CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application claims priority from U.S. provisional patent application 62/315,796 filed on March 31 , 2016. A sequence listing in electronic form is being filed concurrently. The content of the priority application and of the sequence listing are herewith included in their entirety.

TECHNOLOGICAL FIELD

The present disclosure relates to genetically modified host cells (preferably genetically modified yeast host cells) capable of producing and accumulating glutathione as well as processes using same. The genetically modified host cells express a mutated cystathionine beta-synthase protein (Cys4p) having an increased biological activity, a mutated Yapl p having a limited ability of being translocated from the nucleus to the cytoplasm and/or an heterologous threonine aldolase (Glyl p).

BACKGROUND

Glutathione (GSH, L-Y-glutamyl-L-cysteinylglycine) is an ubiquitous non-protein thiol tripeptide which plays a role in several key physiological processes and is increasingly used in the pharmaceutical, cosmetic and food additive industries. This strong antioxidant is of high interest in baking (dough relaxation for example), oenology and brewing (aroma components stabilizer) or as a flavor enhancer ("kokumi" taste) in yeast extracts. Due to GSH's high demand, increasing the efficiency of its production is commercially important. Saccharomyces cerevisiae can be used for GSH microbial synthesis since it is "Generally Regarded As Safe" (GRAS), is a low secretor and is relatively easy to grow at high cell densities on inexpensive substrates.

In the cell, glutathione exists mostly in reduced (GSH) and oxidized (GSSG) forms, and is synthesized from three precursor amino-acids (glutamate, cysteine, and glycine) over two consecutive ATP-dependent reactions. First, the L-y-glutamylcysteine synthetase (Gshl p) converts cysteine into γ-glutamylcysteine, which is then converted into glutathione by the GSH synthetase (Gsh2p). GSH biosynthesis is tightly regulated at three different levels: transcriptional, post-translational, and substrate availability.

The biosynthesis of glutathione (GSH) requires three amino acids: cysteine, glutamate and glycine. During yeast propagation, the addition of cysteine results in increased production of both GSH and the intermediate γ-glutamylcysteine (γ-GC). Co-feeding of cysteine and glycine results in the conversion of a portion of the (γ-GC) to GSH. There are two main pathways leading to the synthesis of glycine in yeast. In one pathway L- threonine aldolase, encoded by GLY1 , produces glycine from L-threonine (which is produced from the glycolytic intermediate oxaloacetate). Glycine may also be formed from L-serine, via two hydroxymethyltransferases, encoded by SHM1 (mitochondrial) and SHM2 (cytosolic). Microbial production of GSH using genetically engineered yeast strains has a potential to satisfy its increasing industrial demand. Conventional methods allow increasing the GSH content in the cells only two-fold compared to the native level, thus increasing further the GSH concentration would allow for improving the efficiency of these products to a considerable extent. The concentration range of GSH in S. cerevisiae is of 0.1 -1 % of the dry cell weight. This varies according to the strain, the growth conditions and the GSH measurement method employed.

It would be highly desirable to be provided with genetically modified host cells capable of producing and accumulating an increase amount of intracellular GSH (preferably mostly in a reduced form) and reducing the amount of intracellular γ-glutamylcysteine or free cysteine, when compared to its corresponding amount in the parental strain. In some embodiments, it would be desirable to reduce or even eliminate supplementation with cysteine or glycine during the GSH production phase (e.g. , the fermentation) with the genetically modified host cells. In some embodiments, it would also be desirable to be provided with processes for making GSH and preparations (raw, semi-purified and purified) comprising GSH using the genetically modified host cells.

BRIEF SUMMARY

The present disclosure concerns a genetically modified host cell (preferably a genetically modified host cell) and its use for the production of GSH. The genetically modified host cell synthesizes more total thiols (also known as total GHS and apparent GSH), which include the free intracellular cysteine, γ-glutamylcysteine (g-GC or γ-GC) and true GSH. Moreover, the genetically modified strain accumulates a higher proportion of true GSH and very little g-GC compared to corresponding wild-type strains.

