AU2003238564A1 - Glutathione production - Google Patents

Description

WO 2004/003217 PCT/AU2003/000837 Glutathione Production Technical Field The present invention relates to methods for the production of glutathione by yeasts, as well as yeast mutants for the production of glutathione and for use in bakery 5 applications. Background Art Antioxidants are routinely used in foods (including animal feeds) for the protection of, for example, lipids and proteins against oxidative damage, and for avoidance of undesirable reactions such as discolouration and browning. They'are also routinely used 10 in the baking industry for control of the rheological properties of dough and the shelf-life of the baked products. Antioxidants are also now increasingly used in personal health-care products, medications and functional foods (to boost daily dietary intake of antioxidants): oxidation of DNA may directly promote cancer; cardiovascular disease is related to the is oxidation of blood lipoproteins which lead to development of atherosclerosis and/or oxidative damage to tissue; and progressive protein oxidation in the eye lens is responsible for the development of cataracts. Studies have shown that increasing intake of oxidants may result in significant reduction of risk of all three of these disease types. Antioxidants also find use in many other fields such as agriculture, aquaculture, 20 paints, and fermentation media. Thousands of synthetic and natural antioxidants have been evaluated for the food and pharmaceutical industries, however, synthetic antioxidants are falling into Worldwide disfavour due to toxicological problems and consumer reluctance, despite their typically lower cost of production. Even some natural antioxidants are falling into disfavour where 25 these are derived from animal sources (such as cysteine, often included in bread improvers for dough conditioning, and the most viable source of which is bird feathers or human hair). Glutathione, being a natural product, typically derived from non-animal sources, and with known biochemical pathways for utilisation within mammalian bodies and having known pathways for removal from mammalian bodies, is an increasingly 30 preferred antioxidant for use in foods, health care products and medications. The growing or potential markets existing in the pharmaceutical, therapeutic, personal health-care and food/nutritional markets for antioxidants has resulted in increased demand for glutathione and its derivatives.

WO 2004/003217 PCT/AU2003/000837 2 Although glutathione biosynthesis and degradation have been well studied (Fig. 1 provides a schematic of the glutathione biosynthetic pathway), the genetic mechanisms influencing intra/intercellular glutathione homeostasis have not been fully elucidated. Commercial production of glutathione has traditionally relied on yeast, in particular 5 selected strains of Saccharomyces or Candida species, and involves growing the yeast for extended periods of up to 5 days. The major proportion of the glutathione produced by the yeast is intracellular but is typically released by heating the harvested concentrated cream yeast (-18-22% solids) up to 70-80*C for 10-15 minutes, and during extraction the glutathione would be expected to concentrate to 10-15% of the dry extract solids. The 10 glutathione may then be further fractionated from the extracted solids, typically by chromatographic methods, but the 15% glutathione extracts are typically used without further purification at least in the food industry due to the prohibitive costs that would be associated with further purified product. These existing methods however suffer the following disadvantages: requirement is for significant amounts of heat/energy to extract the glutathione from the yeast; the need to isolate the glutathione from a large amount of other cellular components released from the yeast during the heating process; and the potential contamination of the yeast culture by other organisms (such as lactic acid bacteria, coliforis and wild yeasts) during the lengthy growth period typically used during commercial production. 20 Therefore, there is a need for an improved process for obtaining glutathione from yeast which can reduce the associated production costs and possibly even result in a cleaner product. Summary of the Invention The present invention relates to the finding that certain yeast mutants when cultured 25 under appropriate conditions release an increased amount of glutathione into the culture medium than the wild-type, and that this will allow for economic recovery of glutathione from the culture medium without the need to heat the yeast and without the need to remove other components that would typically be released from the yeast during heating. Further to this, the present invention also relates to novel mutant yeast strains which 30 secrete increased amounts of glutathione into their surrounding culture medium, relative to the wild-type yeast, and the use of these strains for the production of glutathione, including in breadmaking processes and fermentation of beverages.

WO 2004/003217 PCT/AU2003/000837 3 1. Processes of producing glutathione According to a first embodiment of the invention, there is provided a process for the production of glutathione, wherein said process comprises culturing a mutant yeast strain 5 under conditions promoting glutathione production, and wherein said yeast strain has one or more genetic mutations that result in increased secretion of glutathione into the culture medium relative to the parental strain. The glutathione secreted into the culture medium can, optionally, be isolated from the culture medium by techniques well known to those of skill in the art. It has been to surprisingly found that yeast mutants unable to synthesise or which have a reduced ability to synthesise certain metabolites and/or essential growth factors, such as amino acids or their precursors, secrete increased amounts of glutathione into the surrounding culture medium. Thus, according to one aspect of the process of the invention, the yeast strain is 15 incapable of the synthesis of one or more metabolites and/or essential growth factors which are included in the culture medium in limiting amounts. According to another aspect of the process of the invention, the yeast strain has a mutation that reduces the ability of the strain to synthesise one or more proteins, metabolites and/or essential growth factors which may optionally be included in the 20 culture medium in limiting amounts, depending on the capacity of the yeast strain to synthesise said proteins, metabolites and/or essential growth factors. Typically the metabolite(s) and/or essential growth factor(s) for which the yeast is deficient, or for which it has a reduced ability for synthesis, is an amino acid or a precursor or metabolite thereof. 25 Even more typically, the metabolite(s) and/or essential growth factor(s) for which the yeast is deficient, or for which it has a reduced ability for synthesis, is leucine, isoleucine and/or valine, or precursors or metabolites thereof, and more typically is leucine or precursors or metabolites thereof. It has also been found that a mutation in any one of a number of cellular processes 30 in yeast may lead to increased secretion of glutathione by the yeast into the surrounding culture medium. Thus, according to another aspect of the invention, the yeast strain has a mutation selected from the following groupings, which may overlap: WO 2004/003217 PCT/AU2003/000837 4 i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome or mutation or deletion of the mitochondrial genome; ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and 5 NAD(P); iii) mutation in a gene or genes affecting the assimilation and metabolism of nitrogen in the cell; iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA 10 pathway; v) mutation in a gene or genes affecting endosomal function; vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole transportation pathway or plasma membrane to endosome to vacuole traffic; vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated is proteolysis via the 26S proteosome; viii) mutation in a gene or genes affecting transportation of glutathione across the yeast cell membrane; ix) mutation in a gene or genes affecting glutathione degradation; and x) mutation in a gene or genes involved in vacuolar function. 20 The yeast strain may have more than one mutation within any one or more of the above groups (i) to (x). Yeast strains 'which could be used in the process of the present invention may include yeast selected from the genera Saccharomyces, Candida, Kluyveronyces, Pichia, Rhodotorula, Hansenula, Debaryomyces, Torulopsis or the fission yeast genus 25 Schizosaccharomyces. However, according to a preferred aspect of the methods of the invention, the yeast strain is a Saccharoinyces species, and more preferably a strain of Saccharonyces cerevisiae. According to a preferred aspect of the process according to the invention, the yeast strain has mutations in two or more of gene groups (i) to (x) listed above. Even more 30 preferably, such a yeast strain will also be a mutant for the synthesis of one or more proteins, metabolites and/or essential growth factors, wherein the mutant is unable to synthesise said one or more proteins, metabolites and/or essential growth factors or has a restricted ability for synthesis of said one or more metabolites and/or essential growth WO 2004/003217 PCT/AU2003/000837 5 factors. Typically the one or more metabolites and/or essential growth factors are amino acids or precursors or metabolites thereof More typically the one or more metabolites and/or essential growth factors are selected from leucine, isoleucine or valine or precursors or metabolites thereof, and even more typically from leucine or precursors or 5 metabolites thereof. According to another aspect of the invention, the yeast strains for use in the process according to the invention may have at least one mutation selected from groups (i) to (x) as described above, in addition to genetic manipulation resulting in overexpression of the glutathione synthesis pathway. Such manipulations resulting in, for example, 10 overexpression of gammaglutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2) or GSHI and GSH2. According to another preferred aspect of the process of the invention, the conditions under which the yeast strain is cultured include maintaining the yeast in aerobic growth which provides for increased glutathione production and secretion. 15 According to another preferred aspect of the process of the invention, the conditions under which the yeast strain is cultured include reduced pH, typically a pH of less than about 6, which has also been found to result in increased glutathione production and secretion. Typically the pH of the culture medium is between about 2.5 and 5, more advantageously between about 3 and 4.5, even more advantageously between about 3 and 20 4, and even more preferably about 3.5. According to another preferred aspect of the process of the invention, the conditions under which the yeast strain is cultured include the presence of monovalent cations, which has also been found to result in increased glutathione production and secretion. Typically, the monovalent cations are selected from sodium, potassium, rubidium and 25 caesium, preferably sodium or potassium and even more preferably potassium. The monovalent cation is typically provided as a salt, preferably as the chloride, and the concentration of the salt in the culture medium is typically from about 50mM to 500mM, more typically about 50 to 350mM, more typically from about 100 to 25OmM, even more typically from about 100 to 200mM, and preferably about 150mM. 30 According to a second embodiment of the invention, there is provided a process for the production of glutathione comprising culturing a yeast strain under conditions promoting glutathione production, wherein the culture medium comprises myo-inositol. Typically the resulting glutathione is isolated from the culture medium.

WO 2004/003217 PCT/AU2003/000837 6 According to a preferred aspect of this embodiment, the process is a process according to the invention utilising a mutant yeast strain as described above. Typically, where myo-inositol is included in the culture medium in a process of the invention, the concentration of myo-inositol is from about 0.01mM to 100mM (1.8mg/L 5 to 18000mg/L), more typically about 0.1 to 10mM, more typically from about 0.2 to 5mM, even more typically from about 0.5 to 2mM, and more typically about 1mM. According to a preferred aspect of the processes of the invention, the culture medium comprises myo-inositol and elevated levels of a carbon source. Typically the carbon source used in processes of the invention is selected from 10 fermentable sugars, more typically glucose or fructose or a combination thereof, and/or from oligosaccharides which are homo- or hetero- oligomers comprising fermentable sugar moieties, such as sucrose or maltose, even more typically sucrose. Alternatively, the carbon source may be a non-fermentable carbon source, more typically ethanol, glycerol, lactate, galactose or raffinose. 15 Typically, a carbon source is included in a culture medium at a concentration of about 1-2 %w/v. Where elevated carbon source levels are to be included in the culture medium in combination with myo-inositol, the concentration of the carbon source in the initial, uninoculated, culture medium, is typically greater than about 2% w/v, more typically between about 2% and 10% w/v, more typically between about 3% and 8% w/v; 20 more typically between about 3% and 6% w/v, and even more typically about 4% w/v. According to a preferred aspect of a process of the invention, the process comprises growth of the yeast strain by batch-wise culture. If desired, the glutathione may then be extracted from the culture medium by any of a number of known methods, such as chromatographic methods. 25 Alternatively, a process of the invention comprises growth of the yeast by continuous culture, allowing for continuous harvesting of culture medium and therefore recovery of secreted glutathione. According to yet another aspect of a process of the invention, the process comprises dough preparation. Doughs prepared by this process, or baked products derived 30 therefrom are also provided. According to yet another aspect of a process of the invention, the process comprises preparation of a fermented product. Fermented products prepared by said process are also provided.

WO 2004/003217 PCT/AU2003/000837 7 2. Yeast strains for glutathione production and/or baking or fermentation applications The invention also relates to novel strains obtained by any fonn of directed mutagenesis, consisting of generating, preferably in industrial strains of yeasts, 5 particularly baker's yeast, or in the starting haploids that served for construction of the industrial strains, mutations, monogenic or not, giving the required phenotype in the strains. This includes strains selected after conventional mutation treatment, for example using chemical/physical agents or molecular biological techniques or standard selection recombination methods to generate multiple mutants. 10 Thus, according to a third embodiment of the invention, there is provided a mutant yeast strain having at least two mutations selected from the following groupings, which may overlap: i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the 15 mitochondrial genome or mutation or deletion of the mitochondrial genome;ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and NAD(P)+; iii) mutation in a gene or genes affecting the assimilation and metabolism of nitrogen in the cell; 20 iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA pathway; v) mutation in a gene or genes affecting endosomal function; vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole 25 transportation pathway or plasma membrane to endosome to vacuole traffic; vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated proteolysis via the 26S proteosome; viii) mutation in a gene or genes affecting transportation of glutathione across the yeast cell membrane; 30 ix) mutation in a gene or genes affecting glutathione degradation; x) mutation in a gene or genes involved in vacuolar function. The yeast strain may have more than one mutation within one of the above groups (i) to (x).

