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WO2007095682A1 - Méthodes et micro-organismes pour la modulation de la conversion de composés soufrés non volatils en thiols volatils - Google Patents

Méthodes et micro-organismes pour la modulation de la conversion de composés soufrés non volatils en thiols volatils Download PDF

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WO2007095682A1
WO2007095682A1 PCT/AU2007/000199 AU2007000199W WO2007095682A1 WO 2007095682 A1 WO2007095682 A1 WO 2007095682A1 AU 2007000199 W AU2007000199 W AU 2007000199W WO 2007095682 A1 WO2007095682 A1 WO 2007095682A1
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volatile
micro
sulfur
organism
carbon
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Isak Stephanus Pretorius
Jan Hendrik Swiegers
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Australian Wine Research Institute Ltd
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Australian Wine Research Institute Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • 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.
  • 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.
  • 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).
  • 4MMP 4-mercapto-4-methylpentan-2-one
  • MHA 3-mercaptohexyl acetate
  • 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.
  • the volatile thiols are almost non-existent in the grape juice and develop during fermentation.
  • 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.
  • 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.
  • the present invention arises from studies into the overexpression of the tnaA gene in a commercial wine yeast.
  • 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.
  • 4MMP 4-mercapto-4-methylpentan-2-one
  • 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:
  • 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:
  • 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:
  • the present invention also provides a method of detecting a precursor of a volatile thiol compound in a sample, the method including:
  • 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.
  • 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.
  • 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.
  • an isolated enzyme may be a substantially purified form of the enzyme, or an extract containing the enzyme.
  • 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.
  • the nucleic acid content may be altered by introducing or transferring a nucleic acid into the cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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- mannos
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • Figure 1 shows plasmids constructed and used in this study.
  • Figure 2 shows the biochemical mechanism through which cysteine- ⁇ -lyase enzymes convert grape-derived, non-volatile, cysteinylated thiol precursors into aromatic thiols in wine.
  • Figure 3 shows the cysteine- ⁇ -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; A 340 ). Reactions were allowed to proceed for 75 min and 90 min after which the A3 4 0 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(GS 1 ZJ) were fermented at 3O 0 C 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.
  • 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
  • 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.
  • 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.
  • a product such as a wine product, a food product, a beverage, or a food or beverage additive.
  • the present invention also provides a product with an altered release of a volatile thiol compound produced by the above described method.
  • 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • the carbon-sulfur lyase is a cysteine-S-conjugate ⁇ -lyase.
  • These enzymes which are part of the large carbon-sulfur lyase enzyme family, involve the cleavage of a carbon-sulfur bond in a ⁇ -elimination reaction.
  • the mechanism of cysteine-conjugate lyase enzymes is shown in Figure 1.
  • the cysteine-S-conjugate ⁇ -lyase is a tryptophanase.
  • the tryptophanase is a tryptophanase from a micro-organism.
  • micro-organisms examples include bacteria such as Eschericia sp.,
  • 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 spectacularus; Proteus Vulgaris; Halobacterium sp.; Desulfitobacterium hafniense; and Treponema denticola.
  • bacteria including Eschericia coli, Thermoanaerobacter ethanolicus; Symbiobacterium thermophilum; Photobacterium profundum; Haemophilus somnus; Vibrio spectacularus; Proteus Vulgaris; Halobacterium sp.; Desulfitobacterium hafniense; and Treponema denticola.
  • Tryptophanase enzymes from other organisms 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
  • 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.
  • 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.
  • 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.
  • cysteine-S-conjugate lyases examples 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).
  • CTH Homo sapiens cystathionase
  • 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.
  • plant derived products include wine products such as wine, grape must, grape juice, and other products such as beer, whiskey, sake and cider.
  • 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.
  • 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:
  • the present invention also provides a product with altered aroma produced by this method.
  • the product is a wine product.
  • 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).
  • 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.