According to a first aspect, the present disclosure provides a process of making glutathione. Broadly the process comprises fermenting a substrate with a genetically modified host cell to obtain a fermented mixture comprising glutathione. The genetically modified host cell has (i) a first heterologous nucleic acid molecule coding for a mutated cystathionine beta-synthase protein (Cys4p) having an increased biological activity when compared to a wild-type Cys4p, (ii) a second heterologous nucleic acid molecule coding for a mutated Yapl p having a reduced ability of being translocated from the nucleus to the cytoplasm when compared to a wild-type Yapl p and/or (iii) a third heterologous nucleic acid molecule coding for a threonine aldolase protein (Glyl p). In an embodiment, the genetically modified host cell has the first heterologous nucleic acid molecule and at least one of the second heterologous nucleic acid molecule or the third heterologous nucleic acid molecule. In an embodiment, the mutated Cys4p is a fragment of the wild-type Cys4p. For example, the mutated Cys4p can be obtained by deleting one or more C-terminal amino acid residue(s) from the wild-type Cys4p. In still another example, the mutated Cys4p can be obtained by deleting the regulatory domain from the wild-type Cys4p. In an embodiment, the mutated Cys4p consists of the amino acid sequence of SEQ ID NO: 2. In still another embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild- type Cys4p is replaced by the first heterologous nucleic acid molecule. In yet another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the first heterologous nucleic acid molecule. In a further embodiment, the mutated Yapl p is substantially located in the nucleus of the genetically modified host cell. In still another embodiment, the mutated Yapl p has at least one amino acid substitution when compared to the wild-type Yapl p. For example, the at least one amino acid substitution can be located in a domain corresponding to a cysteine-rich domain of the wild-type Yapl p. In yet another example, the mutated Yapl p can be obtained by substituting a cysteine residue with an hydrophilic amino acid residue (such as, for example, an aspartic acid residue) in the domain corresponding to the cysteine-rich domain of the wild-type Yapl p. In still another example, the substituted cysteine residue of the mutated Yapl p is located at a position corresponding to residue 626 of SEQ ID NO: 3. In still another embodiment, the mutated Yap1 p comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In an embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild-type Yapl p is replaced by the second heterologous nucleic acid molecule. In another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the second heterologous nucleic acid molecule. In a further embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the third heterologous nucleic acid molecule. In still yet another embodiment, the genetically modified host cell comprises the first heterologous nucleic acid molecule and the second heterologous nucleic acid molecule; the first heterologous nucleic acid molecule and the third heterologous nucleic acid molecule; the second heterologous nucleic acid molecule and the third heterologous nucleic acid molecule; or the first heterologous nucleic acid molecule, the second heterologous nucleic acid molecule and the third heterologous nucleic acid molecule. In an embodiment, the process further comprises processing the fermented mixture into a yeast extract or a yeast hydrolysate. In still another embodiment, the process further comprises purifying or inactivating the genetically modified host cell from the fermented mixture. In yet another embodiment, the process further comprises purifying the glutathione from the fermented mixture. In another embodiment, the genetically modified host cell is a genetically modified yeast host cell. For example, the genetically modified yeast host cell can be from the genus Saccharomyces. In another example, the genetically modified yeast host cell is from the species Saccharomyces cerevisiae.