WO 2004/003217 PCT/AU2003/000837 8 Yeast strains which are contemplated by the present invention include, but are not necessarily limited to yeast selected from the genera Saccharomyces, Candida, Kluyveronyces, Pichia, Rhodotorula, Hansenula, Debaryomyces, Torulopsis or the fission yeast genus Schizosaccharonyces. However, according to a preferred aspect of 5 this embodiment of the invention, the yeast strain is a Saccharomyces species, and more preferably a strain of Saccharonyces cerevisiae. According to a preferred aspect of this embodiment of the invention, the yeast strain has mutations in one or more of mutation groups (i) to (x) listed above and will also be a mutant for the synthesis of one or more proteins, metabolites and/or essential growth 10 factors, wherein said mutant is unable to synthesise said one or more proteins, metabolites and/or essential growth factors or has a restricted ability to synthesise said one or more proteins, metabolites and/or essential growth factors. Typically the one or more metabolites and/or essential growth factors are amino acids or precursors or metabolites thereof. More typically the one or more metabolites and/or essential growth factors are is selected from leucine, isoleucine or valine or precursors or metabolites thereof, and even more typically is leueine or precursors or metabolites thereof. According to a preferred aspect of this embodiment of the invention, the yeast strain may have at least one mutation selected from groups (i) to (x) as described above, in addition to genetic manipulation resulting in overexpression of the glutathione synthesis 20 pathway. Such manipulations resulting in, for example, overexpression of gammaglutamylcysteine synthetase (GSHI), glutathione synthetase (GSH2) or GSH1 and GSH2. According to a fourth embodiment of the invention, there is provided a yeast strain herein described as BSO4ycfl. The BSO4 mutation has been identified as a defect in the 25 HAC1 gene (YFL031W ). According to a fifth embodiment of the invention, there is provided a method of preparing a dough comprising combining a yeast strain according to the invention with other dough components. Doughs prepared by this method, and baked products derived therefrom, are also provided. 30 According to a sixth embodiment of the invention, there is provided a method of producing a fermented product comprising adding to the unfermented precursor component(s) of said product a yeast strain according to the invention. Fermented products obtained by this method are also provided.

WO 2004/003217 PCT/AU2003/000837 9 3. Compositions comprising glutathione obtained by the process of the invention, and uses thereof According to a seventh embodiment of the invention, there is provided glutathione obtained by a process of the invention. The glutathione may be provided as a 5 concentrated form of the culture medium or it may be purified to any desired degree. The glutathione may be used in a wide variety of applications including, but not restricted to personal health care, pharmaceuticals, nutraceuticals, cosmetics, food (including bakery and fermentation technology) and animal feeds, agriculture, aquaculture, paints, and fermentation media. For pharmaceutical applications the 10 glutathione is preferably provided as a purified compound, typically greater than 60% pure, more typically greater than 70% pure, more typically greater than 80% pure, even more typically greater than 90% pure, and more preferably greater than 95% pure. According to an eighth embodiment of the invention, there is provided a personal health care composition comprising glutathione obtained by a process of the invention is and a pharmaceutically or topically acceptable carrier. According to a ninth embodiment of the invention, there is provided a pharmaceutical composition comprising glutathione obtained by a process of the invention and a pharmaceutically acceptable carrier. According to a tenth embodiment of the invention, there is provided a food or 20 nutraceutical composition comprising glutathione obtained by a process of the invention in combination with one or more food components. The food/nutraceutical composition may be selected from liquids, semi-solids and solids. According to an eleventh embodiment of the invention, there is provided a dough or bread improving composition comprising glutathione obtained by a process of the 25 invention and a suitable carrier. The carrier may be selected from a wide variety of bakery acceptable ingredients, including flour and/or sugar and the composition may also include other bread improving ingredients such as enzymes (including cellulases, glucanases, amylases, xylanases, arabinoxylanases, dextrinases, maltases, etc.). The composition may be in the form of a powder, granulate or liquid. 30 According to a twelfth embodiment of the invention, there is provided an animal feed additive comprising glutathione obtained by a process of the invention and a suitable carrier. The carrier may be selected from a wide variety of acceptable animal feed ingredients, such as flour (including wheat, corn or soy), and the composition may also WO 2004/003217 PCT/AU2003/000837 10 include other animal feed additives including those which improve the digestibility of the food such as enzymes (including cellulases, glucanases, amylases, xylanases, arabinoxylanases, dextrinases, maltases, etc.). The composition may be in the form of a powder, granulate or liquid. 5 According to a thirteenth embodiment of the invention, there is provided an animal health care composition comprising glutathione obtained by a process of the invention and a veterinary acceptable carrier. According to a fourteenth embodiment of the invention, there is provided a method for preventing oxidative damage in the circulation or tissues of a mammal, said method 10 comprising administering to said mammal an effective amount of a composition comprising glutathione obtained by a process of the invention. According to a fifteenth embodiment of the invention, there is provided a method of protecting a food product from oxidative deterioration comprising adding to said food product an effective amount of glutathione obtained by a process of the invention or a is composition comprising it. Food products prepared by said method are also provided. The food product may be liquid, semi-solid or solid. According to a sixteenth embodiment of the invention, there is provided a method of preparing a dough comprising combining dough components with an effective amount of glutathione obtained by a process of the invention. Doughs prepared by this method, 20 or baked products derived therefrom are also provided. Brief Description of the Drawings Figure 1 shows a representation of the biosynthetic pathway for glutathione in yeast. Figure 2 shows intracellular and extracellular glutathione production with time 25 after inoculation into fresh medium for a mutant strain as compared to the parental strain. Figure 3 is a graph illustrating glutathione production (intracellular and extracellular) with time after inoculation into fresh medium for a deletion mutant (Avps27) of yeast strain BY4743 (Winzeler E.A. et al., (1999), Science 285: 901-906) as compared to the parental strain. 30 Figure 4 is a bar chart showing increased glutathione secretion by the dominant mutant RAS2 Val19 as compared to ras2 and the parental strain. Figure 5 illustrates potential interactions between cellular compartments/ components, associated genes/mutations and glutathione secretion (relative to the parental WO 2004/003217 PCT/AU2003/000837 11 strain - values in brackets represent the ratio of glutathione secreted by the mutant to that secreted by the parental strain). Figure 6 illustrates potential interactions between mitochondrial respiratory chain components, associated genes/mutations and glutathione secretion (relative to the parental 5 strain - values in brackets represent the ratio of glutathione secreted by the mutant to that secreted by the parental strain). Figure 7 is a graph illustrating extracellular glutathione vs pH for a mutant yeast strain as compared to the parental strain. Figure 8 is a bar chart of extracellular glutathione vs pH for a deletion mutant as 10 compared to the parental strain. Figure 9 shows two bar charts - one for extracellular glutathione and the other for corresponding intracellular glutathione produced at pH 3.5 or 6.0 for deletion mutants of yeast strain BY4743 as compared to the parental strain. Figure 10 provides two bar charts illustrating comparative glutathione productions, 15 both intracellular and extracellular for a mutant in the presence of different monovalent salts as compared to the parental strain. Figure 11 is a bar chart showing increased extracellular glutathione levels produced by a wild-type haploid strain grown on SD medium, SD medium supplemented with 200mg/L myo-inositol, SD medium supplemented with 4% w/v glucose, and SD medium 20 supplemented with 200mg/L myo-inositol and 4% w/v glucose. Figure 12 is a bar chart illustrating extracellular glutathione for two yeast single mutants and a double mutant relative to the parental strain. Figure 13 is a bar chart showing extracellular glutathione levels produced by a mutant yeast strain having the combined deletion of HGT1 and loss of mitochondrial 25 respiratory function (petite cells). Figure 14 is a bar chart showing extracellular glutathione levels produced by wild type haploid strains (CY4 and BY4742), single mutants thereof, and diploids obtained by mating the haploids. Definitions 30 The term "Yeast" encompasses any group of unicellular fungi that reproduce asexually - by budding or fission - and sexually - by the production of ascospores. Yeast cells may occur singly or in short chains, and some species produce a mycelium. Typically the yeast will be a member of the genera Saccharonyces, Candida, WO 2004/003217 PCT/AU2003/000837 12 Kluyveromyces, Pichia, Rhodotorula, Hansenula, Debaryonyces, Torulopsis or the fission yeast genus Schizosaccharomyces. However, typically, the yeast is a Saccharomyces species, more typically a strain of Saccharoinyces cerevisiae, and even more typically an industrial baker's yeast strain. 5 "Increased secretion of glutathione into the culture medium relative to the wild type" as referred to herein means secretion of at least 50% more, preferably at least 100% more glutathione by the mutant, relative to the parental strain when grown as described in Example 1 herein. The glutathione secretion by the mutant relative to the wild-type may be expected to vary depending on the growth conditions. 10 The term "mutation" encompasses any mutation which results in a "functional" deficiency, irrespective of how the genes have been mutated. Mutations may typically include deletion mutations, point mutations, insertion or substitution mutations, frame shift mutations or any other method that results in inactivation of a gene (including RNAi approaches to selectively inactivating gene expression). The terms "mutant yeast", is "mutant strain" and "mutant yeast strain" as used herein have corresponding meanings. As used herein, the term "aerobic growth" refers to the growth phase in which yeast is grown in the presence of oxygen. In batch growth of yeast in culture flasks on a given amount of fermentable sugar, aerobic growth on ethanol occurs after the 'diauxic shift' when all the fermentable sugars have been consumed, consumption of sugars to produce 20 ethanol stops and the yeast's physiology alters to adapt to growth on ethanol by respiration. In commercial scale fermenters, yeast is typically grown with exponential sugar feeding rates, after the yeast has started to efficiently consume the ethanol although ethanol is also produced during such an 'aerobic' yeast fermentation, this is generally consumed at a greater rate than it is produced and this growth pattern is also 25 encompassed within the term 'aerobic growth' as used herein. The term "isolated", where used in relation to glutathione, indicates that the material in question has been removed from a cell culture, and associated impurities either reduced or eliminated. Essentially, the 'isolated' material is enriched with respect to other materials extracted from the same source (ie., on a molar basis it is more abundant than 30 any other of the individual species extracted from a given source), and preferably a substantially purified fraction is a composition wherein the 'isolated' material comprises at least about 60percent (on a molar basis) of all molecular species present. Generally, a substantially pure composition of the material will comprise more than about 80 to 90 WO 2004/003217 PCT/AU2003/000837 13 percent of the total of molecular species present in the composition. Most preferably, the 'isolated' material is purified to essential homogeneity (contaminant species cannot be detected in appreciable amounts). An "effective amount", as referred to herein, includes a sufficient, but non-toxic 5 amount of substance to provide the desired effect. The "effective amount" will vary from application to application (such as from dough preparation to use in pharmaceutical compositions) and even within applications (such as from subject to subject in pharmaceutical applications, and from dough to dough in baking applications). For any given case, an appropriate "effective amount" may be determined by one of ordinary skill 10 in the art using only routine experimentation. The term "carbon source", as referred to herein, includes carbohydrates which can be taken up by yeast cells and converted to energy through fermentative and/or aerobic growth pathways. Typically, the carbon source is a fermentable sugar, typically glucose or fructose or a combination thereof, and/or from oligosaccharides which are homo- or is hetero- oligomers comprising fermentable sugar moieties, such as sucrose or maltose, even more typically sucrose. Typically the carbon source is selected from glucose, fructose and/or sucrose (which in commercial sugar sources such as molasses typically occur together), although these are initially utilised through the fermentative pathway to produce primarily ethanol, which is then utilised through the oxidative pathway. 20 In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. Best Mode of Performing the Invention 1. Processes for the production of glutathione 25 The present invention relates to a finding that certain types of mutation in yeasts can result in significantly increased secretion of glutathione relative to a parental strain (examples provided in Figs. 2 to 4), which typically secrete only a small fraction of the glutathione produced. Thus the present invention relates to a process for the production of glutathione, wherein said process comprises culturing a mutant yeast strain under 30 conditions promoting glutathione production, and optionally isolating glutathione from the culture medium, and wherein said yeast strain has one or more genetic mutations that WO 2004/003217 PCT/AU2003/000837 14 result in increased secretion of glutathione into the culture medium relative to a parental strain. It has been surprisingly found that yeast mutants unable to synthesise or which have a reduced ability to synthesise certain amino acids secrete increased amounts of 5 glutathione into the surrounding culture medium. This increased secretion, relative to a parental strain, can be reduced if not eliminated by supplementing the yeast with a compensating amount of the required amino acid or by transforming the strain back to a leucine-synthesising phenotype. According to one aspect, the yeast strain is incapable of the synthesis of one or 10 more metabolites and/or essential growth factors which are included in the culture medium in limiting amounts. According to another aspect, the yeast strain has a mutation that reduces the ability of the strain to synthesise one or more proteins, metabolites and/or essential growth factors which may optionally be included in the culture medium in limiting amounts, 15 depending on the capacity of the yeast strain to synthesise said proteins, metabolites and/or essential growth factors. Typically the metabolite(s) and/or essential growth factor(s) for which the yeast is deficient, or for which it has a reduced ability for synthesis, is an amino acid or a precursor or metabolite thereof. 20 Even more typically, the metabolite(s) and/or essential growth factor(s) for which the yeast is deficient, or for which it has a reduced ability for synthesis, is leucine, isoleucine and/or valine or precursors or metabolites thereof, and more typically is leucine or precursors or metabolites thereof. Typically the metabolite which the strain is unable to synthesise, or which it has a 25 reduced ability for the synthesis of, is included in the growth medium at sub-optimal levels, typically approximately half-optimal levels. It has also been found that a mutation in a number of pathways in yeast may lead to increased secretion of glutathione by the yeast into the surrounding culture medium. Thus, according to another aspect, the yeast strain has a mutation in one or more of 30 the following groupings, which may overlap: i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome or mutation or deletion of the mitochondrial genome; WO 2004/003217 PCT/AU2003/000837 15 ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and NAD(P)*; iii) mutation in a gene or genes affecting the assimilation and metabolism of nitrogen in the cell; 5 iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA pathway; v) mutation in a gene or genes affecting endosomal function; vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole 10 transportation pathway or plasma membrane to endosome to vacuole traffic; vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated proteolysis via the 26S proteosome; viii) mutation in a gene or genes affecting transportation of glutathione across the yeast cell membrane; 15 ix) mutation in a gene or genes affecting glutathione degradation; and x) mutation in a gene or genes involved in vacuolar function. The yeast strain may also have more than one mutation within one of the above groups (i) to (x). Figures 5 and 6 illustrate potential ways in which some of the above listed mutation 20 types may affect the secretion of glutathione from yeast cells. Yeast strains which could be used in a process of the present invention may include yeast selected from the genera Saccharomyces, Candida, Kluyveromyces, Pichia, Rhodotorula, Hansenula, Debaryonyces, Torulopsis or the fission yeast genus Schizosaccharonyces. However, according to a preferred aspect of the methods of the 25 invention, the yeast strain is a Saccharomyces species, more preferably a strain of Saccharomyces cerevisiae and even more preferably an industrial strain of baker's yeast which can better withstand the conditions to which yeast are exposed during industrial scale fermentations. According to another aspect, the yeast is a mutant strain of Saccharomyces 30 cerevisiae which has at least a mutation in a gene encoding a component of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome, wherein said gene is selected from the following: YBL09W (ATPJ); YBR03W (COX]); YBR037C (SCOI); YBR191W (RPL21a); WO 2004/003217 PCT/AU2003/000837 16 YBR220C; YBR268W (MRPL37); YCRO46C (IMG]); YDLO69C (CBS]); YDL107W (MSS2); YDL202W (MRPL11); YDR079W (PET1O); YDR175C (RSM24); YDR197W (CBS2); YDR204W (COQ4); YDR298C (ATP5); YDR322W (MRPL35); YDR337W (MRPS28); YDR462W (MRPL28); YDR529C (QCR7); YER017C (AFG3); YER141W 5 (COX15); YER153C (PET122); YER154W (OXA1); YFL034W (MRPL7); YGRO62C (COX18); YGR171C (MSM1); YGR220C (MRPL9); YGR257C; YHLO04W (MRP4); YHL038C (CBP2); YHRO11W (DIA4); YHR051W (COX6); YHR120w (MSH1); YHR147C (MRPL6); YIL006W; YIL018W (RPL2B); YIL065C (FISJ); YIL070C (MAM33); YIL093C (RSM25); YIL098C (FMCJ); YIR021W (MRS1); YJL063C 10 (MRPL8); YJL102W (MEF2); YJL166W (QCR8); YJL209W (CBPJ); YJR144W (MGM1O1); YKL03C (MRP17); YKL032C (IX1?); YKR06C (MRPL13); YLL009C (COX17); YLL018C-A (COX19); YLR067C (PET309); YLR139C (SLS1); YLR295C (HSP60); YLR369W (SSQI); YML078W (CPR3); YMR064W (AEP]); YMR072W (ABF2); YMR150C (IMP]); YMR193W (MRPL24); YMR228W (MTFJ); YNL177C; 15 YNR036C; YNR037C (RSM19); YNR045W (PET494); YOL009C (MDM12); YOLO33W (MSEJ); YOLO95C (HMIJ); YOR026W (BUB3); YPL132W (COX11); YPL183W-A; YPRO04C; YPR166C(MRP2); and YPR191W(QCR2). According to another aspect, the yeast is a mutant strain of Saccharonyces cerevisiae which has at least a mutation in a gene affecting the levels of NADH and 20 NAD*, wherein said gene is selected from genes encoding enzymes which catalyse the synthesis of glycerol, ethanol and/or genes the expression of which suppress or result in competition for the GLN1, GLT1 glutamate synthesis pathway. Typically these genes are selected from YIL053W (RHR2), YOR375C (GDHJ) and YNL229c (URE2). According to another aspect, the yeast is a mutant strain of Saccharomyces 25 cerevisiae which has at least a mutation in a gene affecting the assimilation and metabolism of nitrogen in the cell, wherein said gene is selected from: YDR300C (PRO1); YDR448W (ADA2); YEL009C (GCN4); YEL062W (NPR2); YGL227W (VID30); YGR252W (GCN5); YNL106C (INP52); YNL229C (URE2); YOR375C (GDH1); and YPL254W(HFIl). 30 According to another aspect, the yeast is a mutant strain of Saccharonyces cerevisiae which has at least a mutation in a gene encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA WO 2004/003217 PCT/AU2003/000837 17 pathway, and wherein said gene is selected from YOL081 W (IRA2); YOR360C (PDE2); and YNL098C (R AS2; RAS2Val19 dominant mutation). According to another aspect, the yeast is a mutant strain of Saccharonyces cerevisiae which has at least a mutation in a gene affecting endosomal function, wherein 5 said gene is selected from: YCLO08C (VPS23; STP22); YDR456W (NHX1); YJR102C (VPS25); YKL02W (DID4); YKL041W (VPS24); YKR035W-A (DID2); YLRO25W (VPS32/SNF7); YLRJ19W (SRN2/VPS37); YLR417W (VPS36); YMR077C (VPS20); YNR06W(VPS27); YPLO65W(VPS28); and YPR173C(VPS4). According to another aspect, the yeast is a mutant strain of Saccharomyces 10 cerevisiae which has at least a mutation in a gene affecting endoplasmic reticulum function, the Golgi to endosome to vacuole transportation pathway, or vacuolar function wherein said gene is selected from: YFL031W (HAC1), YDR027C (LUVJ/VPS54); YDR323C (PEP7/VPS19); YDR484W (VPS52/SAC2); YBR131W (CCZ1); YDR486C (VPS60); YHRO12W (VPS29); YJL154C (VPS35); YLRIJ48W (VA C1/PEP3/VPSJ8); 15 YMLOOIW(YPT7); and YOR036W(PEPJ2/VPS6). According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae which has at least a mutation in a gene affecting ubiquitin levels and ubiquitin mediated proteolysis via the 26S proteosome, wherein said gene is selected from: YBR173C (UMPJ); YER151C (UBP3); YFROJOW (UBP6); YHL01C (PRS3); 20 YKL213C (DOA1); YNR051C (BRE5); YPL03W(ULA1); and YPLO74W(YTA6). According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae which has at least a mutation in a gene involved in transportation of glutathione across the yeast cell membrane, wherein said gene is YDR135C (YCF1) or YJL212C (HGTI). 25 According to a preferred aspect, the yeast strain has mutations in two or more of groups (i) to (x) listed above. Examples of such mutations are described in paragraph 2.1 below. Even more preferably, such a yeast strain will also be a mutant for the synthesis of one or more proteins, metabolites and/or essential growth factors, wherein the mutant is 30 unable to synthesise said one or more proteins, metabolites and/or essential growth factors or has a restricted ability for synthesis of said one or more metabolites and/or essential growth factors. Typically the one or more metabolites and/or essential growth factors are amino acids or precursors or metabolites thereof. More typically the one or more WO 2004/003217 PCT/AU2003/000837 18 metabolites and/or essential growth factors are selected from leucine, isoleucine or valines or precursors or metabolites thereof, and even more typically from leucine or precursors or metabolites thereof. According to another aspect, the yeast strains for use in a process according to the 5 invention may have at least one mutation selected from groups (i) to (x) as described above, in addition to genetic manipulation resulting in overexpression of the glutathione synthesis pathway. Such manipulations resulting in, for example, overexpression of gammaglutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2) or GSH1 and GSH2. 10 According to another preferred aspect, the conditions under which the yeast strain is cultured include maintaining the yeast in aerobic growth which provides for increased glutathione production and secretion. According to another preferred aspect, the conditions under which the yeast strain is cultured include reduced pH, typically a pH of less than about 6, which has also been 15 found to result in increased glutathione production and secretion. Typically the pH of the culture medium is between about 2.5 and 5, more advantageously between about 3 and 4.5, even more advantageously between about 3 and 4, and even more preferably about 3.5. Figures 7 to 9 illustrate results of extracellular glutathione levels produced by representative strains at either pH 3.5 or pH 6.0 or intermediate values (the culture 20 conditions being as described in Example 3). According to another preferred aspect, the conditions under which the yeast strain is cultured include the presence of monovalent cations, which has also been found to result in increased glutathione production and secretion. Typically, the monovalent cations are selected from sodium, potassium, rubidium and caesium, preferably sodium or potassium 25 and even more preferably potassium. The monovalent cation is typically provided as a salt, preferably as the chloride, and the concentration of the salt in the culture medium is typically from about 50mM to 500mM, more typically 50 to 350mM, more typically from 100 to 250mM, even more typically from 100 to 200mM, and preferably about 150mM. Mutations that are likely to affect the natural metabolism/homeostasis of these cations are 30 also expected to play a role in glutathione homeostasis and are contemplated by the present invention. Figure 10 illustrates extracellular glutathione levels produced by the mutant strain BSO4 and the wild-type when grown without, or in the presence of NaCl, KCl, RbCl or CsCL (the culture conditions being as described in Example 3).