  • volatile thiols are of particular importance to the varietal character as it imparts box tree, passionfruit, grapefruit, gooseberry, and guava aromas.
  • 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.
  • 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.
  • 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.
  • the product includes an endogenous non-volatile sulfur compound and/or an exogenous non-volatile sulfur compound.
  • 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.
  • the Vitis vinifera grape variety is Sauvignon Blanc, Riesling, Semillon, Chenin Blanc, Colombard, Cabernet Sauvignon, Merlot, Riesling, Gewurztraminer, Alsace Muscat, Manseng and Arvine.
  • the present invention may be used to modulate the aroma of a product from Vitis vinifera.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • extract as used throughout the specification is to be understood to mean a cell derived product that has carbon-sulfur lyase activity.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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
  • Kloeckera Kluyveromyces
  • Metschnikowia Pichia
  • 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.
  • the fungi is a yeast, such as Saccharomyces cerevisiae.
  • yeasts 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.
  • suitable bacteria examples include 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.
  • 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.
  • 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.
  • the micro-organism may be mutated so as to result in an increased expression and/or activity of a carbon-sulfur lyase.
  • the micro-organism is a micro-organism involved in the production of a beverage.
  • 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.
  • 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.
  • the micro-organism may be a sugar-fermenting micro-organism.
  • the micro-organisms are isolated micro-organisms.
  • 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.
  • the present invention provides a micro-organism including an exogenous nucleic acid encoding a carbon-sulfur lyase or functional part thereof.
  • the micro-organism is a sugar- fermenting micro-organism.
  • the exogenous nucleic acid introduced into the micro-organism encodes a cysteine-S-conjugate ⁇ -lyase, or an active part thereof.
  • the cysteine-S-conjugate ⁇ -lyase is a tryptophanase, such as a tryptophanase encoded by a tnaA gene, or an active part or variant thereof.
  • the tnaA gene is from E.coli, or an active part of variant thereof.
  • 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.
  • BLAST algorithm can be used for determining the extent of homology between two nucleotide sequences
  • 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.
  • 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.
  • the present invention provides a fungus including a nucleic acid including a nucleotide sequence encoding an exogenous tryptophanase gene, or an active part thereof.
  • the fungus is a yeast, such as Saccharomyces cerevisiae.
  • the tryptophanase is an E.coh tnaA gene.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid encoding a carbon-sulfur lyase encodes a cysteine- S-conjugate ⁇ -lyase, or an active part thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transcriptional control sequence such as a promoter
  • 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.
  • promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to the promoter.
  • 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.
  • 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.
  • 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- ⁇ , and ribosomal protein S7 Nucleotide sequences surrounding the translational initiation codon may also be modified to affect expression in the micro-organism.
  • genes in the glycolytic pathway such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, fructose-bisphosphate aldolase, phosphogluco
  • 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.
  • termination regions are known and function satisfactorily in a variety of hosts.
  • 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 ⁇ -intererferon and ⁇ -2 interferon are also known to function in yeast.
  • Termination control regions may also be derived from various genes native to the preferred hosts.
  • a termination site may be unnecessary; however, it is most preferred if included.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fermented product may be a wine or a beer.
  • the product is a Sauvignon Blanc wine.
  • the present invention also provides a method of detecting a precursor of a volatile thiol compound in a sample, the method including:
  • This method may be used to detect or assay for a precursor of a volatile thiol compound in a sample.
  • 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.
  • plant products contain volatile thiols bound as cysteine S- conjugate precursors.
  • plant derived products include, for example, wine products (eg wine, grape must, grape juice) and other products such as beer, whiskey, sake and cider.
  • 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.
  • 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.
  • 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.
  • the enzyme may be added directly to the sample and the enzymatic reaction allowed to occur in solution.
  • the carbon-sulfur lyase is a cysteine-S-conjugate ⁇ -lyase, such as a tryptophanase.
  • tryptophanase enzymes are as discussed previously herein.
