound; and

(ii) assaying for production of a volatile thiol compound by the carbon-sulfur lyase enzyme; wherein the production of a volatile thiol compound by the carbon-sulfur lyase enzyme is indicative of the presence of the precursor of a volatile thiol compound in the sample. The present invention also provides a fermented product produced by the above described method.

The present invention also provides a fungus including a nucleic acid encoding an E.coli tnaA gene, or a functional part thereof.

The present invention also provides an isolated nucleic acid including a transcriptional control sequence functional in a eukaryotic cell operably linked to a nucleic acid encoding a carbon-sulfur lyase.

Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.

The term "carbon-sulfur lyase" as used throughout the specification is to be understood to mean any polypeptide or protein that catalyzes the cleavage of a carbon-sulfur bond by means other than hydrolysis or oxidation. The term includes within its scope any naturally occurring carbon-sulfur lyase enzymes, a fragment/part of the enzyme that retains enzymatic activity, or a variant of the enzyme that retains enzymatic activity, or a synthetic analogue of any of the preceding enzymes.

The term "isolated" as used throughout the specification is to be understood to mean an entity, for example a nucleic acid, an enzyme, a polypeptide, cell, micro-organism, plasmid , or vector, which is semi-purified, purified and/or removed from its natural environment. For example, an isolated enzyme may be a substantially purified form of the enzyme, or an extract containing the enzyme.

The term "micro-organism" as used throughout the specification is to be understood to mean a micro-organism that has the capacity to express a functional carbon-sulfur lyase enzyme, including a micro-organism such as a bacterium or a fungus (eg a yeast, a mold). A "genetically altered micro-organism" is a micro-organism that has had its nucleic acid content (genomic and/or extra-genomic) altered by artificial manipulation, or a cell that is a progeny or derivative of the originally altered cell. For example, the nucleic acid content may be altered by introducing or transferring a nucleic acid into the cell. In this case, the introduced nucleic acid may for example be an oligonucleotide or a polynucleotide, such as a gene encoding a carbon-sulfur lyase enzyme (or a functional part thereof), a plasmid or a vector, an anti-sense nucleic acid, a siRNA or a ribozyme.

Other methods of genetically altering an organism include random or directed mutagenesis.

Methods for genetically altering organisms are known in the art, for example as in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference.

In this regard, the process of transformation will be understood to be the process by which exogenous DNA enters a recipient cell. It may occur under natural or artificial conditions using various methods known in the art, including transformation with calcium chloride or calcium phosphate, phage or viral infection, mating, electroporation, lipofection, and particle bombardment. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, or cells which transiently express the inserted DNA or RNA for limited periods of time. Methods for introducing exogenous DNAs into cells are described for example in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference.

The nucleic acid content of a micro-organism may also be altered, for example, by such techniques as mutagenesis including random mutagenesis (including chemical mutagenesis, UV mutagenesis and transposon-mediated mutagenesis), site-directed mutagenesis, and phage or virus mediated mutagenesis. Methods for mutagenesis of micro-organisms are also known in the art, for example as described in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference.

The term "variant" as used throughout the specification is to be understood to mean an amino acid sequence of a polypeptide or protein that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids. The term also includes within its scope any insertions/deletions of amino acids to a particular polypeptide or protein. A "functional variant" will be understood to mean a variant that retains the functional capacity of a reference protein or polypeptide.

The term "nucleic acid" as used throughout the specification is to be understood to mean to any oligonucleotide or polynucleotide. The nucleic acid may be DNA or RNA, and may be single stranded or double stranded. The nucleic acid may be any type of nucleic acid, including a nucleic acid of genomic origin, cDNA origin (ie derived from a mRNA), viral origin, or of synthetic origin.

In this regard, an oligonucleotide or polynucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups to facilitate the function of the nucleic acid. The oligonucleotide or polynucleotide may be modified at any position on its structure with constituents generally known in the art. For example, an oligonucleotide may include at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyliydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta D- mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3- amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.

The oligonucleotide or polynucleotide may also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In addition, the oligonucleotide or polynucleotide may include at least one modified phosphate backbone, such as a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or any analogue thereof.

The term "amplification" or variants thereof as used throughout the specification is to be understood to mean the production of additional copies of a nucleic acid sequence. For example, amplification may be achieved using polymerase chain reaction (PCR) technologies (for example as described in Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., herein incorporated by reference, and The Nucleic Acid Protocols Handbook ed. by Ralph Rapley (2000) Humana Press New Jersey, which is herein incorporated by reference) or by other methods of amplification, such as rolling circle amplification on circular templates, such as described in Fire, A. and Xu, S-Q. (1995) Proc. Natl. Acad. Sci 92:4641-4645, herein incorporated by reference.

Brief Description of the Figures

Figure 1 shows plasmids constructed and used in this study.

Figure 2 shows the biochemical mechanism through which cysteine-β-lyase enzymes convert grape-derived, non-volatile, cysteinylated thiol precursors into aromatic thiols in wine.

Figure 3 shows the cysteine-β-lyase activity measured for a Saccharomyces cerevisiae laboratory strain (∑1278b) and a strain [∑1278b(CSLi)] transformed with the CSLl gene cassette. Reactions were run for 60 min after which lactic dehydrogenase and NADH were added to indirectly measure pyruvate release. Pyruvate is converted to lactate resulting in the consumption of NADH (absorbance measured at 340 nm; A₃₄₀). Reactions were allowed to proceed for 75 min and 90 min after which the A3₄0 values were measured. At a 100 min, Ellman's reagent was added which reacts with thiols to form a yellow coloured complex (absorbance measured at 412 nm; A_4I2).

Figure 4 shows release of the volatile thiol 4MMP from the precursor Cys-4MMP. Yeast strains VINl 3 and VrNO(GS¹ZJ) were fermented at 3O⁰C for 2 days in SCD medium (containing 2% glucose) and 16 mg/1 Cys-4MMP.

Figure 5 shows release of the volatile thiol 3MH from the precursor Cys-3MH. VIN13 and VINO(CSZJ) were fermented at 28°C for 2 days in SCD medium (containing 4% glucose) and 2 mg/1 (A) and 0.5 mg/1 Cys-3MH. No 3MH and 3MHA could be detected in the VIN 13 ferments.

Figure 6 shows mean ratings for aroma attributes for the Sauvignon Blanc wine produced using the VIN 13 and VINO(C₁SZJ) strains (n=2 fermentation replicates x 14 judges x 3 presentation replicates), ns = not significant * P<0.05, *** P<0.001.

General Description of the Invention

As described above, in one embodiment the present invention provides a method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound, the method including either or both of the following steps: (i) exposing the non-volatile sulfur compound to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and

(ii) exposing the non-volatile sulfur compound to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound. This embodiment of the present invention is directed to a method of modulating the conversion of a non-volatile sulfur compound to a volatile thiol compound.

In one embodiment, the method may be used to alter the aroma of a product, such as a wine product, a food product, a beverage, or a food or beverage additive.

Carbon-sulfur lyases are a class of enzymes that catalyze the cleavage of a carbon-sulfur bond by means other than hydrolysis or oxidation, for example as described in Cooper et al. (2006) Amino Acids. 30: 1-15, herein incorporated by reference.

An appropriate carbon-sulfur lyase capable of converting the non-volatile sulfur compound into a volatile thiol compound may be selected by a person skilled in the art. Enzymes from other organisms, for example micro-organisms such as bacteria or fungi, may be identified by a person skilled in the art, for example by use of the BLAST algorithm to determine the extent of homology between two nucleotide sequences (blastn) or the extent of homology between two amino acid sequences (blastp). BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al. (1990) J. MoI. Biol. 215:403-410, herein incorporated by reference.

Examples of micro-organisms include bacteria such as Eschericia sp., Thermoanaerobacter sp.; Symbiobacterium sp; Photobacterium sp; Haemophilus sp.; Vibrio sp.; Proteus sp; Halobacterium sp.; Desulfitobacterium sp; and Treponema sp, and fungi. Examples of suitable fungi include yeasts, such as Saccharomyces cerevisiae (all strains), Saccharomyces bayanus and species of Brettanomyces and its sexual ('perfect') equivalent Dekkera; Candida; Cryptococcus; Debaryomyces; Hanseniaspora and its asexual counterpart Kloeckera; Kluyveromyces; Metschnikowia; Pichia; Rhodotorula; Saccharomyces; Saccharomycodes; Schizosaccharomyces; and Zygosaccharomyces , and molds (eg Aspergillus) Other yeasts include yeasts that are used in beer, whiskey, sake, cider production, or any other yeast used in the processing of plant derived raw material or extracts.

In one embodiment, the carbon-sulfur lyase is a cysteine-S-conjugate β-lyase. These enzymes, which are part of the large carbon-sulfur lyase enzyme family, involve the cleavage of a carbon-sulfur bond in a β-elimination reaction. The mechanism of cysteine-conjugate lyase enzymes is shown in Figure 1.

In one embodiment, the cysteine-S-conjugate β-lyase is a tryptophanase.

In a further embodiment, the tryptophanase is a tryptophanase from a micro-organism.

Examples of micro-organisms include bacteria such as Eschericia sp.,

Thermoanaerobacter sp.; Symbiobacterium sp; Photobacterium sp; Haemophilus sp.;

Vibrio sp.; Proteus sp; Halobacterium sp.; Desulfitobacterium sp; and Treponema sp, fungi such as yeasts (eg Saccharomycotina, Taphrinomycotina, and

Schizosaccharomycetes) and molds (eg Aspergillus).