WO 2004/003217 PCT/AU2003/000837 19 The addition of myo-inositol to the culture medium has also been found to result in increased glutathione production and secretion by yeast strains. Thus, according to a second embodiment of the invention, there is provided a process for the production of glutathione comprising culturing a yeast strain under 5 conditions promoting glutathione production, wherein the culture medium comprises myo-inositol. Typically the glutathione is isolated from the culture medium. According to a preferred aspect of this embodiment, the process is a process according to the invention utilising a mutant yeast strain as described above. 10 Typically, where myo-inositol is included in the culture medium in processes of the invention, the concentration of myo-inositol is from about 0.01mM to 100mM (1.8mg/L to 18000mg/L), more typically about 0.1 to lOmM, more typically from about 0.2 to 5mM, even more typically from about 0.5 to 2mM, and more typically about 1mM. A synergistic effect of myo-inositol and elevated levels of carbon source, such as is glucose or the resulting ethanol, on the production of glutathione by yeast cells has also been found. Therefore, according to a preferred aspect of the processes of the invention, the culture medium comprises myo-inositol and elevated levels of a carbon source. Typically the carbon source is selected from fennentable sugars, more typically 20 glucose or fructose or a combination thereof, and/or from oligosaccharides which are homo- or hetero- oligomers comprising fermentable sugar moieties, such as sucrose or maltose, even more typically sucrose. Other mono- and oligosaccharides (such as galactose, xylose, lactose, glucosyl sucrose oligosaccaharides such as raffinose and stachyose) or sugar alcohols (such as mannitol, xylitol) are also contemplated where yeast 25 strains are capable of utilising these sugars. Alternatively, the carbon source may be a non-fermentable carbon source, more typically ethanol. The ethanol' may be added as such to the other culture medium ingredients or, more typically, result from the fermentation of sugars by the yeast culture. Typically, a carbon source is included in a culture medium at a concentration of 30 about 1-2 %w/v. Where elevated carbon source levels are to be included in the culture medium in combination with myo-inositol, the concentration of this substrate in the initial, uninoculated, culture medium, is typically greater than about 2% w/v, more WO 2004/003217 PCT/AU2003/000837 20 typically between about 2% and 10% w/v, more typically between about 3% and 8% w/v; more typically between about 3% and 6% w/v, and even more typically about 4% w/v According to a preferred aspect, the yeast strain is grown as a batch-wise culture. If desired, the glutathione may then be extracted from the culture medium by any of a 5 number of known methods, such as chromatographic methods. Alternatively, the yeast may be grown under continuous culture conditions, allowing for continuous harvesting of culture medium and therefore recovery of secreted glutathione. According to yet another aspect of the process of the invention, the process relates 10 to dough preparation. Methods of preparing doughs/baked products are well known in the art. Yeast is typically combined with the other dough components (typically flour, salt, shortening, bread improvers and other additives) as approximately 1-2% of flour weight, although this may vary depending on the type of dough and fermentation type (such as sponge-and-dough, rapid dough/mechanical dough preparation, high-sugar 15 doughs). Although the mutant yeast may make up the total yeast component of the dough, it may also be added as a proportion only of the total yeast component of the dough, a standard commercial baker's yeast making up the remaining amount. Doughs prepared by such processes, or baked products derived therefrom are also provided. According to yet another aspect of the process of the invention, the process is part 20 of fermentation of a beverage, typically beer or wine. Antioxidants are routinely added to fermented beverages so as to inhibit oxidation of the alcohol (or other components) - a process according to this aspect provides the benefit of avoiding the need to add exogenous antioxidants to the brew. Processes for the production of fermented products are well known to those skilled in the art, and amounts of yeast to be added vary 25 significantly amongst targeted products. The mutant strain may comprise all or a portion only of the total yeast component to be added. Processes according to the invention for the production of glutathione will comprise any suitable technique known to those in the art. Typically the process will be carried out in fermenters, more typically industrial scale fermenters such as are already in use for the 30 commercial production of baker's yeast. For example, for batch-wise commercial production of glutathione, a seed culture of the mutant yeast will be produced for inoculation into a fermenter containing a suitable culture medium typically comprising from about 1-2% total fermentable sugars as well as WO 2004/003217 PCT/AU2003/000837 21 a suitable nitrogen source (such as urea) and a phosphate source (such as monoammoniumphosphate) and optionally growth factors such as vitamins (for example, biotin), and/or such as a metabolite or growth factor which the mutant yeast strain is unable to synthesise or for which the mutant yeast strain has a restricted ability for 5 synthesis. In the latter case, the metabolite/growth factor is maintained at sub-optimal concentrations in the fermentation medium, typically at about half-optimal levels. Typically, once ethanol production. has ceased and the ethanol content of the culture medium has dropped to about 0.1-0.3% v/v, an exponential feeding protocol is started by increasing rate of feeding of a sugar source containing, typically, approximately 18-30% 1o total fermentable sugars. The sugar feeding rate is kept at a rate whereby ethanol consumption predominantly exceeds ethanol production (except for the option of a sugar pulse, depending on the desired growth protocol and target activity of the yeast). A suitable nitrogen source and phosphate source are added in pre-determined amounts throughout the fermentation, the amounts depending on the final total yeast solids and the 15 target protein content (typically between 40 to 60% Kjehldal protein). Metabolites and/or growth factors, if the mutant yeast strain is unable to synthesise one or more of these or has a restricted ability for the synthesis, will also be added throughout the fermentation at sub-optimal levels so as to maintain growth. Other additives, such as anti-foam are added if required. 20 Since intracellular glutathione has been found in these studies to overaccumulate prior to secretion, and in many of the strains tested, altered glutathione metabolism was triggered by amino acids limitation (particularly leucine, isoleucine and valine), growth of selected strains under a continuous state of low-leucine (or other parameters) is expected to provide a means of increasing glutathione production further. This could be achieved 25 via the use of a continuous fed-batch culture system. This approach would maintain cells under optimal conditions to facilitate/maximise glutathione production.2. Yeast strains for glutathione production The invention also relates to novel strains obtained by any form of directed mutagenesis, consisting of generating, preferably in industrial strains of yeasts, 30 particularly baker's yeast, or in the starting haploids that served for construction- of the industrial strains, mutations, monogenic or not, giving the required phenotype in the strains. This includes strains selected after conventional mutation treatment, for example WO 2004/003217 PCT/AU2003/000837 22 using chemical/physical agents or molecular biological techniques or standard selection recombination methods to generate multiple mutants. 2.1 Yeast mutants with increased glutathione secretion The present invention therefore also relates to a mutant yeast strain having at least 5 two mutations selected from the following groupings, which may overlap: i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome, or mutation or deletion of the mitochondrial genome; ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and 10 NAD(P)*; iii) mutation in a gene or genes affecting the assimilation and metabolism of nitrogen in the cell; iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA 15 pathway; v) mutation in a gene or genes affecting endosomal function; vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole transportation pathway or plasma membrane to endosome to vacuole traffic; vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated 20 proteolysis via the 26S proteosome; viii) mutation in a gene or genes affecting transportation of glutathione across the yeast cell membrane; ix) mutation in a gene or genes affecting glutathione degradation; and x) mutation in a gene or genes involved in vacuolar function. 25 The yeast strain may have more than one mutation within one of the above groups (i) to (x). For example, two different mutations in group (ii) genes may be contemplated such as a combination of a mutation affecting glycerol synthesis and a mutation in a gene the expression of which suppress or result in competition for the GLN1, GLT1 glutamate synthesis pathway. 30 According to one aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which the genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome are selected from the following: YBL09W(ATP1); YBR03W(COX); YBR037C (SCOI); WO 2004/003217 PCT/AU2003/000837 23 YBR191W (RPL21a); YBR220C; YBR268W (MRPL37); YCR046C (IMG1); YDLO69C (CBS]); YDLJ07W(MSS2); YDL202W(MRPL1I); YDR079W(PETOO); YDR175C (RSM24); YDR197W (CBS2); YDR204W (COQ4); YDR298C (ATP5); YDR322W (MRPL35); YDR337W (MRPS28); YDR462W (MRPL28); YDR529C (QCR7); 5 YER017C (AFG3); YER141W (COX15); YER153C (PET122); YER154W (OXAJ); YFL034W (MRPL7); YGRO62C (COX18); YGR171C (MSMJ); YGR220C (MRPL9); YGR257C; YHL04W (MRP4); YHL038C (CBP2); YHRO1W (DIA4); YHR051W (COX6); YHR120w (MSHJ); YHR147C (MRPL6); YIL006W; YIL018W (RPL2B); YIL065C (FIS]); YIL070C (MA M33); YIL093C (RSM25); YIL098C (FMCJ); YIRO21W 10 (MRS1); YJL063C (MRPL8); YJL102W(MEF2); YJL166W(QCR8); YJL209W(CBP1); YJR144W (MGM101); YKLO03C (MRP17); YKL032C (IXR1); YKR06C (MRPL13); YLL009C (COX] 7); YLL018C-A (COX19); YLR067C (PET309); YLR139C (SLS1); YLR295C (HSP60); YLR369W (SSQ1); YML078W (CPR3); YMR064W (AEP]); YMR072W (ABF2); YMR150C (IMP]); YMR193W (MRPL24); YMR228W (MTF1); 15 YNL177C; YNR036C; YNR037C (RSM19); YNR045W (PET494); YOL009C (MDM12); YOL033W (MSEJ); YOLO95C (HMII); YOR026W (BUB3); YPL132W (COXi1); YPL183W-A; YPRO04C; YPR166C (MRP2); and YPR191W(QCR2). Mutations which result in mitochondrial respiratory deficiency (petite mutations) are also contemplated. According to another aspect, the yeast is a mutant strain of Saccharonyces 20 cerevisiae in which the genes affecting the levels of NADH and NAD* are selected from genes encoding enzymes which catalyse the synthesis of glycerol, ethanol and/or genes the expression of which suppress or result in competition for the GLN1, GLT1 glutamate synthesis pathway. Typically these genes are selected from YIL053W(RHR2), YOR375C (GDH1) and YNL229c (URE2). 25 According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which the genes affecting the assimilation and metabolism of nitrogen in the cell are selected from: YDR300C (PRO1); YDR448W (ADA2); YEL009C (GCN4); YELO62W (NPR2); YGL227W (VID30); YGR252W (GCN5); YNL106C (INP52); YNL229C (URE2); YOR375C (GDH1); and YPL254W(HF1f). 30 According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which the genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA pathway are selected WO 2004/003217 PCT/AU2003/000837 24 from YOL081W(IRA2); YOR36OC (PDE2); and YNLO98C (RAS2; RAS2Val19 dominant mutation - see Figure 11). According to another aspect, the yeast is a mutant strain of Saccharonyces cerevisiae in which the genes affecting endosomal function are selected from: YCLO08C 5 (VPS23; STP22); YDR456W (NHXI); YJR102C (VPS25); YKLO02W (DID4); YKL041W (VPS24); YKR035W-A (DID2); YLR025W (VPS32/SNF7); YLR119W (SRN2/VPS3 7); YLR417W (VPS36); YMR077C (VPS20); YNRO06W (VPS27); YPLO65W (VPS28); and YPR173C (VPS4). Particularly those genes defined as class E compartment genes (Class E vps genes) are of interest. 10 According to another aspect, the yeast is a mutant strain of Saccharonyces cerevisiae in which the genes affecting endoplasmic reticulum function, the Golgi to endosome to vacuole transportation pathway or vacuolar function are selected from: YFL031W (HAC), YDR027C (LUV/IVPS54); YDR323C (PEP7/VPS9); YDR484W (VPS52/SAC2); YBR131W (CCZ); YDR486C (VPS60); YHRO12W (VPS29); YJL154C 1s (VPS35); YLR1148W (VAC/PEP3VPS18); YMLOO1W (YPT7); and YOR036W (PEP12/VPS6). According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which the genes affecting ubiquitin levels and ubiquitin-mediated proteolysis via the 26S proteosome are selected from: YBR173C (UMP1); YER151C 20 (UBP3); YFRO1OW(UBP6); YHLOI1C (PRS3); YKL213C (DOAl); YNR051C (BRE5); YPL03W(ULAI); and YPL074W(YTA6). According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which a gene involved in transportation of glutathione across the yeast cell membrane is YDR135C (YCFI) or YJL212C (HGTJ). 25 According to another aspect, the yeast is a mutant strain of Saccharomyces cerevisiae in which a gene involved in glutathione degradation is pep3, pep12, or pep7. According to another aspect, the yeast is a mutant strain of Saccharoinyces cerevisiae in which a gene involved in vacuolar function is pep3, pepI 2 , or pep 7. According to a preferred aspect, the yeast strain has mutations in two or more of 30 groups (i) to (x) listed above and will also be a mutant for the synthesis of one or more metabolites and/or essential growth factors, wherein the mutant is unable to synthesise said one or more metabolites and/or essential growth factors or has a restricted ability for synthesis of said one or more metabolites and/or essential growth factors. Typically the WO 2004/003217 PCT/AU2003/000837 25 one or more metabolites and/or- essential growth factors are amino acids or precursors or metabolites thereof. More typically the one or more metabolites and/or essential growth factors are selected from leucine, isoleucine or valines or precursors or metabolites thereof, and even more typically from leucine or precursors or metabolites thereof 5 Double mutants which are contemplated by the present invention include, but are not restricted to: - endosomal function (Class E vps or other protein sorting) mutation (as defined in group (v) above) + Ras/c-AMP/PKA mutation (as defined in group (iv) above), examples being: yklO02w (did4) + yor360c (pde2); yklO02w (did4) + yolO8lw 10 (ira2); and yklO02w (did4) + RAS2val19; - mutation affecting vacuolar function or Golgi to endosome to vacuole transport (as defined in group (vi) above) + Ras/c-AMP/PKA pathway mutation (as defined in group (iv) above), examples being: ylr148w (vacl/pep3/vps18) + yor360c (pde2); ylr1148w (vacl/pep3/vpsl8) + yol081w (ira2); ylr1148w; (vacl/pep3/vps18) + 1s RAS2vaZ19; yor036w (pep12/vps6) + yor360c (pde2); yor036w (pepl2/vps6) + yol08]w (ira2); and yor036w (pep12/vps6) + RAS2va19; - endosomal function (Class E vps or other protein sorting) mutation (as defined in group (v) above) + nitrogen assimilation pathway mutation (as defined in group (iii) above), examples of the latter mutation class being: ydr300c (prol); ydr448w 20 (ada2); yelO09c (gcn4); yelO62w (npr2); yg1227w (vid3O); ygr252w (gcn5); ynl106c (inp52); yn1229c (ure2); yor375c (gdhl); and ypl254w (ifil), and examples of some such crosses being: yklO02w (did4) + yn1229c (ure2); yklO02w (did4) + yor3 75c (gdhl); and yklO02w (did4) +ydr300c (pro] ); - endosomal function (Class E vps or other protein sorting) mutation (as 25 defined in group (v) above) + mutation that affects NADH levels (as defined in group (iii) above), an example being yklO02w (did4) + yil053w (rhr2); - endosomal function (Class E vps or other protein sorting) mutation (as defined in group (v) above) + mitochondrial mutation (as defined in group (i) above), examples being: ykl002w (did4) + yklO03c (mrpl 7) and other mitochondrial respiratory 30 chain mutants such as ykO02w (did4)+ ypr004c; ykIO02w (did4) + yhrO1w (dia4); - endosomal function (Class E vps or other protein sorting) mutation (as defined in group (v) above) + mutation in ubiquitin mediated protein degradation (as defined in group (vii) above), an example being yklO02w (did4) + ykl213c (doal); and WO 2004/003217 PCT/AU2003/000837 26 - endosomal function (Class E vps or other protein sorting) mutation (as defined in group (v) above) + glutathione transport mutant (as defined in group (viii) above), an example being yklO02w (did4)+ yj1212c (optl/hgtl); - Ras/c-AMP/PKA pathway mutation (as defined in group (iv) above) + 5 mitochondrial mutation (as defined in group (i) above), an example being yor360c (pde2) +ykO03c (mrp17); - Ras/c-AMP/PKA pathway mutation (as defined in group (iv) above) + glycerol biosynthesis/ NADH metabolism mutation (at the same time increasing GLTl and GLN1 activity, as defined in group (iii) above), an example being yor360c (pde2) + 10 yil053w (rhr2); - Ras/c-AMP/PKA pathway mutation (as defined in group (iv) above) + nitrogen assimilation pathway mutation (as defined in group (iii) above), examples being: yor360c (pde2) + ynl229c (ure2); yor360c (pde2) + yor375c (gdh1); and yor360c (pde2) +ydr300c (prol); 15 - Ras/c-AMP/PKA pathway mutation (as defined in group (iv) above) + ubiqitin mutation (as defined in group (vii) above), an example being yor360c (pde2) + ykl2l3c (doal); and - mitochondrial mutation (as defined in group (i) above) + nitrogen assimilation pathway mutation (as defined in group (iii) above, an example being yklO03c (mrp1 7) + 20 ynl229c (ure2); - mitochondrial/petite mutation as defined in group (i) above + glutathione transport mutant (as defined in group (viii) above), an example being ? + yj1212c (opt1/hgt1).' - mutation affecting endoplasmic reticulum function as defined in group (iv) 25 above + glutathione transport mutation as defined in group (viii) above, an example being BSO4 mutation (yfl031w (hac)) + ydrl35c (ycfl). Mutants with three or more mutations are also contemplated by the present invention and may include, but are not restricted to: did4 + pde2 + ure2; did4 + pde2 + ure2 + mrp1 7; and pde2 + glycerol mutant + ure2. 30 Other multiple mutants which are contemplated by the present invention are yeast strains having at least one mutation selected from groups (i) to (x) as described above, in addition to genetic manipulation resulting in overexpression of the glutathione synthesis pathway. Such manipulations resulting in, for example, overexpression of WO 2004/003217 PCT/AU2003/000837 27 gammaglutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2) or GSH1 and GSH2. Some of the mutants described above can also be grouped by reference to their glutathione secretion in response to external pH, or in response to amino acid availability 5 (particularly availability of the branched chain amino acids leucine, isoleucine and valine), or their ability to utilise glutathione as a sole nitrogen source (cells defective in glutathione degradation and/or transport). Several of the mutants described herein have been found to oversecrete glutathione due to defects in glutathione degradation. This was tested by their ability to grow using glutathione as a sole nitrogen source. Although a 10 failure to grow under these conditions could also result from blocked uptake of glutathione, this is less likely since these cells overaccumulate GSH intracellularly prior to secretion (when they are grown on standard SD medium). They are also hypersensitive to thiol-specific reducing agent dithiothreitol (DTT) indicating that their primary defect is a failure to degrade excess cytoplasmic glutathione. 15 Strains having two or more mutations selected within each of, or amongst the groupings described above are also contemplated according to the invention. Particularly, it is envisaged that, for example, a double mutant generated with the combination of: a leucine more-responsive mutation and a leucine less-responsive mutation; a pH more responsive and a pH less-responsive mutation; or of two different glutathione 20 utilisation/transportation defective mutations; may produce a strain with greater glutathione production and/or secretion than either of the single mutants. Although, for the most part, glutathione secretion by other mutants investigated was highly dependent on external pH, examples of mutants with glutathione secretion less dependent on external pH are: yj1153c (ino1); yol108c (ino4); and ylr226w (bur2). 25 Examples of mutants with glutathione secretion highly dependent on branched chain amino acid availability are: yn1229c (ure2); yhl023c; yol138c; yelO62w (npr2); yolO27c; yir19w (vps37); yolO50c; yjl056c (zapl); ybr003w (cox1); ynr005c; yclO08c (vps23); yjrlO2c (vps25); yor375c (gdhl); yolO04w (sin3); ydr486c (vps60); ydr276c (pmp3); yj1188c (bud19); ylr4l7w (vps36); yklO02w (vps2); ykr035w-a (did2); ypr004c; 30 ylr025w (vps32); yfrOlOw (ubp6); and ykl213c (doal). Examples of mutants with glutathione secretion less dependent on branched chain amino acid availability are: ylrI148w (pep3); ylr396c (vps33); yorO36w (pep12); ydr323c (pep7); ydr027c (luvi); ydr484w (sac2); yfr019w (fabl); ykrOOc (vps13); ydr495c WO 2004/003217 PCT/AU2003/000837 28 (vps3); yn1297c (non 2 ); yor07Oc (gyp]); yjl102w (meJ2); yol08Iw (ira2); yj1153c (ino]); yo108c (ino4); yir114c (efr4); yjl095w (bccl); yhr03Oc (npkl); ydr264c (akrl); yjl042w (mhpl); yal047c (spc72); yclO07c (cwh36); ydlO74c (brel); yerl16c (sx8); ynr03 6 c; ybr056w; yjl1 76c (swi3); and yilO29c. 5 Examples of mutants which are likely to be defective in glutathione degradation and/or transport are: Common Locus name Function yol081w ira2 GTPase-acting protein for Rasip & Ras2p yn1229c ure2 Regulator nitrogen catabolite repression yjr102c vps25 ESCRT-ll complex ylr417w vps36 ESCRT-11 complex ypIO02c vps2 2 ESCRT-l complex yklO02w vps2 ESCRT-1II complex ylr025w vps3 2 ESCRT-I complex ymr077c vps20 ESCRT-III complex ykr035w-a did2 Endosomal protein sorting ypri 73c vps4 AAA-ATPase of ESCRTcomplexes ydr027c luvi Subunit (Sac2p-Vps53p-Luvlp) complex ydr484w sac2 Subunit (Sac2p-Vps53p-Luvlp) complex ydr323c pep7 FYVE domain-containing Vac. Inherit ydr495c vps3 Vacuolar sorting protein and segregation ygl227w vid3O Vacuolar import and degradation yil01 7c vid28 Vacuolar import and degradation yll040c vps13 Protein involved in vacuolar sorting ylr1148w pep3 Class C complex, vacuolar biogenesis y1r396c vps33 Class C complex, vacuolar biogenesis ymi097c vps9 Protein involved in vacuolar sorting yor036w pep12 SNARE-Syntaxin of the late endosome ygl124c mon1 Vacuolar protein sorting yg1223c cod3 Component of Sec34p-Sec35p complex ykl212w sac] Phosphoinisotide phosphatase yer51c ubp3 Ubiquitin-specific protease yal047c spc72 Cytoplasmic plaque of spindle pole body yclO07c cwh36 Generation of mannoprotein layer yhr03Oc ipkl Serine/threonine protein kinase yj1095w bckl Serine/threonine protein kinase yn1225c Component of spindle pole body yor043w whi2 DNA repair protein ybr036c csg2 Ca 2 + homeostasis protein (CUP) family ygr2l7w cchl Voltage-gated Ca 2 channel ybr279w pafi Protein associated with RNA polymerase 11 ybr289w snf5 Component of SWI-SKF complex ydr448w ada2 Component of SAGA & ADA complexes ygr252w gcn5 Component of SAGA & ADA complexes ylr226w bur2 Regulation of transcription yolOR4w sin3 Component of histone deacetylase B WO 2004/003217 PCT/AU2003/000837 29 yp1254w hfil Component of the ADA complex ydr264c akr1 Pheromone signaling pathway yil053w rhr2 D,L-glycerol phosphate phosphatase ynl280c erg24 C-14 sterol reductase yplO22w rad1 Nucleotide excision repairosome yal024c Itel Required for termination of M phase ydlO23c Protein of unknown function ygl1 07c Protein of unknown function yil029c Protein of unknown function yil041w Protein of unknown function yil077c Protein of unknown function yil097w fyv1O Protein of unknown function yill1 Ow Protein of unknown function ymrl23w pkrl Protein of unknown function yolO27c Protein of unknown function Many of the mutants listed above are defective in vacuolar function, where glutathione degradation is known to occur. Glutathione breakdown is therefore a mechanism that leads to increased glutathione production. Double mutants such as ure2 pep2, ure2 inol, inol pep3, vps22 ino1, ure2 vps3 7, 5 io] vps37, amongst others, are contemplated by the present invention. The present invention also relates to a yeast double mutant strain herein described as BSO4ycfl (the glutathione secretion of which, relative to the single ras2 mutation or the parental strain, is illustrated in Figure 12: growth conditions, media and timing as described example 1). The BSO4 mutation has been detected as a defect in the HAC1 10 gene. The present invention also relates to a method of preparing a dough comprising dough components with a yeast strain according to the invention. Doughs prepared by this method, and baked products derived therefrom, are also provided. The present invention also relates to a method of producing a fermented product 15 comprising adding to the unfermented precursor component(s) of said product a yeast strain according to the invention. Fermented products obtained by this method are also provided. 2.2 Generation of mutants Generation of mutant strains according to the invention and/or for use in processes 20 according to the invention may be generated by any one of a wide range of methods known to those of skill in the art and such as are described in well known texts such as "Methods in Yeast Genetics" (1997) (Alison Adams, Daniel E. Gottschling, Chris A.