  • the precursor compound may be endogenously present in the sample and/or may be an exogenous compound added to a sample.
  • 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.
  • 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 ⁇ -lyase-catalyzed cleavage of cysteine conjugates. J Agric Food Chem 52: 110-116 (herein incorporated by reference). The synthesis of [ 2 Hi 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).
  • 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.
  • 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.
  • E. coli strain K-12 strain W1485F ' (Klena and Schnaitman, 1994) was used for the cloning of the tnaA tryptophanase gene, while strain DH5 ⁇ (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 trpl ⁇ ::hisG leu2A::hisG his3A::hisG) (Van Dyk D, Hansson G, Pretorius IS, Bauer FF. 2003.
  • 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 30 0 C 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.
  • sulfometuron methyl resistant yeast transformants For the selection of sulfometuron methyl resistant (Sm R ) yeast transformants, YPD and SCD media were supplemented with 100 ⁇ g/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, 10 6 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.
  • Sm R sulfometuron methyl resistant yeast transformants
  • genomic DNA from E. coli K- 12 strain W1485F " was used as template DNA with the following two primers: tnaA- F(£c ⁇ RI) 5'-GACTGAATTCATGGAAAACTTTAAACATCTCCCTG-S ' (SEQ ID NO.3) and tnaA-R(X/r ⁇ I) 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.
  • pHVXII Volschenk H, ViIj oen M, Grobler J, Petzold B, Bauer FF, Subden R, Young RA, Lonvaud-Funel A, Denayrolles M, Van Vuuren H
  • the PGKl P -tna A-PGKl 7 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, Whybosch 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
  • 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 6xlO 8 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 5O 0 C.
  • the cells were spun down and taken up in 800 ⁇ l of cell-suspension buffer [10 mM Tris (pH 7.2); 20 mM NaCl; 50 mM EDTA] and equilibrated at 50 0 C.
  • a volume of 10 ⁇ l Lyticase (Sigma-Aldrich) solution (12 mg/ml in 10 mM Tris, pH 8.0) was added and immediately combined with 800 ⁇ l 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 ⁇ l 1 M Tris (pH 7.5); 50 mM EDTA; 2.5 ml 0.5 M EDTA] and incubated for 1 h at 37 0 C 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 50 0 C 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 4 0 C 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.
  • 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 ⁇ l breaking buffer [2% (v/v) Triton X-IOO; 100 rriM Tris-Cl, pH 6.8.] and 200 ⁇ l 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 ⁇ l Cys-4MMP (10 mM), 20 ⁇ l pyridoxal 5'-phosphate (1 niM) and 830-875 ⁇ l assay buffer (20 mM phosphate buffer, pH 7.0; 1 mM EDTA). Volumes of 5-50 ⁇ l 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 ⁇ l of L-lactic dehydrogenase enzyme (EC 1.1.1.27; 5 units/ ⁇ l) was added to the reactions followed immediately by an addition of 100 ⁇ l NADH (3 mM).
  • YPD was inoculated with strains VIN 13 and VINO(C 1 S 1 Li). Grape juice was autoclaved and 200 ml aliquots inoculated with the yeast strains and incubated at 3O 0 C for 24 h reaching optical densities (measured at 600 nm; OD ⁇ OO ) 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.
  • the gas chromatograph was fitted with a Sol GelWax (SGE) fused silica capillary column, 15 m x 0.32 mm i.d., 0.5 ⁇ m 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 ⁇ m 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 40 0 C, was held at this temperature for 4 min, then increased to 280 0 C at 5°C/min, and held at this temperature for 5 min.
  • the injector was held at 220 0 C and the transfer line at 26O 0 C.
  • the sample was extracted at 40 0 C 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.
  • SIM Selective Ion Monitoring
  • the ions monitored in SIM runs were: m/z 81, 96, 108, 141 and 142 for [ 2 Hi 0 ]-4MMP, 55, 75, 89, 99 and 132 for 4MMP, 60, 62, 92, 109 and 144 for.[ 2 H 10 ]-3MH, 75, 90, 103, and 118 for [ 2 H 5 ]-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.