In a specific embodiment, the tryptophanase is a bacterial tryptophanase, including a tryptophanase selected from the group of bacteria including Eschericia coli, Thermoanaerobacter ethanolicus; Symbiobacterium thermophilum; Photobacterium profundum; Haemophilus somnus; Vibrio splendidus; Proteus Vulgaris; Halobacterium sp.; Desulfitobacterium hafniense; and Treponema denticola.

Tryptophanase enzymes from other organisms, for example micro-organisms such as bacteria or fungi, may be identified by a person skilled in the art, for example by use of the BLAST algorithm to determine the extent of homology between two nucleotide sequences (blastn) or the extent of homology between two amino acid sequences

(blastp). BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al. (1990) /. MoI. Biol. 215:403-410, herein incorporated by reference.

In one specific embodiment, the tryptophanase is an Eschericia coli tryptophanase (see for example Deeley, M.C. and Yanofsky,C. (1981). "Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12" J. Bacteriol. 147(3): 787- 796, herein incorporated by reference). Accession numbers for E. coli tryptohanase tnaA (P0A853) are K00032 and X15974 (nucleotide sequences) and AAA24676.1 and (CAA34096.1 (amino acid sequences). The nucleotide sequence of the E.coli tnaA gene is designated SEQ ID NO.1. The amino acid sequence is designated SEQ ID NO.2.

In the case of the E. coli tryptophanase enzyme introduced into a micro-organism in the various relevant embodiments of the present invention, in one embodiment the nucleic acid includes a nucleotide sequence of SEQ ID NO: 1, or the nucleic acid includes a nucleotide sequence encoding a polypeptide including an amino acid sequence of SEQ ID NO:2, or a variant thereof, or a functional part thereof.

Examples of other cysteine-S-conjugate lyases include gamma-cystathionase (which produces sulfane sulfur from the disulfide-containing cystein S-conjugates present in allium extracts; for example NM OO 1902 - Homo sapiens cystathionase (cystathionine gamma- lyase) (CTH), transcript variant 1, mRNA; NM 153742 - Homo sapiens cystathionase (cystathionine gamma-lyase) (CTH), transcript variant 2, mRNA; and NM_017074 - Rattus norvegicus CTL target antigen (Cth), mRNA); and alliinase (which releases sulfur compounds from alliin (S-allyl cystein sulphoxide); IUBMB Enzyme Nomenclature EC 4.4.1.4).

Many plant derived products contain volatile thiols bound as cysteine S-conjugate precursors, and conversion of the non-volatile precursor to a volatile thiol product contributes to the aroma of a product. Examples of plant derived products include wine products such as wine, grape must, grape juice, and other products such as beer, whiskey, sake and cider.

However, it will be appreciated that even though the present invention is generally described in reference to the modulation of conversion of a non-volatile sulfur into a volatile thiol associated with a plant derived product, such as a wine, the present invention is not to be limited to the modulation of conversion of thiol compounds from plant derived products. As described above, in one embodiment the present invention may be used to alter the aroma of a product.

Accordingly, in another embodiment the present invention provides a method of modulating the aroma of a product including a non-volatile sulfur compound, the method including either or both of the following steps:

(i) exposing the product to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and

(ii) exposing the product to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; wherein the aroma of the product is modulated by the conversion of the non-volatile sulfur compound to a volatile thiol compound by the carbon-sulfur lyase.

The present invention also provides a product with altered aroma produced by this method.

In one embodiment, the product is a wine product. In one specific embodiment, the wine product is a wine (all grape varieties), such as a wine product produced from the genus Vitis, including the subgenus Euvitis (V. vinifera, V. riparia, V. berlandieri and V. aestivalis) and Muscadinia (V. rotundifolia, V. popenoei and V. mansoiana).

In this regard, some of the most potent aroma compounds found in wine are volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-l-ol (3MH) and 3-mercaptohexyl acetate (3MHA). 4MMP, for example, has the lowest sensory detection threshold of any yeast modified metabolite described, and plays a central role in the aroma of wine. For example, in wine made from Vitis vinifera var. Sauvignon Blanc, volatile thiols are of particular importance to the varietal character as it imparts box tree, passionfruit, grapefruit, gooseberry, and guava aromas.

In one embodiment, the present invention is used to modulate one or more aromas in a wine product, including aromas such as described as passionfruit, box tree, cat urine, broom, grapefruit, gooseberry, and guava. A person skilled in the art is able to identify and distinguish such aromas. It will also be appreciated that such aromas may have alternative descriptors, as is recognised in the art.

Examples of volatile thiol compounds and their non volatile precursors present in grapes are as follows:

4MMP (4-mercapto-4-methylpentan-2-one) and its non-volatile precursor Cys-4MMP

(4-(4-methylpentan-2-one)-L-cysteine); 3MHA (3-mercaptohexyl acetate) and its non-volatile pre-precursor Cys-3MH (3-

(hexan-l-ol)-L -cysteine); the direct precursor of 3MHA is 3MH (3-mercaptohexan-l- ol);

3MH (3-mercaptohexan-l-ol) and its non-volatile precursor Cys-3MH precursor (S-3-

(hexan-l-ol)-L -cysteine; and 4MMP0H (4-mercapto-4-methylpentan-2-ol) and its non-volatile precursor S-4-(4- methylpentan-2-ol)-L-cysteine.

It will be appreciated that in the various embodiments of the present invention that the non-volatile sulfur compound may be endogenously present in a product and/or may be an exogenous non-volatile sulphur compound added to a product. In this regard, it may desirable to spike a product with one or more non-volatile sulfur compounds, so as to produce desired aromas in the product after their conversion to volatile thiol compounds, by the methods of the present invention.

Accordingly, in another embodiment the product includes an endogenous non-volatile sulfur compound and/or an exogenous non-volatile sulfur compound. In one embodiment, the modulation of conversion of a non- volatile sulfur compound to a volatile thiol compound in the various embodiments of the present invention occurs in a product from a grape of the genus Vitis, including Vitis vinifera and its varieties. In one specific embodiment, the Vitis vinifera grape variety is Sauvignon Blanc, Riesling, Semillon, Chenin Blanc, Colombard, Cabernet Sauvignon, Merlot, Riesling, Gewurztraminer, Alsace Muscat, Manseng and Arvine.

Thus, in one embodiment the present invention may be used to modulate the aroma of a product from Vitis vinifera.

In one embodiment, the method is used to modulate the aroma of a wine product, such as modulating one or more of the passionfruit, box tree, cat urine, broom, grapefruit, gooseberry, and guava aromas of a Sauvignon Blanc variety.

However, it will be appreciated that the aroma of other grape and non-grape products are included within the scope of the present invention, including modulating the aroma of other plant products (eg non-wine products and fruit products). In addition, it will be further appreciated that the aroma of a product may be modulated by modulating the aroma of additive for a product, and that the aroma of a final product may therefore be modulated by addition of the additive to the product.

In one embodiment, the non-volatile sulfur is 4-(4-methylpentan-2-one)-L-cysteine and the volatile thiol compound is 4-mercapto-4-methylpentan-2-one, and the method is used to modulate the passionfruit and/or box tree aroma of a wine product.

In this regard, the following aromas are associated with each of the following volatile thiol compounds are shown in Table 1.

Table 1

Accordingly, in one embodiment the present invention may be used to modulate one or more of the above aromas indicated in Table 1 by modulating the release of the associated volatile thiol compounds.

For example, the non-volatile sulfur compound may be 4-(4-methylpentan-2-one)-L- cystein and the volatile thiol compound is 4-mercapto-4-methylpentan-2-one and the methods of the present invention are used to modulate one or more of the passionfruit, box tree and broom aromas of a wine product; the non-volatile sulfur compound may be 3-(hexan-l-ol)-L-cysteine and the volatile thiol compound is 3-mercaptohexan-l-ol and the methods of the present invention are used to modulate one or more of the grapefruit, passionfruit, guava and gooseberry aromas of a wine product; the non-volatile sulfur compound may be 3-(hexan-l-ol)-L-cysteine) and the volatile thiol compound is 3- mercaptohexyl acetate and the methods of the present invention are used to modulate one or more of the passionfruit, guava and gooseberry aromas of a wine product; and the non-volatile sulfur compound may be 4-mercapto-4-methylpentan-2-ol and the volatile thiol compound is 4-(4-methylpentan-2-ol)-L-cysteine and the methods of the present invention are used to modulate the citrus zest aroma of a wine product.

Modulation of the conversion of a non-volatile sulfur compound to a volatile thiol compound in the various embodiments of the present invention is achieved by either exposing the non-volatile sulfur compound to a genetically altered micro-organism having an increased expression and/or activity of a carbon-sulfur lyase enzyme, and/or exposing the non-volatile sulfur compound to an extract derived from the genetically altered micro-organism which has carbon-sulfur lyase activity. In this regard, the term "extract" as used throughout the specification is to be understood to mean a cell derived product that has carbon-sulfur lyase activity. For example, the extract is any mixture, fraction, preparation, purified or semi purified component, or concentrate which retains carbon-sulfur lyase activity. Methods for producing cell extracts and purifying enzymes are known in the art, for example as described in Protein Purification Protocols (2nd Ed.) ed. by Paul Cutler 2004 Humana Press Inc. New Jersey, herein incorporated by reference, and Tominaga et al (1998) J. Agric. Food Chem. 46:5215-5219, herein incorporated by reference.

In one embodiment, the present invention provides a method of modulating the aroma of a wine product including a non-volatile sulfur compound, the method including exposing the product to an isolated enzyme having a carbon-sulfur lyase enzyme activity capable of converting the non-volatile sulfur compound to a volatile thiol compound, wherein the aroma of the product is modulated by the conversion of the non- volatile sulfur compound to a volatile thiol compound by the carbon-sulfur lyase.