WO 2004/003217 PCT/AU2003/000837 30 Kaiser, Tim Steams, eds., Cold spring Harbour Laboratory Press) and "Molecular Cloning", 2nd Edition (1989) (Sambrook, J., E.F. Fritsch and T. Maniatis, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989). Typically, the mutational techniques may include any method which results in a 5 mutation which results in a "functional" deficiency, irrespective of how the genes have been mutated. Mutations may typically include deletion mutations, point mutations, insertion or substitution mutations, frame-shift mutations or any other method that results in inactivation of a gene (including RNAi approaches to selectively inactivating gene expression) or chemical/physical means. Suitable techniques may include mutagenic 10 techniques (using mutagens such as UV, X-ray, y-ray, ethylmethanesulfonate, N-methyl N'-nitro-N-nitrosoguanidine) or recombinant DNA techniques, chemical/physical agents, molecular biological techniques (including PCR methods to generate deletants, site directed mutagenesis protocols), or standard selection recombination methods to generate multiple mutants. Multiple mutations can be generated either by successive application of 15 mutagenic techniques or by recombination of single mutations of strains using standard hydridization techniques involving mating, diploid isolation, sporulation and recombination, or by processes of recombination. 2.3 Compositions comprising glutathione produced by the process of the invention, and uses thereof 20 The present invention also relates to a glutathione obtained by the process of the invention. The glutathione may be provided as a concentrated form of the culture medium or it may be purified to a desired degree. The glutathione may be used in a wide variety of applications as a catalyst, reactant or reductant/antioxidant., Fields of application include, but are not restricted to personal 25 health care, pharmaceuticals, nutraceuticals, cosmetics, food (including bakery and fermentation technology) and animal feeds, agriculture, aquaculture, paints, and fermentation media. For pharmaceutical purposes the glutathione is preferably provided as a purified compound, typically greater than 60% pure, more typically greater than 70% pure, more typically greater than 80% pure, even more typically greater than 90% pure, 30 and more preferably greater than 95% pure.