  • the repeatability of the analysis was determined at two concentrations for each analyte (100 ⁇ g/1 and 500 ⁇ g/1 for 4MMP, 50 ⁇ g/1 and 716 ⁇ g/1 for 3MH, and 50 ⁇ g/1 and 562 ⁇ g/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 ⁇ g/1), 5% (at 500 ⁇ g/1) and 5 ⁇ g/1; for 3MH, 1% (at 50 ⁇ g/1), 9% (at 716 ⁇ g/1), and 10 ⁇ g/1, and for 3MHA, 1% (at 50 ⁇ g/1), 8% at (at 562 ⁇ g/1), and 1 ⁇ g/1.
  • Lychee 4 tsp canned Lychee syrup (Admiral Lychees m light syrup)
  • Lantana/tomato leaf 4-6 leaves lantana vine per glass
  • Solvent/nail polish remover 0.05 ml glacial acetic acid m 100 ml BW
  • 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 22-24 0 C. 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).
  • ANOVA analysis of variance
  • cysteine- ⁇ -lyase was produced by the ⁇ 1278b yeast transformant.
  • 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- ⁇ -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.
  • the 'immediate' A 340 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.
  • 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.
  • 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 A 340 more than halves compared to its already low reading at the 60 min time point.
  • VIN13(CSLi) 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 ⁇ g/1) described above, no 3MH could be detected in the VIN 13 control ferment. On the other hand, the VINB(GS 1 ZJ) transformant released substantial amounts of 3MH, accompanied by smaller amounts of 3MHA, from the Cys-3MH precursor ( Figure 5).
  • 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 ⁇ -lyase activity cleaving of cysteine to ammonia and pyruvate.
  • the VIN 13 (CSLl) transformant produced a wine with lower volatile acidity compared to the VIN 13 host strain.
  • 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.
  • 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.
  • 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- ⁇ -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.
  • expression of the tnaA gene was directed by the S.
  • 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.
  • 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.

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Abstract

La présente invention concerne une méthode de modulation de la conversion d'un composé soufré non volatil en un thiol volatil. La méthode inclut l'une des deux étapes suivantes, ou les deux : (i) exposition du composé soufré non volatil à un micro-organisme génétiquement modifié présentant une augmentation de l'expression et/ou de l'activité d'une enzyme carbone-soufre lyase capable de convertir le composé soufré non volatil en thiol volatil ; et (ii) exposition du composé soufré non volatil à un extrait dérivé d'un micro-organisme génétiquement modifié présentant une augmentation de l'expression et/ou de l'activité d'une enzyme carbone-soufre lyase capable de convertir le composé soufré non volatil en thiol volatil.