According to a second aspect, the present disclosure provides a fermented substrate, a yeast extract, a yeast hydrolysate, a purified genetically modified host cell and/or an inactivated genetically modified host cell obtainable or obtained by the process described herein. According to a third aspect, the present disclosure concerns a process for increasing glutathione accumulation in a genetically modified host cell. Broadly, the process comprises introducing a first heterologous nucleic acid molecule, a second heterologous nucleic acid molecule and/or a third heterologous nucleic acid molecule in a parental yeast host cell to generate the genetically modified host cell. In the process, the first heterologous nucleic acid molecule codes for a mutated cystathionine beta-synthase protein (Cys4p) having an increased biological activity when compared to a wild-type Cys4p. In addition, the second heterologous nucleic acid molecule codes for a mutated Yapl p having a limited ability of being exported in the cytoplasm when compared to a wild-type Yapl p. Further, the third heterologous nucleic acid molecule codes for an heterologous threonine aldolase (Glyl p). In an embodiment, the genetically modified host cell has the first heterologous nucleic acid molecule and at least one of the second heterologous nucleic acid molecule or the third heterologous nucleic acid molecule. In an embodiment, the mutated Cys4p is a fragment of the wild-type Cys4p. For example, the mutated Cys4p can be obtained by deleting one or more C-terminal amino acid residue from the wild-type Cys4p. In still another example, the mutated Cys4p can be obtained by deleting the regulatory domain from the wild-type Cys4p. In an embodiment, the mutated Cys4p consists of the amino acid sequence of SEQ ID NO: 2. In still another embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild-type Cys4p is replaced by the first heterologous nucleic acid molecule. In yet another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the first heterologous nucleic acid molecule. In a further embodiment, the mutated Yapl p is substantially located in the nucleus of the genetically modified host cell. In still another embodiment, the mutated Yapl p has at least one amino acid substitution when compared to the wild-type Yapl p. For example, the at least one amino acid substitution can be located in a domain corresponding to a cysteine-rich domain of the wild-type Yapl p. In yet another example, the mutated Yapl p can be obtained by substituting a cysteine residue with an hydrophilic amino acid residue (such as, for example, an aspartic acid residue) in the domain corresponding to the cysteine-rich domain of the wild-type Yapl p. In still another example, the substituted cysteine residue of the mutated Yapl p is located at a position corresponding to residue 626 of SEQ ID NO: 3. In still another example, the mutated Yapl p comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In an embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild-type Yap1 p is replaced by the second heterologous nucleic acid molecule. In another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the second heterologous nucleic acid molecule. In yet another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the third heterologous nucleic acid molecule. In another embodiment, the genetically modified host cell is a genetically modified yeast host cell. For example, the genetically modified yeast host cell can be from the genus Saccharomyces. In another example, the genetically modified yeast host cell is from the species Saccharomyces cerevisiae.

According to a fourth aspect, the present disclosure concerns a genetically modified host cell obtained by the process described herein.

According to a fifth aspect, the present disclosure concerns a genetically modified host cell comprising at least two of (i) a first heterologous nucleic acid molecule encoding a mutated cystathionine beta-synthase protein (Cys4p) having an increased biological activity when compared to a wild-type Cys4p; (ii) a second heterologous nucleic acid molecule encoding a mutated Yapl p having a reduced ability of being translocated from the nucleus to the cytoplasm when compared to the wild-type Yapl p; and/or (iii) a third heterologous nucleic acid molecule encoding a threonine aldolase (Glyl p). In an embodiment, the genetically modified host cell has the first heterologous nucleic acid molecule and at least one of the second heterologous nucleic acid molecule or the third heterologous nucleic acid molecule. In an embodiment, the mutated Cys4p is a fragment of the wild-type Cys4p. For example, the mutated Cys4p can be obtained by deleting one or more C-terminal amino acid residue from the wild-type Cys4p. In still another example, the mutated Cys4p can be obtained by deleting the regulatory domain from the wild-type Cys4p. In an embodiment, the mutated Cys4p consists of the amino acid sequence of SEQ ID NO: 2. In still another embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild-type Cys4p is replaced by the first heterologous nucleic acid molecule. In yet another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the first heterologous nucleic acid molecule. In a further embodiment, the mutated Yapl p is substantially located in the nucleus of the genetically modified host cell. In still another embodiment, the mutated Yapl p has at least one amino acid substitution when compared to the wild-type Yapl p. For example, the at least one amino acid substitution can be located in a domain corresponding to a cysteine-rich domain of the wild-type Yapl p. In yet another example, the mutated Yapl p can be obtained by substituting a cysteine residue with an hydrophilic amino acid residue (such as, for example, an aspartic acid residue) in the domain corresponding to the cysteine-rich domain of the wild-type Yap1 p. In still another example, the substituted cysteine residue of the mutated Yapl p is located at a position corresponding to residue 626 of SEQ ID NO: 3. In still another example, the mutated Yapl p comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In an embodiment, in the genetically modified host cell, at least one copy of the native nucleic acid molecule coding for the wild-type Yapl p is replaced by the second heterologous nucleic acid molecule. In another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the second heterologous nucleic acid molecule. In yet another embodiment, the genetically modified host cell comprises at least one, at least two, at least three or at least four copies of the third heterologous nucleic acid molecule. In another embodiment, the genetically modified host cell is a genetically modified yeast host cell. For example, the genetically modified yeast host cell can be from the genus Saccharomyces. In another example, the genetically modified yeast host cell is from the species Saccharomyces cerevisiae.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Figure 1 provides the relative total thiol content of a control non-modified S. cerevisiae strain (parental strain) and various genetically modified S. cerevisiae strains SIL005, SIL016, SIL064, SIL010, SIL019, SIL020, SIL022, SIL024, SIL030, SIL078, SIL043, SIL070, SIL085, SIL068 and SIL090. Values are relative to the parental (wild-type, parental, not genetically modified) S. cerevisiae strain. Values are average of duplicates.