WO 2004/003217 PCT/AU2003/000837 31 Thus, the present invention also relates to a personal health care composition comprising glutathione obtained by the process of the invention and a pharmaceutically or topically acceptable carrier. The present invention also relates to a pharmaceutical composition comprising 5 glutathione obtained by the process of the invention and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may be used in the treatment of, for example, cancer, cardiovascular disease (such as atherosclerosis), oxidative damage to tissue (such as aging, or progressive protein oxidation in the eye lens), respiratory distress syndrome, toxicology, AIDS, and liver disease. 1o The present invention also relates to a food or nutraceutical composition comprising glutathione obtained by the process of the invention in combination with one or more food components. The food/nutraccutical composition may be selected from liquids, semi-solids and solids. The present invention also relates to a dough or bread improving composition 15 comprising glutathione obtained by the process of the invention and a suitable carrier. The carrier may be selected from a wide variety of bakery acceptable ingredients, including flour and/or sugar and the composition may also include other bread improving ingredients such as enzymes (including cellulases, glucanases, amylases, xylanases, arabinoxylanases, dextrinases, maltases, etc.). The composition may be in the form of a 20 powder, granulate or liquid. The present invention also relates to an animal feed additive comprising glutathione obtained by the process of the invention and a suitable carrier. The carrier may be selected from a wide variety of acceptable animal feed ingredients, such as flour (including wheat, corn or soy), and the composition may also include other animal feed 25 additives including those which improve the digestibility of the food such as enzymes (including cellulases, glucanases, amylases, xylanases, arabinoxylanases, dextrinases, maltases, etc.). The composition may be in the form of a powder, granulate or liquid. The present invention also relates to an animal health care composition comprising glutathione obtained by the process of the invention and a veterinary acceptable carrier. 30 The present invention also relates to a method for preventing oxidative damage in the circulation or tissues of a mammal, said method comprising administering to said mammal an effective amount of a composition comprising glutathione obtained by the process of the invention.

WO 2004/003217 PCT/AU2003/000837 32 The present invention also relates to a method of protecting a food product from oxidative deterioration comprising adding to said food product an effective amount of glutathione obtained by the process of the invention or a composition comprising it. Food products prepared by said method are also provided. The food product may be liquid, 5 semi-solid or solid. The present invention also relates to a method of preparing a dough comprising combining dough components with an effective amount of glutathione obtained by the process of the invention. Doughs prepared by this method, or baked products derived therefrom are also provided. 10 Examples Example 1 Identification of yeast mutants for glutathione production A deletion library of yeast strains derived from yeast strain BY4743, and as described in Winzeler E.A. et al., (1999), Science 285: 901-906 was purchased from 15 EUROSCARF Saccharomyces cerevisiae (www.rz.unifranlcfurt.de/FB/fbl6/mikro/euroscarf). According to the Winzeler E.A. et al reference, these mutants are deletion strains according to the following procedure: two long oligonucleotide primers are synthesized, each containing (3[prime] to 5[prime]) 18 or 19 bases of homology to the antibiotic resistance cassette, KanMX4 (Ul, Dl), a unique 20 20-bp tag sequence, an 18-bp tag priming site (U2 or D2), and 18 bases of sequence complementary to the region upstream or downstream of the yeast ORF being targeted (including the start codon or stop codon; see http://sequence www.stanford.edu/group/yeast/yeast deletion project/new deletion strategy.html). These 74-mers are used to amplify the heterologous KanMX4 module, which contains a 25 constitutive, efficient promoter from a related yeast strain. Ashbya gosspii, fused to the kanamycin resistance gene, nptl. Because oligonucleotide synthesis is 3[prime] to 5[prime] and the fraction of full-size molecules decreases with increasing length, improved targeting is achieved by performing a second round of PCR using primers bearing 45 bases of homology to the region upstream and downstream of a particular 30 ORF. Transformation with the PCR product results in replacement of the targeted gene upon selection for G418 resistance. The unique 20-mer tag sequences are covalently linked to the sequence that targets them to the yeast genome, creating a permanent association and genetic linkage between a particular deletion strain and the tag sequence.