PCT/AU2007/000199 2006-02-24 2007-02-23 Méthodes et micro-organismes pour la modulation de la conversion de composés soufrés non volatils en thiols volatils Ceased WO2007095682A1 (fr)

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AU2006900917 2006-02-24
AU2006900917A AU2006900917A0 (en) 2006-02-24 Methods and Micro-Organisms for Modulating the Conversion of Non-Volatile Sulfur Compounds to Volatile Thiol Compounds

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WO2010070838A1 (fr) * 2008-12-18 2010-06-24 メルシャン株式会社 Procédé de préparation d'une solution contenant du 3-mercaptohexan-1-ol et un alcool
JP2011094044A (ja) * 2009-10-30 2011-05-12 Ogawa & Co Ltd 持続性香気賦与剤
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EP2554650A1 (fr) 2011-08-01 2013-02-06 Pernod-Ricard Procédé de production de boisson alcoolisée ayant une saveur fruitée
EP3336166A1 (fr) * 2016-12-15 2018-06-20 Anheuser-Busch InBev S.A. Procédé de préparation d'un composant de boisson comprenant des thiols volatils, composant de boisson obtenu selon ledit procédé et utilisation d'un tel composant de boisson
CN112118741A (zh) * 2018-03-26 2020-12-22 丹斯塔发酵股份公司 增加植物中硫醇前体含量的方法
CN114829618A (zh) * 2019-10-17 2022-07-29 伯克利发酵科学公司 基因工程化的酵母细胞及其使用方法
EP4291627A4 (fr) * 2021-02-10 2025-02-19 Omega Yeast Labs, LLC Matériels et procédés pour le brassage de la bière

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WO2010070838A1 (fr) * 2008-12-18 2010-06-24 メルシャン株式会社 Procédé de préparation d'une solution contenant du 3-mercaptohexan-1-ol et un alcool
WO2010070836A1 (fr) * 2008-12-18 2010-06-24 メルシャン株式会社 Boisson sans alcool, extrait de péricarpe de raisin et leurs méthodes de production
JP2011094044A (ja) * 2009-10-30 2011-05-12 Ogawa & Co Ltd 持続性香気賦与剤
JP2011125256A (ja) * 2009-12-17 2011-06-30 Mercian Corp Ｓ−３−（ヘキサン−１−オール）−ｌ−システインの製造方法
AU2012292135B2 (en) * 2011-08-01 2016-03-31 Pernod Ricard Method of producing an alcoholic beverage having a fruity flavor
CN103814121A (zh) * 2011-08-01 2014-05-21 珀诺德·里卡德公司 生产具有果味的酒精饮料的方法
WO2013017581A1 (fr) 2011-08-01 2013-02-07 Pernod Ricard Procédé de production d'une boisson alcoolique à saveur fruitée
EP2554650A1 (fr) 2011-08-01 2013-02-06 Pernod-Ricard Procédé de production de boisson alcoolisée ayant une saveur fruitée
US9758752B2 (en) 2011-08-01 2017-09-12 Pernod Ricard Method of producing an alcoholic beverage having a fruity flavor
BE1025839B1 (nl) * 2016-12-15 2019-07-30 Anheuser-Busch Inbev S.A. Werkwijze voor het bereiden van een drankcomponent omvattend vluchtige thiolen, drankcomponent verkregen door dergelijke werkwijze en gebruik van dergelijke drankcomponent
WO2018109186A1 (fr) * 2016-12-15 2018-06-21 Anheuser-Busch Inbev S.A. Procédé de préparation de constituant de boisson comprenant des thiols volatils, constituant de boisson obtenu par un tel procédé, et utilisation d'un tel constituant de boisson
EP3336166A1 (fr) * 2016-12-15 2018-06-20 Anheuser-Busch InBev S.A. Procédé de préparation d'un composant de boisson comprenant des thiols volatils, composant de boisson obtenu selon ledit procédé et utilisation d'un tel composant de boisson
CN112118741A (zh) * 2018-03-26 2020-12-22 丹斯塔发酵股份公司 增加植物中硫醇前体含量的方法
CN114829618A (zh) * 2019-10-17 2022-07-29 伯克利发酵科学公司 基因工程化的酵母细胞及其使用方法
JP2022553039A (ja) * 2019-10-17 2022-12-21 バークリー ファーメンテーション サイエンス，インコーポレーテッド 遺伝子学的に改変された酵母細胞およびこれの使用の方法
EP4045670A4 (fr) * 2019-10-17 2023-11-08 Berkeley Fermentation Science Inc. Cellules de levure génétiquement modifiées et leurs procédés d'utilisation
US12503675B2 (en) 2019-10-17 2025-12-23 Berkeley Fermentation Science, Inc. Genetically engineered yeast cells and methods of use thereof
EP4291627A4 (fr) * 2021-02-10 2025-02-19 Omega Yeast Labs, LLC Matériels et procédés pour le brassage de la bière

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