In one embodiment, the enzyme is isolated from a genetically altered micro-organism having an increased expression and/or activity of the carbon-sulfur lyase enzyme.

The genetically altered micro-organism in the various embodiments of the present invention may be for example a fungus or a bacterium,

Examples of suitable fungi include yeasts, such as Saccharomyces cerevisiae (all strains), Saccharomyces bay anus and species of Brettanomyces and its sexual ('perfect') equivalent Dekkera; Candida; Cryptococcus; Debaryomyces; Hanseniaspora and its asexual counterpart Kloeckera; Kluyveromyces; Metschnikowia; Pichia; Rhodotorula; Saccharomyces; Saccharomycodes; Schizosaccharomyces; and Zygosaccharomyces, and molds such as Aspergillus.

Other yeasts include yeasts that are used in beer, whiskey, sake, cider production, or any other yeast used in the processing of plant derived raw material or extracts. In the case of modulating the conversion of a non-volatile sulfur compound to a volatile thiol compound during fermentation of wine, in one embodiment the fungi is a yeast, such as Saccharomyces cerevisiae. Examples of commercially available Saccharomyces cerevisae strains for fermentation of wine products include VL3, EG9, VLl, 522d, VIN13, VIN7, NTl 16, VL2, X5, ECl 118, QA23 and L2056.

Examples of suitable bacteria including lactic acid bacteria genera, Lactobacillus, Leuconostoc, Oenococcus and Pediococcus, bacteria used in beer, sake, whiskey, cider production, or bacteria used in the processing of plant derived raw material or extracts.

In one embodiment, the micro-organism is a micro-organism involved in the production of a beverage. In one specific embodiment, the micro-organism is sugar-fermenting micro-organism.

The increased expression and/or activity of the carbon-sulfur lyase in a micro-organism may be achieved by a suitable method that results in an alteration of the genomic or extra-genomic nucleic acid content of the micro-organism. For example, the increased expression and/or activity of the carbon-sulfur lyase may be achieved by the introduction of a nucleic acid encoding a carbon-sulfur lyase (or an active part thereof) into the micro-organism, so as to genetically alter the micro-organism. Alternatively, the micro-organism may be mutated so as to result in an increased expression and/or activity of a carbon-sulfur lyase. Methods for genetically altering micro-organisms are known in the art and have been previously discussed herein.

As described above, in one embodiment the micro-organism is a micro-organism involved in the production of a beverage. In one specific embodiment, the microorganism is sugar-fermenting micro-organism. The micro-organisms may be used to modulate the aroma of a product, including modulating the aroma of wine product, such as a Sauvignon Blanc wine.

Accordingly, in another embodiment the present invention provides a genetically altered micro-organism, the micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme. As discussed above, the micro-organism may be a sugar-fermenting micro-organism. In one embodiment, the micro-organisms are isolated micro-organisms.

In one embodiment, the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of an exogenous nucleic acid encoding a carbon-sulfur lyase (or part thereof) into a micro-organism.

Accordingly, in another embodiment the present invention provides a micro-organism including an exogenous nucleic acid encoding a carbon-sulfur lyase or functional part thereof. As described above, in one embodiment the micro-organism is a sugar- fermenting micro-organism.

In one embodiment, the exogenous nucleic acid introduced into the micro-organism encodes a cysteine-S-conjugate β-lyase, or an active part thereof. In one embodiment, the cysteine-S-conjugate β-lyase is a tryptophanase, such as a tryptophanase encoded by a tnaA gene, or an active part or variant thereof. In one specific embodiment, the tnaA gene is from E.coli, or an active part of variant thereof.

In one embodiment, the tryptophanase has greater than 50% identity to that encoded by the E. coli tnaA gene, typically greater than 75% identity, such as having greater than 90% identity. In one specific embodiment, the tryptophanase has greater than 95% identity to that encoded by the E. coli tnaA gene.

In this regard, various algorithms exist for determining the degree of homology between any two proteins or any two nucleotide sequences. For example, the BLAST algorithm can be used for determining the extent of homology between two nucleotide sequences

(blastn) or the extent of homology between two amino acid sequences (blastp). BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al. (199Oj J. MoI. Biol. 215:403-410, herein incorporated by reference. In one embodiment, the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding the tnaA gene (or a variant or an active fragment thereof) into a micro-organism.

In another embodiment, the present invention provides a fungus including a nucleic acid including a nucleotide sequence encoding an exogenous tryptophanase gene, or an active part thereof.

Examples of fungi are as previously described herein. In one embodiment, the fungus is a yeast, such as Saccharomyces cerevisiae.

In one embodiment, the tryptophanase is an E.coh tnaA gene.

In one specific embodiment, the present invention provides a Saccharomyces cerevisiae cell including a nucleic acid encoding an E.coli tnaA gene, or a variant or functional part thereof.

Methods for the isolation of nucleic acid sequences and their cloning into a suitable expression vector are known in the art, for example as described in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference. The recombinant molecule may then be introduced into the cell and the cloned nucleic acid expressed.

Methods for introducing nucleic acids into cells are known in the art and include transformation using calcium phosphate, phage or viral infection, electroporation, lipofection, and particle bombardment. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, or cells which transiently express the inserted DNA or RNA for limited periods of time. Methods for introducing exogenous DNAs into cells are known in the art, for example as described in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference. The present invention also provides a product including a genetically altered microorganism, or an extract derived from the genetically altered micro-organism, as described herein. In a preferred form, the product is a wine product.

The present invention also provides an extract having carbon-sulfur lyase activity, or an isolated carbon-sulfur lyase enzyme, produced from a micro-organism in the various embodiments of the present invention.

In the case of a nucleic acid introduced into a cell to express a desired product, generally the nucleic acid encoding the desired product is introduced into the microorganism in conjunction with other elements necessary for the expression of the product from the nucleic acid.

For example, a nucleic acid expressing a carbon-sulfur lyase may be introduced into a micro-organism so as to result in increased expression and/or activity of the carbon- sulfur lyase in the micro-organism.

Expression systems and expression vectors containing regulatory sequences that direct expression of proteins are known in the art, and can be used to construct chimeric genes for production of any of the gene products of a carbon-sulfur lyase. These chimeric genes may then be introduced into appropriate micro-organisms via transformation to provide expression of the encoded proteins.

The present invention also provides isolated nucleic acids including a nucleotide sequence encoding carbon-sulfur lyases. The nucleic acids may be isolated nucleic acids.

For example, in one embodiment, there is provided an isolated nucleic acid including a transcriptional control sequence functional in a eukaryotic cell operably linked to a nucleic acid encoding a carbon-sulfur lyase.

In one embodiment, the nucleic acid encoding a carbon-sulfur lyase encodes a cysteine- S-conjugate β-lyase, or an active part thereof. In one specific embodiment, the cysteine-S-conjugate lyase is a tryptophanase, such as a tryptophanase encoded by a tnaA gene (eg the tnaA gene from E. coli), or an active part thereof.

The term "transcriptional control sequence" is to be understood to mean a nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence. The transcriptional control sequence of the present invention may comprise any one or more of, for example, a leader, promoter, enhancer or upstream activating sequence.

In one embodiment, the transcriptional control sequence is a promoter. A "promoter" as referred to herein, encompasses any nucleic acid that confers, activates or enhances expression of an operably connected nucleotide sequence in a cell. The promoter may be a constitutive promoter or an inducible promoter.

In the case of a yeast promoter for example, the promoter may be a constitutive promoter or an inducible promoter, such as the galactose promoter GALl or a promoter for a heat shock protein. A suitable yeast promoter is the PGKl promoter.

As used herein, the term "'operably connected" refers to the connection of a transcriptional control sequence, such as a promoter, and a nucleotide sequence of interest in such as way as to bring the nucleotide sequence of interest under the transcriptional control of the transcriptional control sequence. For example, promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to the promoter. In the construction of heterologous transcriptional control sequence/nucleotide sequence of interest combinations, it is generally preferred to position the promoter at a distance from the transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, ie. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. The present invention also provides plasmids or vectors including the nucleic acids of the present invention, and cells including the nucleic acids or plasmids/vectors. In this case, the cells may be prokaryotic cells (eg. E.coli, lactic acid bacteria genera, Lactobacillus, Leuconostoc, Oenococcus and Pediococcus, bacteria used in beer, sake, whiskey, cider production, or bacteria used in the processing of plant derived raw material or extracts) or eukaryotic cells (eg yeast). The plasmids, vectors and cells may be isolated plasmids, vectors and cells.

Vectors or DNA cassettes useful for the transformation of suitable host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products, the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation and a region 3' of the DNA fragment that controls transcriptional termination. It is most preferred when both control regions are derived from genes from the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Initiation control regions or promoters which are useful to drive expression of the encoded genes in the host cell are known in the art. Expression in a host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species. The transcriptional initiation regulatory regions can be obtained, for example, genes in the glycolytic pathway (such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, fructose-bisphosphate aldolase, phosphoglucose-isomerase, phosphoglycerate kinase), regulatable genes (such as acid phosphatase, lactase, metallothionein, and glucoamylase), the translation elongation factor EFl-α, and ribosomal protein S7 Nucleotide sequences surrounding the translational initiation codon may also be modified to affect expression in the micro-organism. If the desired polypeptide is poorly expressed, the nucleotide sequences of exogenous genes can be modified to include an efficient translation initiation sequence to obtain optimal gene expression. This can be done, for example, by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous gene. Alternatively, the consensus translation initiation sequence in the host may be used.