WO 2004/003217 PCT/AU2003/000837 33 The mutants were screened for glutathione production, both intracellular and extracellular after growth in the following medium and under the following conditions. Growth Medium (SD minimal medium) (mass per litre water) 5 D-glucose 20g ammonium sulphate 5g yeast nitrogen base (without amino acids, without ammonium sulphate) 1.7g (Purchased from Difco) Additional growth supplements 10 L-leucine 0.131g L-isoleucine 0.066g L-valine, 0.059g L-histidine 0.209g uracil 0.022g 15 * The medium was sterilised by autoclaving at 121'C for 15min. The additional supplements leucine, isoleucine, valine, histidine and uracil were prepared separately as a sterile 100x stock solution and were added to the growth medium after autoclaving to give the final quantity per litre of each ingredient shown above. Growth parameters/conditions 20 * Culture vessel: Standard 24 well flat bottomed plastic culture plate manufactured by Sarstedt e Growth temperature 30'C * Growth period 48h e Orbital shaker speed 500rpm 25 e Volume of growth medium per culture plate well lml * Inoculating cultures were pre-grown in the above medium for 48h and were used to inoculate 24 well cultures containing the same medium at a starting culture density of approximately 2 x 10 4 cells per ml. Growth conditions 30 The sterilised medium was aliquoted into 24 well plastic culture plates and inoculated via the addition of the appropriate inoculating culture. The cultures were WO 2004/003217 PCT/AU2003/000837 34 shaken (500rpm) at 30"C for 48h and the optical density of the culture was measured at 600nm. A 500microlitre aliquot of each culture was transferred to a 1.5ml Eppendorf microcentrifuge tube which was centrifuged for 30 seconds at 1000g. A 100microlitre of the clarified culture medium was taken to allow quantification of extracellular glutathione 5 content. Intracellular and extracellular glutathione were determined by a method adapted from that reported by Vandputte C. et al., Cell Biology and Toxicology (1994) vol 10: 415-421: Sample preparation 1. Spin down cells for 30s at 10OOg (4oC) for culture up to lmL or for larger 10 cultures (10-50ml) spin down cells 5mim at 5000rpm in an SS34 rotor (44C). 2. Take a sample of the culture medium for extracellular glutathione quantification using the protocol described later in this section. 3.For the quantification of intracellular GSH was the pellet with an equal volume (equal to the harvest volume) of ice-cold PBS pH 7.4 and centrifuge as above. 15 4. Lyse the cell pellet by the addition of 400ul ice-cold 1.3% (w/v) 5-sulfosalicylic acid/ 8mM hydrochloric acid (4'C) and add glass beads to facilitate breakage using a mini bead beater (breakage time 1min ant high speed) or vortex vigorously for 2min. 5. Centrifuge the cell lysate for 5min at 8000g (4*C) to clarify solution. The sample is ready to assay. 20 6. Dilute sample as required Assay reaction mixture contents 143mM NaH 2

PO

4 6.3mM EDTA pH 7.4 400mg/L 5,5'-dithion-bis(2-nitrobenzoic acid) 25 100mg/L NADPH Glutathione assay procedure Add as 4 parts reaction mixture per 1 part unknown/sample. Start the reaction by the addition of 40microlitres of 0.85units/ml glutathione reductase enzyme (purchased from Sigma chemical Company). 30 Monitor the reaction at 410nm wavelength. Compare the change in absorbance to suitably prepared standards containing a known quantity of glutathione. Compare the quantity of glutathione produced divided by the number of cells isolated in the sample.

WO 2004/003217 PCT/AU2003/000837 3b Following normalisation of the data in this way GSH levels may be compared between strains. Typically glutathione values should be compared for both raw concentrations as well as for concentration normalised to cell number. Glutathione levels produced by respective cultures were adjusted to culture density 5 and then compared to the figure recorded for the wild type/parent strain grown under identical conditions. Yeast strains having the following gene deletions were found to provide elevated accumulation of extracellular glutathione, and the results are also provided in Tables 1 to 10 yalO02w ydr300c ygrO62c yj1042w ylr262c yolO04w yalO24c ydr3221 ygr100w yj1053w ylr268w yolO08w yal047c ydr323c ygr105w yjl063c ylr295c yolO09c yar0O2c-a ydr332w ygrl50c yj1095w ylr322w yol018c yblOO7c ydr33 7w ygr171c yjl102w ylr330w yol02 7c ybl009w ydr448w ygr2l 7w yj1138c ylr342w yolO33w ybl027w ydr456w ygr220c yjl152w ylr357w yol050c yblOOc ydr462w ygr252w yjl154c ylr360w yol081w ybr003w ydr475c ygr257c yj166w y1r369w yolO95c ybr036c ydr484w ygr284c yjll 76c y1r373c yol108c ybr037c ydr486c yIzO04w yj1183w y1r396c yol138c ybr04lw ydr495c yhiOllc yjll 8 8 c ylr4l7w yor0O8c ybr056w ydr497c yhlO23c yjl2Olw ylr439w yor026w ybr059c ydr5l8w yhlO25w yjl204c y1r447c yor036w ybr125c ydr529c yhl031c yjl209w ymnl001w yor043w ybr127c ydr533c yhl038c yjl212c yml048w yor069w ybr131w yelO07w yhr0lOw yjr059w ynlO71c yor070c ybr162c yeIO09c yhrO 11w yjr063w yml078w yor088w ybrl63w yel036c yhrO2w yjrO75w yml097c yor089c ybr173c yel051w yhr030c yjr102c yinr004w yor106w ybrl9lw yelO62w yhr05lw yjrl44w ynr064w yorl32w ybr220c yer0l7c yhrl16w yklO02w ymr066w yor332w ybr268w yer056c yhr120w yklO03c ynr072w yor360c ybr279w yerll6c yhr129c ykl032c ymr077c yor375c ybr289w yer122c yhr147c ykl041w ymrl23w yor384w yclO07c yer141w yhrl 71w ykl212w ymr150c ypl003w yclO08c yerl5lc yhr185c ykl213c ymr151w ypl01 7c ycr046c yer153c yilO06w ykr001c ymrl93w yplO22w ydl023c yer154w yilO08w ykr006c ymr228w ypl037c ydl039c yfl03lw yil017c ykr035c ynl098c ypl058c ydlO69c yfl034w yil018w ykr035w-a ynl106c yplO65w ydlO74c yfrOlOw yil029c ykr054c ynl177c yplO74w ydl077c yfrO9w yil041w yllO09c ynl215w ypl091w ydl107w yglO25c yil053w yllOOc ynl225c ypl120w ydl191w ygl10 7 c yil065c yll018c-a ynl229c yp1132w WO 2004/003217 PCT/AU2003/000837 36 ydl202w ygll15w yil07Oc yll040c ynl280c ypll 49 w ydr01 7c ygl124c yil077c ylrOO6c yn1296w ypl183w-a ydrO27c ygl1 2 7c yil092w ylr025w yn1297c ypl25 4 w ydrO79w ygll 67c yil093c ylr067c ynr005c ypr004c ydr175c ygll68w yilO97w ylr114c ynrOO6w ypr036w ydr197w ygl212w yil098c ylr119w ynr036c ypr099c ydr200c yg1227w yil11Ow ylrl39c ynr037c yprlOOw ydr2O4w yg1237c yirO2]w ylr148w ynr045w yprl59w ydr264c yg1244w yjlO04c ylr193c ynr05Oc ypr166c ydr276c yg1252c yjlO22w y1r226w ynr05lc yprl73c ydr298c ygrO2lw yj1029c ylr26lc yol001W yprl9lw Table 1 - Mitochondrial related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain Mitochondrial related Ratio GSH secreted mutant:parental ybIO09w atp1 6 ybr003w cox1 12 ybr037c sco1. 10 ybrl9lw rpI2la 4 ybr220c 7 ycr046c img1 8 ydI069c cbsl 10 ydIl07w mss2 8 ydrO79w petlO 11 ydr175c rsm24 8.6 ydr197w cbs2 10 ydr204w coq4 8.6 ydr298c atp5 10.2 ydr322w mrpl35 9.7 ydr462w nrpI28 11 ydr529c qcr7 10 yer141w cox15 11 yer153c pet122 9 yer154w oxa1 11 ygr062c cox18 5.5 ygr171c msm1 11 ygr220c mrpl9 1.2 ygr257c 14 yhIO04w mrp4 10 yhrOllw dia4 15.2 yhr051w cox6 7.1 yhr120w msh1 10.6 yhr147c mrpl6 11 yiO06w 6 yiIO18w rpl2b 4 yi1065c fis1 10.3 yiI070c mam33 11 yiIO93c rsm25 6.8 yi1098c fmcI 14 yir02lw mrsl 8.6 yj063c mrpl8 6.4 yjl102w mef2 7.8 yj1166w qcr8 7.8 yjI209w cbpl 8.3 yjr144w mgm101 11 WO 2004/003217 PCT/AU2003/000837 37 ykIO03c mrp17 29 ykI032c ixr1 ykr006c mrpl13 8.1 yllO09c cox17 2 yll018c-a cox19 10 ylr067c pet309 yIr139c ssi y1r295c hsp60 1 ylr369w ssqI ym1078w cpr3 ymr064w aepI 10.8 ymrO72w abf2 9.6 ymr150c imp1 8.1 ymrl93w mrpl24 11.8 ymr228w mif1 8.1 ynl177c 7.6 ynr036c 11 ynr037c rsm19 3.8 ynr045w pet494 10 yoIO09c mdml2 yoI033w msel 8.7 yol095c hmil 9.5 yor026w bub3 5 ypl132w cox11 8.4 ypIl83w-a 12 ypr004c 24.5 ypr166c mrp2 8.4 yprl9lw qcr2 7.1 Table 2 - Ubiquitin related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain Ubiquitin related Ratio GSH secreted _ mutant:parental ybr173c ump1 9.7 yer151c ubp3 3 yfrOlOw ubp6 18 yhl011c prs3 8.2 ykI213c doal 25 ynr05lc bre5 9 ypIO03w ulal 35 ypIO74w yta6 11 Table 3 - Nitrogen assimilation related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain Nitrogen related Ratio GSH secreted mutant:parental ydr300c prol 18 ydr448w ada2 1.4 yeIO09c gcn4 1.7 yeI062w npr2 6 yg1227w vid3O 2.6 ygr252w gcn5 6.8 ynhl06c inp52 1.3 yn1229c ure2 23.7 yor375c gdh1 5 ypI254w hfil 5.1 WO 2004/003217 PCT/AU2003/000837 38 Table 4 - c-AMP related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain c-AMP related Ratio GSH secreted mutant:parental yol081w ira2 25 yor360c pde2 122 Table 5 - Cell wall related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain cell wall Ratio GSH secreted mutant:parental ydrOl7c kcsl 7.3 ydr497c itr1 1.7 yhr03Oc mpk1 5 yjI095w bckl 2.4 yjl152w ino1 14 ylr330w chs5 5 yol108c ino4 13.1 ypr159w kre6 4.8 5 Table 6 - Signal transduction related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain Signal Transduction Ratio GSH secreted mutant:parental yaIO24c Ite1 1.7 ybrO59c akl1 2.7 ybr125c ptc4 1.1 ybr279w paf1 10.4 ybr289w snf5 11 ydr264c akr1 5.1 ydr332w 9 yerll6c slx8 5 ygI115w 1.6 yhI025w snf6 3 yjI138c tif2 6.1 yjI176c swi3 2.6 yjr063w rpa12 7 y1r006c sskl 6.5 ylr357w rsc2 2.2 yoIO04w sin3 6.6 Table 7 - Transporter related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain transporters Ratio GSH secreted mutant:parental yer056c fcy2 6.9 yjl212c optl/hgtl 3.3 yorQ88w yVC1 2.7 ypI058c pdr12 6.8 ygI167c pmrl 8 Table 8 - Membrane potential related mutations - ratio of glutathione secretion by mutant 10 to amount secreted by parental strain Membrane potential Ratio GSH secreted mutant:parental ydr276c pmp3 5.5 yjr059w ptk2 II WO 2004/003217 PCT/AU2003/000837 39 yUl010c psrl 1.6 Table 9 - Protein sorting related mutations - ratio of glutathione secretion by mutant to amount secreted by parental strain Protein sorting/ Protein Ratio GSH secreted folding mutant:parental yaIO02w vps8 7.3 ybr105c vid24 2.3 ybrl3lw ccz1 10.5 ycIO08c vps23 27 (stp22) yd1077c vps39/vam6 5.3 ydr027c luvl/vps54 15.2 ydr323c pep7/vps19 28.1 ydr456w nhx1 5.8 ydr484w vps52/sac2 10.3 ydr486c vps60 19 ydr495c vps3 15.5 ydr5l8w eugl 12 ye1036c anp1 4.8 ye051w vma8 6 yer122c go3 6 yfr0l9w fabl 10.7 ygl67c pmrl 7.8 ygI212w vps43/vam7 5.2 ygr284c erv29 3.9 yh1031c gos1 22 yhrO12w vps29 8.2 yhr171w apg7 3.9 yj1029c vps53 13.4 yjl053w vps26/pep8 6.9 yjI154c vps35 18.8 yjrO75w hoc1 3.6 yjrl02c vps25 29.5 ykO02w did4 37 yk1041w vps24 25.7 ykI212w sac1 3.2 ykrOOc vps1 7.9 ykr035w-a did2 21 yl040c vpsl3 3.1 yr025w vps32/snf7 23 yIr1148w vacl/pep3/vp 21 s18 yrl9w srn2/vps37 25 y1r262c ypt6 2.5 y1r268w sec22 6.6 ylr360w vps38 5.9 ylr373c vid22 3.7 ylr396c vps33 14 yIr4l7w vps36 26.2 ymIOOw ypt7 13.1 ym1071c dor1 4.6 ymIO97c vps9 1.7 ymr004w mvp1 2.9 ynr077c vps20 28.9 ynr006w vps27 33.7 y01018C tig2 1.4 WO 2004/003217 PCT/AU2003/000837 40 yor036w pep 12/vps6 21.2 yor069w vps5 7.5 yorO7Oc gyp1 3.5 yorO89c vps21 8.7 yor106w vam3 6.1 yor132w vps17 2.6 ypIO65w vps28 29.4 ypl120w vps30 3.8 ypl149w apg5 2.7 ybr127c vma2 5.8 yor332w vma4 6 ylr447c vma6 6 ypr036w vma 13 2.4 ygr105w vma2l 6 ypr173c vps4 12 yfl031w hac1 8 Table 10 - Miscellaneous mutations - ratio of glutathione secretion by mutant to amount secreted by pa ental strain Miscellaneous Ratio GSH secreted mutant:parental ya1047c spc72 5 yb!007c sIa1 9.1 ybr041w fatl 1.8 ycO07c cwh36 7 yg1025c pgdl 1.4 yg1127c sohl 2.9 ygr2l7w cchl 2.2 yhr185c pfs1 2.1 y1053w rhr2 7 yj1042w mhp1 10.4 yjIl83w mnnll 2.9 ylr226w bur2 5.9 ym1048w gsf2 1.6 yn1225c cnml 2.4 ynI280c erg24 3.9 yoIOOlw pho80 3.9 yor043w whi2 3.9 yp1022w radl 24 ypI037c egdl 11.4 Example 2 5 Glutathione production (extracellular and intracellular) relative to growth phase The strain designated as BSO4 was grown as per Example 1, but intracellular and extracellular glutathione levels were determined at a number of timed intervals after inoculation into fresh medium. The parental strain was also grown and sampled in the same way. 10 The results are illustrated in Figure 2.