Figure 2 provides the true glutathione (true GSH), cysteine (Cys), γ-L-glutamyl-L-cysteine (v- GC or g-Glu-Cys) and total thiol content of S. cerevisiae strain SIL068 grown in molasses in a 20L fermenter. Values are provided as mg/g of dry weight in function of fermentation conditions. Values are average of duplicates or triplicates. Figure 3 illustrates the effect of total thiols and true GSH on the baguette's length in a baking test. Baguettes were obtained with (1) no additive (control), (2) 40 ppm L-cysteine, (3) 0.25% Fermaid-SR™, (4) 0.5% Fermaid-SR™, (5) 0.071 % SIL068 IDY1 , (6) 0.14% SIL068 IDY1 , (7) 0.083% SIL068 IDY2 or (8) 0.167% SIL068 IDY2. Figure 4 provides cysteine (Cys), γ-L-glutamyl-L-cysteine (g-GC), true GSH and total thiol content of S. cerevisiae strains SIL061 (n = 1) and SIL143 (n = 2) grown on minimal medium supplemented with cysteine. Values are provided as mg/g of dry weight in function of fermentation conditions.

Figure 5 provides cysteine (Cys), γ-L-glutamyl-L-cysteine (g-GC), true GSH and total thiol content of P. pastoris strains X33, SIL148 and SIL150 grown on minimal medium supplemented with cysteine (n = 2). Values are provided as mg/g of dry weight in function of fermentation conditions.

Figure 6 provides cysteine (Cys), γ-L-glutamyl-L-cysteine (g-GC), true GSH and total thiol content of P. pastoris strains X33, SIL151 and SIL153 grown on minimal medium supplemented with cysteine (n = 2). Values are provided as mg/g of dry weight in function of fermentation conditions.

Figure 7 provides the amino acid sequence alignment of wild-type Cys4p from Saccharomyces cerevisiae (SEQ ID NO: 6), Saccharomyces bayanus (SEQ ID NO: 8), Cyberlindnera jadinii (SEQ ID NO: 12), Torulaspora delbrueckii (SEQ ID NO: 14), Zygosaccharomyces bailii (SEQ ID NO: 16), Scheffersomyces (Pichia) stipitis (SEQ ID NO: 19), Kluyveromyces lactis (SEQ ID NO: 20) and Pichia pastoris (SEQ ID NO: 22). The catalytic and regulatory documents are boxed and identified. The essential active-site residues are threonine-81 , serine-82, threonine-85, glutamine-157 and tyrosine-158 when using the sequences of S. cerevisiae (Aitken et a/., 2004). The amino acid alignment was generated using the CLUSTAL® OMEGA software using default parameters except for order which was changed from aligned to input.

Figure 8 provides the amino acid sequence alignment of wild-type Yapl p from Saccharomyces cerevisiae strain (SEQ ID NO: 7), Saccharomyces bayanus (SEQ ID NO: 9), Saccharomyces kudriavzevii (SEQ ID NO: 1 1), Cyberlindnera jadinii (SEQ ID NO: 13), Torulaspora delbrueckii (SEQ ID NO: 15), Zygosaccharomyces bailii (SEQ ID NO: 17), Schizosaccharomyces pombe (SEQ ID NO: 18), Kluyveromyces lactis (SEQ ID NO: 21) and Pichia pastoris (SEQ ID NO: 23). The basic region (DNA binding), leucine zipper and cysteine-rich domain are boxed and identified. The amino acid alignment was generated using the CLUSTAL® OMEGA software using default parameters except for order which was changed from aligned to input.