A large number of termination regions are known and function satisfactorily in a variety of hosts. In the case of a yeast, the termination region may be derived for example from a yeast gene, particularly Saccharomyces , Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3'-regions of some mammalian genes (eg γ-intererferon and α-2 interferon) are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.

Other features that may be manipulated to control gene expression including the nature of the relevant transcriptional promoter and terminator sequences; the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; the final cellular location of the synthesized protein; the efficiency of translation in the host organism; the intrinsic stability of the cloned gene protein within the host cell; and the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the carbon-sulfur lyase.

Methods are known in the art for expressing genes, for example as described in Gene Expression: Reviews and Protocols (2004) 2^nd edition, ed. by Paulina Balbas and Argelia Lorence, Humana Press Inc., New Jersey, herein incorporated by reference.

The exposing of the non-volatile sulfur compound to a genetically altered microorganism having an increased expression and/or increased activity of a carbon-sulfur enzyme may be achieved by a suitable method known in the art. For example, in the case of cysteine bound precursors present in the grape, the yeast may be contacted with grape juice or grape must during the fermentation process.

The exposing of the non-volatile sulfur compound to an extract from a micro-organism may, for example, be achieved by directly exposing the non-volatile sulfur compound to the extract.

In one embodiment, the present invention includes exposing the non-volatile sulfur compound to a crude extract prepared from the micro-organisms of the present invention. Methods for producing a crude extract from the micro-organisms of the present invention are known in the art, as described previously herein.

In another embodiment, the present invention provides exposing the non-volatile sulfur compound to semi-purified or purified carbon-sulfur lyase produced from the micro- organism of the present invention. Methods for purifying the enzymes are known in the art, as described previously herein.

Generally, the over expression of the carbon-sulfur lyase in the micro-organism will lead to an increased conversion of the non-volatile sulfur compound to a volatile thiol compound.

Methods for determining the conversion of a non-volatile sulfur compound to a volatile thiol compound are known in the art.

As described previously, the present invention also provides a product with an altered conversion of a non-volatile sulfur compound to a volatile thiol compound produced by the method of the present invention. For example, the product may be a Sauvignon Blanc wine with increased aromas such passionfruit, grapefruit abox tree.

The present invention also provides a method of fermenting a product including a nonvolatile sulfur compound using a genetically altered sugar- fermenting organism of the present invention. The present invention also provides a fermented product producing by the above described method. For example, the fermented product may be a wine or a beer. In one specific embodiment, the product is a Sauvignon Blanc wine.

(ii) assaying for production of a volatile thiol compound by the carbon-sulfur lyase enzyme; wherein the production of a volatile thiol compound by the carbon-sulfur lyase enzyme is indicative of the presence of the precursor of a volatile thiol compound in the sample.

This method may be used to detect or assay for a precursor of a volatile thiol compound in a sample. For example, the method may be used as a diagnostic test for the presence of chemical compounds in a sample that can be used to modulate the aroma of a product. Examples of products that may be assayed are as previously discussed herein.

Methods are known in the art for assaying the presence of a volatile thiol compound, as described previously herein.

Methods are also known in the art for the use of enzymes for assaying, for example as described in "Enzyme Assays" 2006 ed. Bu Jean-Louis Reymond Wiley-VCH Verlag Gmbh & Co KgaA Weinheim Germany, herein incorporated by reference.

Many products, including plant products, contain volatile thiols bound as cysteine S- conjugate precursors. Examples of plant derived products include, for example, wine products (eg wine, grape must, grape juice) and other products such as beer, whiskey, sake and cider.

Thus, the present method may be used to sample such products. In this regard it will also be appreciated that the term "sample" means any product, extract or derivative that is used be used to detect the presence of, and/or quantify the level of, a precursor of volatile thiol compound. It will also be appreciated that even though this method is generally described in reference to the detecting a precursor of a volatile thiol compound in a plant derived product, the method is not to be limited to detecting the precursors in only plant derived samples.

Examples of suitable carbon-sulfur lyase enzyme are as previously described herein. The enzyme may provided in the form of an extract (eg produced from a microorganism expressing the enzyme), or a semi-purified or purified form of the enzyme. The enzyme may also be, for example, a free enzyme or an enzyme coupled to a solid substrate or another chemical moiety. Methods for producing cell extracts and purifying enzymes are known in the art, for example as described in Protein Purification Protocols (2nd Ed.) ed. by Paul Cutler 2004 Humana Press Inc. New Jersey, herein incorporated by reference, and Tominaga et al (1998) J. Agric. Food Chem. 46:5215-5219, herein incorporated by reference.

In one embodiment, the enzyme is produced from a micro-organism of the present invention.

The exposing of the sample to the enzyme may be performed by a suitable method known in the art. For example, the enzyme may be added directly to the sample and the enzymatic reaction allowed to occur in solution.

In one embodiment, the carbon-sulfur lyase is a cysteine-S-conjugate β-lyase, such as a tryptophanase. Examples of tryptophanase enzymes are as discussed previously herein.

Examples of volatile thiol compounds and their non volatile precursors are as previously described herein.

It will also be appreciated that that the precursor compound may be endogenously present in the sample and/or may be an exogenous compound added to a sample. Finally, standard techniques may be used for recombinant DNA technology, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), herein incorporated by reference and The Nucleic Acid Protocols Handbook ed. by Ralph Rapley (2000) Humana Press, New Jersey, herein incorporated by reference.

Description of Specific Embodiments

Reference will now be made to experiments that embody the above general principles of the present invention. However, it is to be understood that the following description is not to limit the generality of the above description.

Example 1

Chemicals

All the chemicals used were of analytical grade and purchased from Sigma-Aldrich, (St. Louis, MO, USA) unless otherwise stated. 4MMP and Cys-4MMP were synthesized as described by Howell KS, Swiegers JH, Elsey G, Siebert TE, Bartowsky EJ, Fleet GH, Pretorius IS, de Barros Lopes MA. 2004. "Variation in 4-mercapto-4-methyl-pentan-2- one release by different wine strains" FEMS Microbiol Lett 240: 125-129 (herein incorporated by reference) and [²Hi₀]-4MMP, 3-MH, 3-MHA, and [²H₅]-3MHA were synthesized as described by Kotseridis Y, Ray JL, Augier C, Baumes R. 2000. "Quantitative determination of sulfur containing wine odorants at sub-ppb levels - Synthesis of the deuterated analogues. J Agric Food Chem 48:5819-5823 (herein incorporated by reference). Cys-3MH, as a mixture of diastereoisomers, was prepared using the procedure of Wakabayashi H, Wakabayashi M, Eisenreich W, Engel KH. 2004. Stereochemical course of the generation of 3-mercaptohexanal and 3- mercaptohexanol by β-lyase-catalyzed cleavage of cysteine conjugates. J Agric Food Chem 52: 110-116 (herein incorporated by reference). The synthesis of [²Hi_O]-3MH is described in Pardon KH, Granley SD, Capone DL, Swiegers JH, Sefton MA, Elsey GM. 2006. Cysteinylated precursors to flavour: Synthesis of the individual diastereomers of the cysteine conjugate of 3 -mercaptohexanol (3-MH). J Agric Food Chem (submitted).

The synthesis and structures of: (a) 4-mercapto-4-methylpentan-2-one (4MMP) and (b) 4-(4-methylpentan-2-one)-l-cysteine (Cys-4MMP) are shown in the scheme below:

Briefly, thiolacetic acid (11.65 g, 153 mM) was added to mesityl oxide (5.0 g, 51 mM) in tetrahydrofuran (50 mL) containing triethylamine (1 mL) and the mixture was stirred at room temperature overnight. After this time, the mixture was diluted with ether (50 mL) and washed successively with water, 10% sodium hydroxide solution (*2), water, and dried with Na2SO4. The solvent was removed by gentle distillation to give the thioacetate as an orange oil (8.4 g, 94%) and purity was confirmed by NMR.

Thioacetate (1.44 g, 8.3 mM) in ether (25 mL) was stirred at room temperature with hydrazine hydrate (0.83 g, 16.6 mM). After 2 h, the mixture was washed with water and dried. After concentration, the residue was chromatographed on silica gel (5% etheπpentane) to give the thiol as a colourless oil (0.78 g, 71%). The purity of 4MMP was determined by NMR.

The grape precursor to 4MMP, Cys-4MMP, was synthesised using a modification of the method of Tominaga et al. (1998) J. Agric. Food Chem. 46: 5215-5219 (herein incorporated by reference) as shown in the scheme above. Mesityl oxide (700 mg, 71 mM) and pyridine (1.13 g, 142 mM) were added to a solution of 1-cysteine (875 mg, 72 mM) in water (15 mL). The mixture was stirred at room temperature for 48 h before being filtered. The filtrate was concentrated under reduced pressure to give a white solid (1.05 g, 66%). Purity was confirmed by NMR.

Example 2

Microbial strains, media and culture conditions

E. coli strain K-12 strain W1485F^' (Klena and Schnaitman, 1994) was used for the cloning of the tnaA tryptophanase gene, while strain DH5α (Gibco BRL / Life Technologies, Gaithersburg, MD, USA) was used for the transformation and amplification of plasmid DNA. S. cerevisiae ∑1278b derived strain YHUM272a (MATa ura3-52 trplΔ::hisG leu2A::hisG his3A::hisG) (Van Dyk D, Hansson G, Pretorius IS, Bauer FF. 2003. Cellular differentiation in response to nutrient availability: The repressor of meiosis, Rmelp, positively regulates invasive growth in Saccharomyces cerevisiae. Genetics 165: 1045-1058, herein incorporated by reference) and commercial wine yeast strain VIN 13 (Anchor Yeast, Cape Town, South Africa) were used as host strains for the overexpression of the tnaA gene cassettes.