WO 2004/003217 PCT/AU2003/000837 41 A mutant having a deletion in the VPS27 gene was grown as per Example 1, but intracellular and extracellular glutathione levels were determined at 15, 17, 19, 21, 23, 25, 27, 29, 32, 36 and 44 hours after inoculation into fresh medium. The parental strain was also grown and sampled in the same way. 5 The results are illustrated in Figure 3. Example 3 Glutathione production (extracellular and intracellular) relative to pH Several of the above mentioned strains from the BY4743 series were grown as follows. 10 Growth medium: As per SD minimal medium as described in Example 1, except the pH of the growth medium was buffered using a 25mM PIPPS/MES buffer system (PIPPS = piperazine-N,N'-bis(2-ethanesulfonic acid) MES= 2(N-morpholino)ethane sulfonic acid). The pH of the medium was adjusted to either pH 3.5 or pH 6.0 via the addition of ammonium hydroxide, or even a range of pH values were tested for strain BSO4. 15 Growth conditions and quantification of glutathione: The method used was identical to that outlined for the screening of the BY4743 series of deletion mutants. The results (illustrated in Figures 7 to 9) show that extracellular GSH accumulation is greater if the pH of the growth medium is buffered at pH 3.5 vs pH 6.0. The differences observed in extracellular glutathione were determined to not be due to pH 20 dependent degradation of glutathione. * Buffering the pH of the growth medium to pH 3.5 was found to increase the accumulation of extracellular glutathione in stationary phase cultures (48h) when compared to an equivalent culture grown in medium buffered to pH 6.0. To the best of our knowledge the effect of pH on the accumulation of extracellular GSH has not 25 been reported. Subsequent tests, using a broader selection of strains tested in either unbuffered SD medium or in SD medium buffered at pH6.0, identified the fact that different mutations are influenced in their glutathione secretion to different degrees by extracellular pH, although greater glutathione secretion was, except for in one instance, greater in the 30 unbuffered medium. The results are summarised in Table 11 (glutathione levels provided as ptM).

WO 2004/003217 PCT/AU2003/000837 42 Table 11 Locus Gene Total glutathione S.D. Total glutathione SD S.D. name SD medium medium (unbuffered) pH 6.0 BY4743 Parent 4.6 0.4 0.5 0.2 yjl153c inol 40 3 27 3 yol108c ino4 15 3 22 2 yplO02c vps22 50 1 22 4 ygl167c pmrl 30 4 15 4 ylr1148w pep3 50 1 14 2 ylr226w bur2 14 3 12 2 ylrl14c efr4 44 4 12 3 ybr279w pafi 25 1 9 2 ybr289w snf5 19 1 8 1 ylr322w 20 2 6 1 ylr396c vps33 24 5 5 0.3 yhlO25w snf6 13 7 5 0.1 ygl127c sohl 10 7 4 1 ydr323c pep7 31 7 4 1 ydr495c vps3 23 7 4 1 ylr025w vps32 40 7 4 1 ycrO63w bud3l 11 1 4 1 ynl215w 7 1 3.4 0.3 ymr077c vps20 35 5 3.5 0.5 yor036w pep12 35 2 3.0 0.4 ylr261c 18 3 3 1 These results also suggest that the combination of mutations where glutathione production is highly dependent on external pH, with those that are less-dependent on external pH could produce cells that produce even higher levels of glutathione. 5 Example 4 Glutathione production (extracellular and intracellular) in the presence of different monovalent cations To test the effect of various alkali metals on the relative secretion by deletion mutants, Saccharomyces cerevisiae laboratory strains designated CY4 and BSO4 were 10 grown as follows. Growth medium: As per SD minimal medium except the pH of the growth medium was buffered to pH 3.5 using a 25mM 1IPPS/MES buffer system. The growth medium contained either 150mM KCl, RbCl or CsCl. The pH of the medium was adjusted to pH 3.5 via the addition of conc. ammonium hydroxide. The effect of adding combinations of is the above salts was not studied. The effect of the addition of theses salts was also confirmed in unbuffered medium.

WO 2004/003217 PCT/AU2003/000837 43 Growth conditions and quantification of glutathione: the method used was identical to that outlined for the screening of the BY4743 series of deletion mutants. Results: The addition of some alkali metal salts was shown to increase the accumulation of extracellular glutathione 5 . The addition of the following salts at 150mM concentration in the growth medium was also correlated with the increase accumulation of extracellular glutathione (CY4 strain need to reference): KCl, RbCl, CsC1. The results for strain BSO4 are illustrated in Figure 10. Example 5 10 Glutathione production (extracellular) in the presence of leucine, isoleucine and valine In this experiment the extracellular glutathione production was determined for one mutant yol081w (ira2), which for the mass screen work carried a marker for leucine auxotrophy. That is the strain contained a mutation in the leucine biosynthetic pathway 15 (LEU2 gene mutation) and therefore for all our experiments the medium outlined in example 1 was used for the screen, in particular containing Leucine 0.131 g/L isoleucine 0.066g/L and valine 0.059g/L 20 The ira2 mutant was altered to carry the plasmid (vector) Yep13LEU2 to allow the strain to make its own leucine. The data (below) shows glutathione secreted by the ira2 mutant grown with the additional supplements vs the ira2Yep13 transformant grown under identical conditions but without the supplements. Glutathione in the medium was determined after the cultures reached stationary 25 phase using the conditions identical to those outlined in Example 1 (glutathione results provided as tmole/L). GSH expressed as nanomoles of GSH per 3x1 07 cells Parental strain without the Yep 13 plasmid = 0.43 ira2 = 10.6 30 ira2Yepl3 = 0.98 WO 2004/003217 PCT/AU2003/000837 44 This data shows that if the strain can make leucine and leucine content of the culture is not regulated, then the strain secretes less GSH (therefore manipulating leucine in the medium for a strain that can make leucine is likely to have little effect - strains that can make normal levels of leucine are likely to secrete lower levels of GSH). 5 The extracellular GSH following growth in standard medium containing the above mentioned levels of leucine, isoleucine and valine (1X) was also compared to production in medium containing 2X leucine/isoleucine/valine (ie leucine 0.262g/L, isoleucine 0.132g/L and valine 0.118g/L), and to 4X leucine/isoleucine/valine (leucine 0.524g/L, isoleucine 0.264g/L and valine 0.236g/L). 10 Three strains were tested, 2 at all three concentrations of supplements, and 1 at two concentrations. At IX supplements: extracellular GSH per 3x 107 cells. yor360c (pde2) produced 5.4 0.4 ynrOO6w (vps 2 7 ) produced 12.3 0.5 15 ykl002w (did4) produced 14.0 0.8 At 2X supplements: extracellular GSH per 3x10 7 cells. yor360c (pde2) produced 0.47 0.07 ynrOO6w (vps27) produced 3.04 0.3 yklO02w (did4) produced 2.4 0.3 20 At 4X supplements: extracellular GSH per 3x107 cells. yor360c (pde2) produced 0.43 0.03 ynrOO6w (vps27) produced 2.0 0.1 yklOO2w (did4) Not tested The data show that supplementation of the leucine mutants with 2X 25 leucine/isoleucine/valine resulted in a dramatic reduction in GSH secretion by these mutants. Further experiments, using the same growth conditions and media described above, but with a broader range of strains, have indicated that different mutations are influenced in their glutathione secretion to different degrees by branched chain amino acid levels.

WO 2004/003217 PCT/AU2003/000837 45 Table 12 provides data for strains which were strongly responsive to branched chain amino acid levels in the culture medium, and Table 13 provides data for strains which were less responsive to branched chain amino acid levels in the culture medium. Table 12 5 Deletants secreting lower levels of glutathione following growth in medium supplemented with increased branched-chain amino acids (BCAA) Locus Gene name Glutathionea S.D. Glutathionea S.D. Glutathionea S.D. 1X BCAA 2XBCAA 4XBCAA BY4743 parent 5 1' 5 0 4 2 ynl229c ure2 26 7 2.8 0.5 1.9 0.5 yhlO23c 33 3 20 4 . 4.2 0.3 yol138c 34 1 16 1 4.8 0.4 yelO62w npr2 35 2 22 5 5 2 yo027c 30 1 12 1 5 1 ylr119w vps37 38 8 10 1 7 1 yol050c 16 1 12 1 3 0 yjl056c zapi 25 7 15 7 5 1 ybr003w cox] 33 0 23 3 6 2 ynr005c 38 11 12 1 8 1 ycl08c vps23 38 10 14 1 8 2 yjrl02c vps25 40 8 16 0 9 1 yor375c gdh1 12 5 4 0 3 1 yolO04w sin3 37 6 11 1 9 1 ydr486c vps60 37 2 28 3 9 7 ydr276c pinp3 29 6 16 5 7 1 yjl188c bud19 32 6 18 0 8 1 ylr4l7w vps36 36 7 15 1 10 1 ykl02w vps2 52 0 28 3 14 1 ykr035w-a did2 24 9 14 2 7 2 ypr004c 34 2 15 1 9 1 ylrO25w vps32 40 7 19 1 12 2 yfrOlOw ubp6 20 2 11 2 6 1 ykl213c doal 24 5 16 1 7.6 0.1 aExtracellular glutathione concentration in pm. 24 most-responsive deletants shown (based on [GSH secretion in SD medium]/]GSH secretion in SD containing 4X BCAA supplements]) WO 2004/003217 PCT/AU2003/000837 46 Table 13 Glutathione oversecreting deletants less responsive to increased branched chain amino acid (BCAA) supplementation Locus Gene Glutathionea S.D. Glutathionea S.D. Glutathionea S.D. name 1X BCAA 2X BCAA 4X BCAA BY4743 parent 5 1 5 0 4 2 ylr1148w pep3 50 1 48 2 50 2 ylr396c vps33 24 6 30 6 41 6 yor036w pep12 35 2 38 5 28 1 ydr323c pep7 31 1 35 5 26 5 ydrO27c luvi 32 2 31 1 35 3 ydr484w sac2 35 4 32 3 27 1 yfrOl9w fabi 28 0 28 0 26 4 ykrOOc vpsl3 23 3 22 1 25 1 ydr495c vps3 23 1 28 0 18 2 yn1297c mon2 23 4 20 6 18 2 yorO70c gyp1 18 2 19 1 17 0 yjl102w inef2 24 2 22 1 24 3 yol081w ira2 18 3 18 1 17 3 yj1153c ino1 40 3 37 1 39 6 yol108c ino4 14 3 15 2 16 1 ylrl14c efr4 44 4 35 2 36 1 yjl095w bckl 13 2 15 1 18 1 yhr03Oc inpkl 12 2 12 1 15 0 ydr264c akr1 18 1 12 4 19 4 yj1042w mhpl 29 1 26 1 25 3 yal047c spc72 14 1 14 2 12 2 yclO07c cwh36 14 2 12 2 11 1 ydlO74c brel 22 6 16 1 21 4 yerll6c sIx8 15 4 21 0 24 6 ynr036c 23 2 24 3 21 1 ybr056w 15 2 16 0 21 5 yjl176c swi3 10 2 11 1 14 1 yil029c 33 1 26 3 25 1 aExtracellular glutathione concentration in ptm. 24 most-responsive deletants shown 5 (based on [GSH secretion in SD medium]/]GSH secretion in SD containing 4X BCAA supplements]) These results also suggest that the combination of mutations where glutathione production is strongly responsive to branched chain amino acid levels, with those that are less- responsive to branched chain amino acid levels could produce cells that produce 10 even higher levels of glutathione.