Bacterial donor and host strains were grown in Luria-Bertani (LB) medium (Ausubel et al., 1994) at 37°C. Ampicillin-resistant (Ap^R) bacterial transformants were selected on LB medium containing 100 mg/1 ampicillin. Yeast strains were cultivated at 30⁰C in either a rich medium, YPD (containing 1% yeast extract, 2% peptone and 2% glucose), or a synthetic dropout medium, SCD [containing 2% glucose, 0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI, USA)], supplemented with essential amino acids from a 0.13% amino acid stock solution (Sherman F, Fink GR, Hicks J. 1991. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, herein incorporated by reference). For the selection of sulfometuron methyl resistant (Sm^R) yeast transformants, YPD and SCD media were supplemented with 100 μg/ml sulfometuron methyl (Dupont, Wilmington, DE, USA) dissolved in N-N- dimethylformamide. Solid media contained 2% agar (Difco). For the detection of volatile thiol release, 10⁶ yeast cells from an overnight YPD culture were inoculated into an SCD-based medium spiked with either Cys-4MMP (sCD^{Cys 4MMP}) or Cys-3MH (SCD^c>soMH) in varying concentrations. Fermentations were conducted in 250-ml conical flasks fitted with a water lock and side arm septum for sampling.

Example 3

Recombinant DNA methods, plasmid construction and transformation

Standard procedures for isolation and manipulation of DNA were used throughout this study (Ausubel FM, Brent R, Kingston RE (eds). 1994. Current Protocols in Molecular Biology, vol. 13.0.1-13.14.17. Wiley: New York, herein incorporated by reference). Restriction enzymes, T4 DNA-ligase and Expand Hi-Fidelity DNA polymerase (Roche, Mannheim, Germany) were used according to the specifications of the supplier. E.coli was transformed as described by Ausubel et al. (1994), while the lithium acetate method (Gietz RD, St. Jean A, Woods RA, Schiestl RH. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acid Res 20: 1425-1425, herein incorporated by reference) was used for yeast transformations.

To clone and amplify a 1423-bp EcoM-Xhol DNA fragment containing the tnaA gene with the polymerase chain reaction (PCR) method, genomic DNA from E. coli K- 12 strain W1485F^" was used as template DNA with the following two primers: tnaA- F(£cøRI) 5'-GACTGAATTCATGGAAAACTTTAAACATCTCCCTG-S ' (SEQ ID NO.3) and tnaA-R(X/røI) 5 '-GACTCTCGAGTTAAACTTCTTTAAGTTTTGCGGTG- 3' (SEQ ID NO.3). PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and digested with EcoRl and Xhol for cloning into the EcoRl and Xhol sites of the yeast multi-copy episomal plasmid, pHVXII (Volschenk H, ViIj oen M, Grobler J, Petzold B, Bauer FF, Subden R, Young RA, Lonvaud-Funel A, Denayrolles M, Van Vuuren HJJ. 1997. Engineering pathways for malate degradation in Saccharomyces cerevisiae. Nat Biotechnol 1_5:253— 257, herein incorporated by reference), containing the S. cerevisiae phosphoglycerate kinase I gene (PGKl) promoter (PGKl _P) and terminator (PGK1_T). The PGKl _P-tna A-PGKl₇ gene cassette (designated CSLl for cysteine lyase) was isolated as a Hindlll fragment from plasmid pHVXII-GSU (Figure 1) and cloned into the Hindlll site of the yeast single-copy integrating plasmid pDLG42 (kindly provided by Dr DC La Grange, Stellenbosch University, South Africa) containing the ILV2 (SMRl -410) marker gene, which confers resistance to sulfometuron methyl (SMM).

Multi-copy plasmid pHVXII-CSLi was transformed into a laboratory strain of S. cerevisiae, ∑1278b, generating transformant ∑1278b(CSLi). Plasmid pOLG42-CSLl was linearized with Apal and transformed into VIN13. Sm^R VIN 13 transformants were selected, grown on YPD, and reselected on SMM-containing media. Genomic DNA was isolated from the Sm^R transformants grown overnight in YPD and the integration of the CSLl gene cassette into the genome (at the ILV2 locus) of VIN 13 was confirmed by the standard PCR technique (Ausubel et ah, 1994). This transformant was designated

VINO(CSLi).

Example 4

Pulsed-field gel electrophoresis (CHEF)

Yeast cultures were grown overnight in YPD medium. These pre-cultures were used to inoculate 10 ml of YPD shaking overnight at 28°C and 6xlO⁸ cells (3 ml) were sampled. The cells were spun down and taken up in 1 ml of cold 50 mM EDTA (pH 8.0) and kept on ice. A 2% agarose gel solution using low-melting-point agarose (Bio-Rad, Hercules, CA, USA) was prepared using half-strength TBE buffer (Ausubel et ah, 1994) and kept at 5O⁰C. The cells were spun down and taken up in 800 μl of cell-suspension buffer [10 mM Tris (pH 7.2); 20 mM NaCl; 50 mM EDTA] and equilibrated at 50⁰C. A volume of 10 μl Lyticase (Sigma-Aldrich) solution (12 mg/ml in 10 mM Tris, pH 8.0) was added and immediately combined with 800 μl of 2% agarose solution (low-melting-point agarose; Bio-Rad), mixed, and placed into disposable plug moulds and stored in the fridge for 30 min. The solidified plugs were pushed into 10 ml tubes with 5 ml of Lyticase buffer [10 mM Tris (pH 7.2); 250 μl 1 M Tris (pH 7.5); 50 mM EDTA; 2.5 ml 0.5 M EDTA] and incubated for 1 h at 37⁰C without shaking. The Lyticase buffer solution was removed and the plugs washed with washing buffer [20 mM Tris (pH 8.0) and 50 mM EDTA]. A volume of 5 ml of Proteinase K reaction buffer (1 mg Proteinase K dissolved in sterile, double-distilled water; Sigma-Aldrich,) was added and the plugs incubated at 50⁰C without shaking overnight. The plugs were washed four times in 50 ml of washing buffer for 1 h each time with gentle shaking at room temperature and then stored at 4⁰C in washing buffer. A 1% agarose gel (chromosomal grade) was poured using half-strength TBE buffer in the gel and tank. The plugs were carefully loaded and sealed with extra 1% agarose. The CHEF mapper (Bio-Rad) was run using a 'ramp'- 24 h run, 6 V, 120° angle, 60-120 sec switch time program.

Example 5

Cysteine β-lyase assay

Yeast cells were grown overnight in SCD medium, and 1.5 ml of the cell suspension centrifuged in microcentrifuge tubes for 3 min and washed twice with 1 ml of Milli-Q water. Cells were resuspended in 200 μl breaking buffer [2% (v/v) Triton X-IOO; 100 rriM Tris-Cl, pH 6.8.] and 200 μl acid washed glass beads were added after which it was vigorously vortexed for 5 min at 4°C and kept on ice. The assay mixture contained 100 μl Cys-4MMP (10 mM), 20μl pyridoxal 5'-phosphate (1 niM) and 830-875 μl assay buffer (20 mM phosphate buffer, pH 7.0; 1 mM EDTA). Volumes of 5-50 μl of yeast cell lysates were added depending on the density of the cell lysate suspensions and the reaction was allowed to proceed for 60 min. After 60 min, 2 μl of L-lactic dehydrogenase enzyme (EC 1.1.1.27; 5 units/μl) was added to the reactions followed immediately by an addition of 100 μl NADH (3 mM). Absorbance was immediately measured at 340 nm (A3₄0) for all the reactions as an indication of the relative presence of NADH. After the A₃₄₀ measurement at 60 min, the same reactions (now containing L- lactic dehydrogenase and NADH) were allowed to continue and the A3₄0 was measured at 75 min and 90 min to determine the consumption of NADH. After 100 min, 100 μl of 1 mM Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] was added and absorbance measured at 412nm (A₄₁₂) to give an indication of the formation of thiols during the reaction. Example 6

Wine fermentation

YPD was inoculated with strains VIN 13 and VINO(C₁S¹Li). Grape juice was autoclaved and 200 ml aliquots inoculated with the yeast strains and incubated at 3O⁰C for 24 h reaching optical densities (measured at 600 nm; OD_ΘOO) of 7.52 for the VIN 13 culture and 6.17 for the VINB(CSLi) culture. Frozen Sauvignon Blanc clarified juice (obtained from the Riverland, Australia) was thawed, thoroughly mixed and transferred to 3 -litre glass fermentors (Duran-Schott, Stafford, UK) with airlocks and inoculated in duplicate with 30 ml of the VIN 13 culture and 35 ml of the ViN13(CSLi) culture. Wines were fermented at 17°C without stirring and after 14 days [for VIN13(CSLi)] and 16 days (for VIN13) were cold-stabilized for 5 days at 4°C. Wines were racked sulfured at 50 ppm SO₂ and cold-stabilized for a further 10 days after which they were racked again, sulfured at 50 ppm SO₂, filter-sterilized (0.45 μm) and bottled in 500-ml glass reagent bottles with plastic Teflon-seals and stored at 4°C.

Example 7

Gas chromatography/mass spectrometry analysis

Preparation of samples: For 4MMP analyses, an aliquot (100 μl) of a solution of [ H₁0]- 4MMP (9.88 μg/ml) in ethanol was added using a glass syringe (100 μl; SGE, Austin, TA, USA) to the sample (10 ml) containing 2 g of salt (NaCl; BDH Laboratory Supplies, Dorset, UK) and approximately 20 mg ethylene di-amine tetra-acetic acid (EDTA) in a 20 ml SPME vial with a magnetic crimp cap (Gerstel, Baltimore, MD, USA). For analysis of 3MH and 3MHA, aliquots (100 μl) and (50 μl) of a solution of [²H_1O]-3MH (14.32 μg/ml) and [²H₅]-3MHA in ethanol (9.96 μg/ml), respectively, were similarly added. GC/MS analysis: The quantification of the thiols was carried out using headspace solid- phase microextraction/stable isotope dilution analysis coupled with gas chromatography mass spectrometry (HS-SPME-SIDA-GC/MS). Samples were analysed with an Agilent 6890N gas chromatograph (Santa Clara, CA, USA) fitted with a Gerstel MPS2 autosampler and coupled to an Agilent 5973N mass spectrometer. The autosampler was fitted with an automated 65 μm Carbowax-divinylbenzene SPME fibre (Supelco, Bellefonte, PA, USA). The gas chromatograph was fitted with a Sol GelWax (SGE) fused silica capillary column, 15 m x 0.32 mm i.d., 0.5 μm film thickness, connected via a glass a glass connector to a Valco Bond fused silica capillary column with dimensions of 60 m x 0.25 mm i.d. and 0.5 μm film thickness. The carrier gas was helium (BOC gases, Ultra High Purity), and the flow rate was 2.5 ml/min. The oven temperature started at 40⁰C, was held at this temperature for 4 min, then increased to 280⁰C at 5°C/min, and held at this temperature for 5 min. The injector was held at 220⁰C and the transfer line at 26O⁰C. The sample was extracted at 40⁰C for 30 min and desorbed in the inlet for 15 min. The splitter, at 20:1, was opened after 30 sec. Fast injection was done in pulsed splitless mode with an inlet pressure of 45.0 psi maintained until splitting. The injection sleeve (Supelco, 0.75 mm i.d.) was borosilicate glass. Positive ion electron impact spectra at 70 eV were recorded in the range m/z 35-350 for scan runs. For quantification, mass spectra were recorded in the Selective Ion Monitoring (SIM) mode. The ions monitored in SIM runs were: m/z 81, 96, 108, 141 and 142 for [²Hi₀]-4MMP, 55, 75, 89, 99 and 132 for 4MMP, 60, 62, 92, 109 and 144 for.[²H₁₀]-3MH, 75, 90, 103, and 118 for [²H₅]-3MHA, 55, 82, 100 and 134 for 3MH, and 73, 88, 101 and 116 for 3MHA. Selected fragment ions were monitored for 20 ms each. The underlined ion for each compound was the ion typically used for quantitation, having the best signal to noise and the least interference from other wine components. The other ions were used as qualifiers.

Validation: The analytical method was validated by a series of duplicate standard additions of unlabelled analyte (1 to 1000 μg/1, n = 8 x 2 for the analyte) to a 5% aqueous ethanol solution, saturated with potassium hydrogen tartrate, pH adjusted to 3.4 with tartaric acid. The standard addition curves obtained were linear throughout the concentration range, with coefficients of determination (r²) of 0.998, 0.999 and 0.994 and linear regression equations y = 3.02 x - 0.256, y = 4.33 x - 0.178 and y = 2.36 x - 0.0331 for 4MMP, 3MHA and 3MH, respectively. The repeatability of the analysis was determined at two concentrations for each analyte (100 μg/1 and 500 μg/1 for 4MMP, 50 μg/1 and 716 μg/1 for 3MH, and 50 μg/1 and 562 μg/1 for 3MHA) by spiking seven replicate aliquots of the same model wine. The coefficient of variance (or relative Standard deviation) and limits of detection were, respectively, for 4MMP, 2.8% (at 100 μg/1), 5% (at 500 μg/1) and 5 μg/1; for 3MH, 1% (at 50 μg/1), 9% (at 716 μg/1), and 10 μg/1, and for 3MHA, 1% (at 50 μg/1), 8% at (at 562 μg/1), and 1 μg/1.

Example 8

Sensory evaluation

For sensory descriptive analysis, carried out approximately three months after the wines were bottled, 14 trained assessors (seven males and seven females) rated 17 aroma attributes using a consensus descriptive analysis method for the four wines [VTN 13 and VIN13(CSLi), duplicate fermentation replicates] in triplicate, following eight training sessions. The data from three final practice rating training sessions were examined to evaluate the success of the training process prior to commencing the formal sessions. The intensity of each aroma attribute was rated using an unstructured 10 cm line scale with indented anchor points of 'low' and 'high' placed at 1 cm and 9 cm, respectively. A list of the attributes rated and the composition of the reference standards is provided in Table 2.

Table 2. Aroma attributes rated by the sensory panel and the reference standards composition

Aroma attribute Reference standard composition*

Estery/confectionery 0.2 ml ester stock solution (0.5 g isobutyl acetate, 0.09 g ethyl butyrate, 0.2 g ethyl hexanoate, 0.2 g ethyl octanoate, m 100 ml redistilled ethanol)

American grape/artificial 1/3 of a piece of grape flavoured chewing gum (Hubba Bubba) fruit

Floral 0.2 ml of 0.1% v/v 2-phenylethanol stock solution

Pineapple 10 ml canned pineapple juice (Golden Circle)

Passionfruit Small piece of fresh passionfruit skm + pulp m glass

Lychee 4 tsp canned Lychee syrup (Admiral Lychees m light syrup)

Lemon Approx 5 ml Bern lemon squeeze

Lime 10 ml Bickfords lime cordial

Orange peel 2 tsp orange marmalade (Rose's) m 100 ml BW

Grapefruit 1 cm² piece per glass

Grassy 2 tsp freshly cut grass

Box hedge/cat uπne 1 small spπg English Box (4 leaves) per glass

Capsicum lcm² piece of green capsicum

Asparagus/green bean 2 tsp tinned asparagus brine with 2 tsp green bean brine (Edgell)

Lantana/tomato leaf 4-6 leaves lantana vine per glass

Sweaty 0.2 ml hexanoic acid stock (10 g/1) + 0.1 ml 3 -methyl-butyric acid stock (10 g/l) m l00 ml BW

Solvent/nail polish remover 0.05 ml glacial acetic acid m 100 ml BW

*in 100 ml Chenm Blanc, 2004, 21 bag in box wme (11% alc/vol) unless otherwise specified

Samples were assessed in three sessions on three consecutive mornings under sodium lighting in isolated, temperature controlled, ventilated tasting booths. At each formal rating session, each of the judges evaluated the same four samples in a complete block design. Samples (30 ml) were presented for each formal session in a random and balanced order across the judges in coded, covered ISO wine tasting glasses, at 22-24⁰C. Three replicates of each sample were thus assessed. FIZZ software (Version 2.0, Biosystems, Couternon, France) was used for the collection of all data. The data for each attribute were analyzed using a nested analysis of variance (ANOVA) testing for the effects of strain and replicate nested within treatment, using a mixed model treating judges as a random effect (JMP 5.1, SAS Institute, Cary, NC, USA). Example 9

Constitutive expression of a bacterial cysteine- β-lyase encoding gene cassette in yeast

The basic constructs of the multi-copy episomal (pHVXII-CSZJ) and single-copy integrating (pDLG42-CSZJ) plasmids with which S. cerevisiae ∑1278b and VIN 13 were transformed are shown in Figure 1. In both yeast transformants, the E. coli tryptophanase gene (tnaA) was expressed constitutively under control of the yeast PGKl promoter and terminator sequences.

In order to confirm that biologically active cysteine-β-lyase was produced by the ∑ 1278b yeast transformant, an assay was developed to determine the enzyme activity (see above). The assay is based on the formation of pyruvate and thiols (using the chemically synthesized cysteine conjugates as substrates), which are end-products of the reaction. The mechanism of cysteine-β-lyase enzymes on cysteine conjugates is illustrated in Figure 2. Pyruvate was indirectly measured through the formation of lactate in a reaction catalyzed by lactate dehydrogenase resulting in the consumption of NADH (Howell BF, McCune S, Schaffer R. (1979) Lactate-to-pyruvate or pyruvate-to- lactate assay for lactate dehydrogenase: a re-examination. Clin Chem 25:269-272, herein incorporated by reference). Therefore, the reduction in the concentration of NADH (recorded by A₃₄₀ measurements) was correlated to the formation of pyruvate and hence the cysteine-β-lyase activity. At the end of the reaction, Ellman's reagent was added which reacts with thiols to form a yellow complex (recorded by A_4I2 measurements) (Ellman GL. (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70-77, herein incorporated by reference).

After addition of lactate dehydrogenase and NADH at 60 min, the 'immediate' A₃₄₀ value obtained for the CSZJ-expressing crude yeast extract was approximately half that of the wild-type crude yeast extract, thereby indicating that a significant amount of NADH was consumed by the CSLl -expressing crude yeast extract. Interestingly, the concentration of NADH in the wild-type crude extract was even higher than the control without crude yeast extract, indicating that some NADH could have been introduced from the cell. After the 60 min A₃₄₀ measurement, the effect of NADH being oxidised was evident when the reactions containing the added NADH and lactate dehydrogenase were left to proceed and A340 measured at 75 min and 90 min. During this time, the amount of NADH dropped in the reaction containing the wild-type crude yeast extract but not nearly as much as the reaction containing the CSLl crude yeast extract where the A₃₄₀ more than halves compared to its already low reading at the 60 min time point.