WO 2004/003217 PCT/AU2003/000837 47 Example 6 Glutathione production in the presence of myo-inositol Manipulation of myo-inositol content alone or together with additional glucose supplementation (or potentially other carbon sources such as respiratory carbon sources) 5 in the culture medium was found to influence glutathione production. A wild-type haploid laboratory strain, CY4, was grown in SD medium (either by itself (open bars, 2% glucose), or SD medium supplemented by 200mg/L myo-inositol (hatched bars), 4%w/v glucose (final glucose concentration - shaded bars), or both 200mg/L myo-inositol and 4%w/v glucose (solid bars)), and the culture medium assayed 10 for external glutathione as described in Example 1. The data shown are means (± standard deviation) for triplicate measurements from a representative experiment. The results, illustrated in Figure 11, show the effect of increased myo-inositol supplementation, either alone or in combination with glucose, on glutathione production (extracellular levels). While glucose supplementation alone did not appear to affect the 15 amount of extracellular glutathione produced, when glucose supplementation was combined with myo-inositol supplementation, significantly greater amounts of extracellular glutathione were produced relative to growth in SD medium, or SD medium supplemented with myo-inositol alone. Thus, myo-inositol supplementation, optionally combined with elevated levels of 20 carbon source/ substrate can result in elevated extracellular glutathione production by yeast strains. Example 7 Glutathione production by combined/double mutants A number of yeast strains having mutations in two of the genes referred to in Table 25 1 above (and/or having two mutations as identified tables 2 to 10) have also been found to provide elevated extracellular glutathione production. A number of these double mutants provide greater extracellular glutathione production than strains having either mutation alone. Figures 12 to 14 provide examples of this (media and methods as described in Example 1). 30 Figure 12 illustrates the extracellular glutathione levels produced by: - a wild-type (wt) yeast strain; WO 2004/003217 PCT/AU2003/000837 48 - a mutant of the wild-type having the BSO4 mutation (defect in the HAC1 gene, YFL031 W, identified in a BY4742 strain background); - a mutant of the wild-type having the ycfl (ydr135c ) mutation; and - a yeast strain having combined BSO4 and ycfl mutations as HACI then the 5 bso4 ycf1 double mutant can be listed as hac1 ycfl (in place of bso4 ycf1). Figure 13 illustrates the extracellular glutathione levels produced by a yeast strain having the hgt1 mutation (glutathione uptake (re-uptake) mutation), and a yeast strain having combined hgtl and petite (mitochondrial respiratory deficiency) mutations. The data shown are means (± S.D.) for triplicate measurements from a representative 10 experiment. Figure 14 illustrates the extracellular glutathione levels produced by different haploid wild-types (CY4 and BY4742), single mutants thereof, and different diploid crosses. The diploid strain generated by mating a bso4 haploid to a hac1 deletant (hence a double mutant) produces diploid cells that produce higher levels of glutathione relative 15 to either of the respective haploid strains or diploids derived from mutant-wild-type crosses (single mutant diploids. The hac1 deletant is derived from the BY4742 the strain background and BSO4, which carries a mutation in HAC, is derived from the CY4 strain background). The data shown are means (± S.D.) for triplicate measurements from a representative experiment. 20

Claims (73)

1. A process for the production of glutathione, wherein said process comprises culturing a mutant yeast strain under conditions promoting glutathione production, and wherein said yeast strain has one or more genetic mutations that result in increased 5 secretion of glutathione into the culture medium relative to a parental strain.

2. The process of claim 1, wherein said glutathione is isolated from the culture medium.

3. The process of claim 1 or claim 2, wherein the yeast strain has a mutation that reduces the ability of the strain to synthesise one or more proteins, metabolites or 10 essential growth factors.

4. The process of claim 3, wherein said metabolites or essential growth factors are included in the culture medium in limiting amounts.

5. The process of claim 3 or claim 4, wherein said metabolites or essential growth factors are selected from amino acids, or precursors or metabolites thereof. 15

6. The process of claim 5, wherein the yeast is deficient, or has a reduced ability for synthesis of, leucine, isoleucine or valine, or a combination thereof, or precursors or metabolites thereof.

7. The process of claim 1, wherein the yeast strain has a mutation selected from one or more of the following groupings, which may overlap: 20 i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome or mutation or deletion of the mitochondrial genome; ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and NAD(P)*; 25 iii) mutation in a gene or genes. affecting the assimilation and metabolism of nitrogen in the cell; iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA pathway; 30 v) mutation in a gene or genes affecting endosomal function; vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole transportation pathway or plasma membrane to endosome to vacuole traffic; WO 2004/003217 PCT/AU2003/000837 50 vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated proteolysis via the 26S proteosome; viii) mutation in a gene or genes involved in transportation of glutathione across the yeast cell membrane; 5 ix) mutation in a gene or genes involved in glutathione degradation; and x) mutation in a gene or genes involved in vacuolar function.

8. The process of claim 7, wherein said yeast strain has more than one mutation within one of the above groups (i) to (x).

9. The process of claim 7 or claim 8, wherein said yeast strain also has a 10 mutation that reduces the ability of the strain to synthesise one or more proteins, metabolites or essential growth factors.

10. The process of claim 9, wherein said metabolites or essential growth factors are included in the culture medium in limiting amounts.

11. The process of claim 9 or claim 10, wherein said metabolites or essential 15 growth factors are selected from amino acids, or precursors or metabolites thereof.

12. The process of claim 11, wherein the yeast is deficient, or has a reduced ability for synthesis of, leucine, isoleucine or valine, or a combination thereof, or precursors or metabolites thereof.

13. The process of any one of claims 7 to 12, wherein said yeast strain also 20 overexpresses the glutathione synthesis pathway.

14. The process of claim 13, wherein said yeast strain overexpresses gammaglutamylcysteine synthetase (GSHJ), glutathione synthetase (GSH2), or both.

15. The process of any one of claims 1 to 14, wherein the yeast culture is grown aerobically. 25

16. The process of any one of claims 1 to 15, wherein the yeast culture is grown at a pH less than about 6.

17. The process of claim 16, wherein the pH is about 3.5.

18. The process of any one of claims 1 to 17, wherein the yeast culture is grown in the presence of monovalent cations. 30

19. The process of claim 18, wherein said monovalent cations are selected from sodium, potassium, rubidium or caesium or a combination thereof.

20. The process of claim 18 or claim 19, wherein the concentration of said monovalent cations in the culture medium is from about 50mM to 500mM. WO 2004/003217 PCT/AU2003/000837 51

21. The process of claim 18 or claim 19, wherein the concentration of said monovalent cations in the culture medium is about 150mM.

22. The process of any one of claims 1 to 21, wherein the yeast culture is grown in the presence of myo-inositol. 5

23. The process of claim 22, wherein the concentration of the myo-inositol in the culture medium is from about 0.01mM to 100mM.

24. The process of claim 22, wherein the concentration of the myo-inositol in the culture medium is about 1mM.

25. The process of any one of claims 22 to 24, wherein the yeast is also grown in 10 the presence of elevated levels of carbon source.

26. The process of claim 25, wherein said carbon source is selected from fermentable sugars, oligosaccharides which are homo- or hetero- oligomers comprising fermentable sugar moieties, or a combination thereof.

27. The process of claim 26, wherein said carbon source is selected from glucose, 15 fructose, sucrose or a combination thereof

28. The process of claim 25, wherein said carbon source is a non-fermentable carbon source.

29. The process of claim 28, wherein said carbon source is ethanol.

30. The process of any one of claims 25 to 29, wherein the concentration of said 20 carbon source in the initial uninoculated culture medium is greater than 2%w/v.

31. The process of claim 30, wherein the concentration of said carbon source in the initial uninoculated culture medium is from about 2% to 1 0%w/v.

32. The process of claim 30, wherein the concentration of said carbon source in the initial uninoculated culture medium is about 4%w/v. 25

33. A process for the production of glutathione comprising culturing a yeast strain under conditions promoting glutathione production, wherein the culture medium comprises myo-inositol.

34. The process of claim 33, wherein the concentration of the myo-inositol in the culture medium is from about 0.01mM to 100mM. 30

35. The process of claim 33, wherein the concentration of the myo-inositol in the culture medium is about 1mM.

36. The process of any one of claims 33 to 35, wherein the yeast is also grown in the presence of elevated levels of carbon source. WO 2004/003217 PCT/AU2003/000837 52

37. The process of claim 36, wherein said carbon source is selected from fermentable sugars, oligosaccharides which are homo- or hetero- oligomers comprising fermentable sugar moieties, or a combination thereof.

38. The process of claim 37, wherein said carbon source is selected from glucose, 5 fructose, sucrose or a combination thereof.

39. The process of claim 36, wherein said carbon source is a non-fermentable carbon source.

40. The process of claim 39, wherein said carbon source is ethanol.

41. The process of any one of claims 36 to 40, wherein the concentration of said 10 carbon source in the initial uninoculated culture medium is greater than 1 %w/v.

42. The process of claim 41, wherein the concentration of said carbon source in the initial uninoculated culture medium is from about 2% to 10%w/v.

43. The process of claim 41, wherein the concentration of said carbon source in the initial uninoculated culture medium is about 4%w/v. 15

44. The process of any one of claims 1 to 43, wherein said process comprises batch-wise fermentation.

45. The process of any one of claims 1 to 43, wherein said process comprises a fed-batch fermentation.

46. The process of any one of claims 1 to 43, wherein said process comprises a 20 continuous culture fermentation.

47. The process of any one of claims I to 43, wherein said process comprises dough preparation.

48. The process of any one of claims 1 to 43, wherein said process comprises preparation of a fermented product. 25

49. A mutant yeast strain having at least two mutations selected from the following groupings, which may overlap: i) mutation in a gene or genes encoding components of the mitochondrial respiratory chain or nuclear genes encoding proteins that maintain the integrity of the mitochondrial genome or mutation or deletion of the mitochondrial genome; 30 ii) mutation in a gene or genes affecting intracellular levels of NAD(P)H and NAD(P)*; iii) mutation in a gene or genes affecting the assimilation and metabolism of nitrogen in the cell; WO 2004/003217 PCT/AU2003/000837 53 iv) mutation in a gene or genes encoding regulatory components of the Ras/cAMP/PKA pathway or otherwise affecting the activity of the Ras/cAMP/PKA pathway; v) mutation in a gene or genes affecting endosomal function; 5 vi) mutation in a gene or genes affecting the Golgi to endosome to vacuole transportation pathway or plasma membrane to endosome to vacuole traffic; vii) mutation in a gene or genes affecting ubiquitin levels and ubiquitin-mediated proteolysis via the 26S proteosome; viii) mutation in a gene or genes involved in transportation of glutathione across 10 the yeast cell membrane; ix) mutation in a gene or genes involved in glutathione degradation; and x) mutation in a gene or genes involved in vacuolar function.

50. The yeast strain of claim 49, which has more than one mutation within one of the above groups (i) to (x). 15

51. The yeast strain of claim 49 or claim 50, which also has a mutation that reduces the ability of the strain to synthesise one or more proteins, metabolites or essential growth factors.

52. The yeast strain of claim 51, wherein said metabolites or essential growth factors are included in the culture medium in limiting amounts. 20

53. The yeast strain of claim 51 or claim 52, wherein said metabolites or essential growth factors are selected from amino acids, or precursors or metabolites thereof.

54. The yeast strain of claim 53, which is deficient, or has a reduced ability for synthesis of, leucine, isoleucine or valine, or a combination thereof, or precursors or metabolites thereof. 25

55. The yeast strain of any one of claims 49 to 54, which also overexpresses the glutathione synthesis pathway.

56. The yeast strain of claim 55, which overexpresses gammaglutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), or both.

57. The process of any one of claims 1 to 48, wherein the yeast strain is a yeast 30 strain of any one of claims 49 to 56.

58. A method of preparing a dough comprising combining a yeast strain of any one of claims 49 to 56 with other dough components. WO 2004/003217 PCT/AU2003/000837 54

59. A method of producing a fermented product comprising adding to the unfennented precursor component(s) of said product a yeast strain of any one of claims 49 to 56.

60. Glutathione when obtained by a process of any one of claims 1 to 48 or 57. 5

61. The glutathione of claim 60, which is provided as a concentrated form of the culture medium.

62. The glutathione of claim 60, which is isolated from the culture medium.

63. The glutathione of claim 61, which is at least 70% pure.

64. The glutathione of claim 62, which is at least 95% pure. 10

65. A personal health care composition comprising glutathione according to any one of claims 60 to 64 and a pharmaceutically or topically acceptable carrier.

66. A pharmaceutical composition comprising glutathione according to any one of claims 60 to 64 and a pharmaceutically acceptable carrier.

67. A food or nutraceutical composition comprising glutathione according to any 15 one of claims 60 to 64 in combination with one or more food components.

68. A dough or bread improving composition comprising glutathione according to any one of claims 60 to 64 and a suitable carrier.

69. An animal feed additive comprising glutathione according to any one of claims 60 to 64 and a suitable carrier. 20

70. An animal health care composition comprising glutathione according to any one of claims 60 to 64 and a veterinary acceptable carrier.

71. A method for preventing oxidative damage in the circulation or tissues of a mammal, said method comprising administering to said mammal an effective amount of a composition comprising glutathione according to any one of claims 61 to 65. 25

72. A method of protecting a food product from oxidative deterioration comprising adding to said food product an effective amount of glutathione according to any one of claims 60 to 64.

73. A method of preparing a dough comprising combining dough components with an effective amount of glutathione according to any one of claims 60 to 64. 30