Interestingly though, in the reaction without any crude yeast extract, the NADH also dropped from 60 minutes through 75 to 90 min, even more than in the reaction with the crude wild-type yeast extract, indication that chemical oxidation is also taking place.

To confirm that the formation of pyruvate was related to the cleaving of cysteine from cys-4MMP to form 4MMP, pyruvate and ammonium, the qualitative formation of thiols were measured through the addition of Ellman's reagent to the same reactions which have now been running for a 100 min (40 min of that with NADH and lactate dehydrogenase present). The A_4I2 value was approximately double in the CSLl- expressing crude yeast extract reaction compared to the wild-type. In the reaction with no crude yeast extract, the A_4I2 value was relatively high compared to the control containing Ellman's reagent but without cys-4MMP (which had an OD_4I2 reading of zero), the same as the water blank. This indicated the presence of thiols due to spontaneous degradation products and/or thiol impurities in the Cys-4MMP preparation. The reaction containing wild-type crude extract had the same A_4I2 reading as the 'no extract' control, indicating that the possible contribution of SH-containing compounds in the cell extract is not relevant. In spite of the high amount of background reactions with Ellman's reagent with the Cys-4MMP preparation, the CSLl expressing crude yeast extract reaction still had double the A_4I2, indication that it contained significantly more thiols. The results in Figure 3 confirm that cysteine-β-lyase activity was produced in S. cerevisiae transformed with the CSLl gene cassette. Example 10

Enhanced volatile thiol release during fermentation

Most common laboratory strains of S. cerevisiae (e.g., ∑1278b) are unable to ferment grape juice, often containing more than 200 g/1 sugar, to dryness (less than 5 g/1 of sugar at the end of fermentation). Therefore, only the wine yeast strain (and not the laboratory strain) into whose genome the CSLl gene cassette was integrated, VIN13(CSTJ), was used to conduct fermentation trials in a model grape juice medium spiked with the chemically synthesized cysteine conjugate precursors. Analysis of the thiol concentrations after fermentation indicated that the VINO(CSLi) transformant producing the bacterial cysteine-β-lyase had more than a 10-fold increase in 4MMP concentration compared to the VIN 13 control strain (Figure 4).

The ability of VIN13(CSLi) to release 3MH from Cys-3MH was also investigated. Using the HS-SPME-SIDA-GC/MS-based method (with a detection limit of 10 μg/1) described above, no 3MH could be detected in the VIN 13 control ferment. On the other hand, the VINB(GS¹ZJ) transformant released substantial amounts of 3MH, accompanied by smaller amounts of 3MHA, from the Cys-3MH precursor (Figure 5).

Interestingly, the amount of 3MH released was found to be correlated with the total amount of Cys-3MH spiked in the fermentation medium with higher concentrations of precursor leading to higher amounts of 3MH released (Figure 5).

Example 11

Sensory impact of enhanced thiol releasing yeast on wine aroma

To investigate the effect on sensory properties due to the cysteine-β-lyase produced by the modified wine yeast strain [VIN13(C5Li)], Sauvignon Blanc grape juice was fermented with the VIN 13 and VIN13(C5L7) yeast strains. The basic wine composition - notably pH, alcohol and titratable acidity - did not differ substantially between the wines made with the different yeast (Table 3). Table 3. Basic composition for the Sauvignon Blanc wines made using the modified [VIND(C₁S¹Li)] and non-modified yeast (VINl 3) strains

VIN 13 VIN13(CSZJ) pH 3.23 3^23

Alcohol 11.8 12.3

Total acidity (g/1) 6.6 6.5

Glucose and fructose d 6.3 0.8 Volatile acidity (g/1) 0.39 0.11

The fermentation kinetics of the two yeast strains were slightly different as in both replicate fermentations the VIN 13 (CSLl) strain consumed the sugar slightly faster, explaining the extra residual sugar left by VIN 13 after 12 days fermentation. This could be due to the extra nitrogen released by cysteine β-lyase activity cleaving of cysteine to ammonia and pyruvate. Interestingly, the VIN 13 (CSLl) transformant produced a wine with lower volatile acidity compared to the VIN 13 host strain. After fermentation, yeast cells were isolated and it was confirmed by CHEF pulsed-field gel electrophoresis and PCR that the VIN 13 and VIN13(CSLi) strains finished the fermentations.

Data from sensory descriptive analysis (Figure 6) showed that the Sauvignon Blanc wine fermented with VIN13(CSLi) had significantly increased ratings for the passionfruit, lychee, grapefruit, box hedge and sweaty aroma attributes, compared to the

VIN 13 host strain. There were no significant fermentation replicate differences in these attributes. The two wines fermented with VIN 13 and VIN 13 (CSLl) differed most in the passionfruit aroma, and it is noteworthy that the two profiles did not differ significantly in attributes that are not related to the influence of volatile thiols.

Preliminary experiments with Cabernet Sauvignon, Riesling, Viognier and Chardonnay wines made with VIN13-føαA indicate that all had an overpowering passionfruit aroma when assessed informally. Discussion

In this work, yeasts able to set free the untapped thiol aromas in grape juice during winemaking were developed by cloning the E. coli tnaA gene, encoding a tryptophanase with strong cysteine-β-lyase activity, into a S. cerevisiae laboratory strain (∑1278b) and a commercial wine yeast (VIN13). The tnaA gene construct (designated CSLl) was introduced with a multi-copy episomal plasmid into the ∑1278b laboratory strain while it was integrated into the ILV2 locus of the VIN 13 genome. As part of the CSLl gene construct, expression of the tnaA gene was directed by the S. cerevisiae PGKl regulatory sequences in these two yeast transformants, ∑l278b(CSLl) and VrNB(GS¹Li). The yeast transformants expressing carbon-sulfur lyase activity released substantially more volatile thiols (4MMP and 3MH) in model ferments than the control host strain. Wines produced with the VIN 13 (CSLl) wine yeast transformant displayed an intense passionfruit aroma.

By measuring carbon-sulfur lyase activity of the yeast lysate at a pH of 7, the results confirmed that the CSLl -encoded carbon-sulfur lyase was active in vitro.

Based on our results, we suggest that the cysteine conjugate precursors are transported into the yeast cells, the enzymatic activity takes place inside the cells and the thiol is released and removed from the cells, either through diffusion or through active transport. The fact that ferments were analyzed as soon as fermentation was completed suggest that there was little, or no autolysis taking place which could result in enzymes escaping into the medium and resulting in activity.

In this study we also confirmed the ability of yeast to convert 3MH to 3MHA. Fermentation media spiked with Cys-3MH resulted in the formation of significant quantities of 3MHA although no Cys-3MHA was added. Therefore, 3MHA could only be formed by yeast, through the acetylation of 3MH (as shown before in media spiked with 3MH) or even by the acetylation of Cys-3MH, prior to release.

The sensory analysis of the Sauvignon Blanc wine made with VIN 13(CSZJ) clearly indicated that the enhanced release of thiols resulting in an increase in the characteristic volatile thiol aromas of passionfruit, grape fruit and box hedge. An increase in the 'sweaty' aroma attribute was also observed. At high concentrations, volatile thiols can elicit sweaty or cat's urine aromas, especially for those assessors more sensitive to the compound. For most experienced wine assessors who have evaluated these wines, the response has been to strongly prefer the VINO(CSLi) derived wines over the relatively neutral counterpart.

In conclusion, the yeast that has been developed has far-reaching and exciting possibilities for winemakers because it has the potential to provide them with the ability to transform bland-tasting Sauvignon Blanc grape juice, such as the juice studied, into intensely flavoured wines with overpowering varietal aromas of passionfruit, and thus consistently meet the sensory expectations of consumers in certain target markets. This novel yeast is able to increase the concentrations of fruity, aroma-enhancing thiols by a couple of orders of magnitude and gives wine producers an exceedingly powerful mechanism for the future, for example to blend insipid base wines with strongly fruit- flavoured wines produced by this yeast to achieve optimal aroma-determining thiol concentrations.

Finally, it will be appreciated that various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. A method of modulating conversion of a non-volatile sulfur compound to a volatile thiol compound, the method including either or both of the following steps: (i) exposing the non-volatile sulfur compound to a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to volatile thiol compound; and

(ii) exposing the non-volatile sulfur compound to an extract derived from a genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to volatile thiol compound.

2. A method according to claim 1, wherein the modulation of conversion of the non-volatile sulfur compound to a volatile thiol compound is an increased conversion of the non-volatile sulfur compound to a volatile thiol compound.

3. A method according to claim 2, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of an exogenous nucleic acid encoding a carbon-sulfur lyase, or a function part thereof, into the genetically altered micro-organism.

4. A method according to any one of claims 1 to 3, wherein the genetically altered micro-organism is a bacterium or a yeast.

5. A method according to claim 4, wherein the yeast is Saccharomyces cerevisiae.

6. A method according to claim 4, wherein the bacterium is a lactic acid bacterium.

7. A method according to any one of claims 1 to 6, wherein the carbon-sulfur lyase enzyme is a cysteine-S-conjugate β-lyase.

8. A method according to claim 7, wherein the non-volatile sulfur compound is 4-

(4-methylpentan-2-one)-L-cystein and the volatile thiol compound is 4-mercapto-4- methylpentan-2-one.

9. A method according to claim 7, wherein the non-volatile sulfur compound is 3-

(hexan-l-ol)-L-cysteine and the volatile thiol compound is 3-mercaptohexan-l-ol.

10. A method according to claim 7, wherein the non-volatile sulfur compound is 3- (hexan-l-ol)-L-cysteine) and the volatile thiol compound is 3-mercaptohexyl acetate.

11. A method according to claim 7, wherein the non- volatile sulfur compound is 4- mercapto-4-methylpentan-2-ol and the volatile thiol compound is 4-(4-methylpentan-2- ol)-L-cysteine.

12. A method according to any one of claims 7 to 11, wherein the cysteine-S- conjugate β-lyase is a tryptophanase.

13. A method according to claim 12, wherein the tryptophanase is derived from Escherichia coli.

14. A method according to claims 12 or 13, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding a tnaA gene into the genetically altered micro-organism.

15. A method according to claim 14, wherein the nucleic acid introduced into the genetically altered micro-organism includes a nucleotide sequence of SEQ ID NO: 1, or includes a nucleic acid encoding a polypeptide including an amino acid sequence of SEQ ID NO:2, or a variant thereof, or a functional part thereof.

16. A method according to any one of claims 1 to 15, wherein the non-volatile sulphur compound is present in grape juice or wine must.

17. A method according to claim 16, wherein the method is used to modulate the aroma of a wine product.

18. A method according to claim 16, wherein the non-volatile sulfur compound is 4-(4-methylpentan-2-one)-L-cystein and the volatile thiol compound is 4-mercapto-4- methylpentan-2-one and the method is used to modulate the passionfruit and/or box tree aroma of a wine product.

19. A method according to claim 16, wherein the wine product is a wine product derived from a Vitis vinifera grape variety, including a wine product derived from a

Sauvignon Blanc grape variety.

20. A genetically altered micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme.

21. A micro-organism according to claim 20, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding a carbon-sulfur lyase, or a functional part thereof, into the micro-organism.

22. A micro-organism according to claim 21, wherein the nucleic acid introduced into the micro-organism includes a nucleotide sequence of SEQ ID NO: 1, or includes a nucleic acid encoding a polypeptide including an amino acid sequence of SEQ ID NO:2, or a variant thereof, or a functional part thereof.

23. A micro-organism according to any one of claims 20 to 22, wherein the microorganism is a bacterium or a yeast.

24. A micro-organism according to claim 23, wherein the yeast is Saccharomyces cerevisiae.

25. A micro-organism according to claim 23, wherein the bacterium is a lactic acid bacterium.

26. A micro-organism according to any one of claims 20 to 25, wherein the carbon-sulfur lyase enzyme is a cysteine-S-conjugate β-lyase.

27. A micro-organism according to claim 26, wherein the cysteine-S-conjugate β- lyase is a tryptophanase.

28. A micro-organism according to claim 27, wherein the tryptophanase is derived from Escherichia coli.

29. A micro-organism according to any one of claims 20 to 28, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding a tnaA gene into the genetically altered micro-organism.

30. A micro-organism according to any one of claims 20 to 29, wherein the micro- organism is used to modulate the aroma of a wine product.

31. A micro-organism according to claim 30, wherein the wine product is a wine product derived from a Vitis vinifera grape variety.

32. A micro-organism including an exogenous nucleic acid encoding a carbon- sulfur lyase.

33. A micro-organism according to claim 32, wherein the exogenous nucleic acid includes a nucleotide sequence of SEQ ID NO:1, or includes a nucleic acid encoding a polypeptide including an amino acid sequence of SEQ ID NO:2, or a variant thereof, or a functional part thereof.

34. A micro-organism according to claims 32 or 33, wherein the micro-organism is a bacterium or a yeast.

35. A micro-organism according to claim 34, wherein the yeast is Saccharomyces cerevisiae.

36. A micro-organism according to claim 34, wherein the bacterium is a lactic acid bacterium.

37. A micro-organism according to any one of claims 32 to 36, wherein the carbon-sulfur lyase enzyme is a cysteine-S-conjugate β-lyase.

38. A micro-organism according to claim 37, wherein the cysteine-S-conjugate β- lyase is a tryptophanase.

39. A micro-organism according to claim 37, wherein the tryptophanase is derived from Escherichia coli.

40. A micro-organism according to any one of claims 32 to 39, wherein the microorganism is used to modulate the aroma of a wine product.

41. A micro-organism according to claim 40, wherein the wine product is a wine product derived from a Vitis vinifera grape variety, including a Sauvignon Blanc grape variety.

42. A micro-organism according to any one of claims 20 to 41, wherein the microorganism is a micro-organism that is involved in the production of beverages.

43. A micro-organism according to any one of claims 20 to 42, wherein the microorganism is a sugar-fermenting micro-organism.

44. An extract having carbon-sulfur lyase activity, or an isolated carbon-sulfur lyase enzyme, produced from a micro-organism according to any one of claims 20 to 43.

45. A method of modulating the aroma of a product including a non-volatile sulfur compound, the method including either or both of the following steps:

46. A method according to claim 45, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding a carbon-sulfur lyase, or a functional part thereof, into the genetically altered microorganism.

47. A method according to claim 46, wherein the nucleic acid introduced into the genetically altered micro-organism includes a nucleotide sequence of SEQ ID NO: 1, or includes a nucleic acid encoding a polypeptide including an amino acid sequence of SEQ ID NO:2, or a variant thereof, or a functional part thereof.

48. A method according to any one of claims 45 to 47, wherein the genetically altered micro-organism is a bacterium or a yeast.

49. A method according to claim 48, wherein the yeast is Saccharomyces cerevisiae.

50. A method according to claim 49, wherein the bacterium is a lactic acid bacterium.

51. A method according to any one of claims 45 to 50, wherein the carbon-sulfur lyase enzyme is a cysteine-S-conjugate β-lyase.

52. A method according to claim 51, wherein the non-volatile sulfur compound is S-4-(4-methylpentan-2-one)-L-cystein and the volatile thiol compound is 4-mercapto-4- methylpentan-2-one.

53. A method according to claim 52, wherein the non-volatile sulfur compound is S-3-(hexan-l-ol)-L-cysteine and the volatile thiol compound is 3-mercaptohexan-l-ol.

54. A method according to claim 51, wherein the non-volatile sulfur compound is 3-(hexan-l-ol)-L-cysteine) and the volatile thiol compound is 3-mercaptohexyl acetate.

55. A method according to claim 51, wherein the non-volatile sulfur compound is 4-mercapto-4-methylpentan-2-ol) and the volatile thiol compound is S-4-(4- methylpentan-2-ol)-L-cysteine.

56. A method according to any one of claims 51 to 55, wherein the cysteine-S- conjugate β-lyase is a tryptophanase.

57. A method according to claim 56, wherein the tryptophanase is derived from Escherichia coli.

58. A method according to any one of claims 45 to 57, wherein the increased expression and/or activity of the carbon-sulfur lyase is due to the introduction of a nucleic acid encoding a tnaA gene into the genetically altered micro-organism.

59. A method according to any one of claims 45 to 58, wherein the product is a wine product, including grape juice, wine must or wine.

60. A method according to claim 59, wherein the non-volatile sulfur compound is 4-(4-methylpentan-2-one)-L-cystein and the volatile thiol compound is 4-mercapto-4- methylpentan-2-one and the method is used to modulate one or more of the passionfruit, box tree and broom aromas of a wine product.

61. A method according to claim 59, wherein the non-volatile sulfur compound is 3-(hexan-l-ol)-L-cysteine and the volatile thiol compound is 3-mercaptohexan-l-ol and the method is used to modulate one or more of the grapefruit, passionfruit, guava and gooseberry aromas of a wine product.

62. A method according to claim 59, wherein the non-volatile sulfur compound is 3-(hexan-l-ol)-L-cysteine) and the volatile thiol compound is 3-mercaptohexyl acetate and the method is used to modulate one or more of the passionfruit, guava and gooseberry aromas of a wine product.

63. A method according to claim 59, wherein the non-volatile sulfur compound is 4-mercapto-4-methylpentan-2-ol and the volatile thiol compound is 4-(4-methylpentan-

2-ol)-L-cysteine and the method is used to modulate the citrus zest aroma of a wine product.

64. A method according to any one of claims 59 to 63, wherein the wine product is derived from a Vitis vinifera grape variety, including a Sauvignon Blanc variety.

65. A method of modulating the aroma of a wine product including a non-volatile sulfur compound, the method including exposing the product to an isolated enzyme having a carbon-sulfur lyase enzyme activity capable of converting the non-volatile sulfur compound to a volatile thiol compound, wherein the aroma of the product is modulated by the conversion of the non-volatile sulfur compound to a volatile thiol compound by the carbon-sulfur lyase.

66. A method according to claim 65, wherein the enzyme is isolated from a genetically altered micro-organism having an increased expression and/or activity of the carbon-sulfur lyase enzyme.

67. A product with modulated aroma produced by the method according to any one of claims 45 to 66.

68. A method of fermenting a product including a non- volatile sulfur compound, the method including either or both of the following steps:

(i) exposing the product to a genetically altered sugar-fermenting microorganism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound; and (ii) exposing the product to an extract derived from a genetically altered sugar-fermenting micro-organism having an increased expression and/or increased activity of a carbon-sulfur lyase enzyme capable of converting the non-volatile sulfur compound to a volatile thiol compound.

69. A fermented product produced by the method according to claim 68.

70. A fungus including a nucleic acid including a nucleotide sequence encoding an E.coli tnaA gene, or a functional part thereof.

71. An isolated nucleic acid including a transcriptional control sequence functional in a eukaryotic cell operably linked to a nucleic acid encoding a carbon-sulfur lyase.

72. A method of detecting a precursor of a volatile thiol compound in a sample, the method including: (i) exposing the sample to a carbon-sulfur lyase enzyme capable of converting the precursor to a volatile thiol compound; and

73. A method according to claim 72, wherein the sample is a wine product, including grape juice, wine must or wine grape must.