Figure 9 provides the amino acid sequence alignment of wild-type Glyl p from Saccharomyces cerevisiae strain (SEQ ID NO: 24), Saccharomyces bayanus (SEQ ID NO: 25), Saccharomyces kudriavzevii (SEQ ID NO: 26), Cyberlindnera jadinii (SEQ ID NO: 27), Torulaspora delbrueckii (SEQ ID NO: 28), Zygosaccharomyces bailii (SEQ ID NO: 29), Schizosaccharomyces pombe (SEQ ID NO: 30), Kluyveromyces lactis (SEQ ID NO: 31) and Pichia pastoris (SEQ ID NO: 32). The basic region (DNA binding), leucine zipper and cysteine-rich domain are boxed and identified. The amino acid alignment was generated using the CLUSTAL® OMEGA software using default parameters except for order which was changed from aligned to input.

DETAILED DESCRIPTION

The present disclosure concerns genetically modified host cells and their uses in processes for making glutathione during fermentation. The present disclosure also especially concerns genetically modified yeast host cells and their uses in processes for making glutathione during fermentation. In some embodiments, the use of the genetically modified host cells allows for a substantial increase in total thiols and especially in glutathione production and accumulation. Under certain circumstances, the total thiols content of genetically modified host cells is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even higher when compared to corresponding wild-type (i.e., parental or non-genetically modified) cells. In other circumstances, the genetically modified host cells generate a higher proportion of true GSH than its γ-L-glutamyl-L-cysteine byproduct when compared to corresponding wild- type (i.e., parental or non-genetically modified host) cells. In some circumstances, the genetically modified host cells generate less intracellular free cysteine when compared to corresponding wild-type (i.e., parental or non-genetically modified host) cells. In still other circumstances, the genetically modified host cells require less supplementation (for example less cysteine and/or less glycine supplementation) that the corresponding wild-type (i.e., parental or non-genetically modified) cells during fermentation to achieve the same GSH content.

Genetically modified host cells

The present disclosure provides genetically modified host cells capable of making and accumulating more GSH than corresponding parental or non-genetically modified host cells. The genetically modified host cells can be genetically modified yeast host cells. In the context of the present disclosure, the genetically modified host cells have at least one genetic modification allowing the expression of a mutated cystathionine beta-synthase protein Cys4p, a second genetic modification allowing the expression of a mutated Yapl p and/or a third genetic modification allowing the expression of an heterologous threonine aldolase (Glyl p). In some embodiments, the genetically modified host cell also includes further genetic modifications, for example, for expressing an heterologous gamma glutamyl cysteine synthetase 1 protein (Gshl p) and/or an heterologous glutathione synthetase 2 protein (Gsh2p).

The genetically modified host cell can be a yeast host cell. Suitable yeast host cells that can be genetically modified as described herein can be, for example, from the genus Arxula, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Kloeckera, Kluyveromyces, Hanseniaspora, Hansenula, Metschnikowia, Pichia, Phaffia, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Schwanniomyces, Sporobolomyces, Starmerella, Tetrapisispora, Yarrowia or Zygosaccharomyces. In some embodiments, the yeast host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiment, the yeast host cell can be an oleaginous microalgae host cell (e.g. , for example, from the genus Thraustochytrium or Schizochytrium). Suitable yeast species can include, for example, Arxula adeninivorans, Brettanomyces bruxellensis, Candida albicans, Candida colliculosa / Torulaspora Delbrueckii, Candida tropicalis, Candida utilis (Cyberlindnera jadinii), Cryptococcus skinneri, Debaryomyces sp., Debaryomyces hansenii, Debaryomyces polymorphus, Hanseniaspora vinea, Hanseniaspora occidentalis, Hanseniaspora uvarum, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus var drosophilarum, Kluyveromyces thermotolerans, Kluyveromyces wickerhamii, Kluyveromyces fragilis, Metschnikowia pulcherrima (Candida pulcherrima), Metschnikowia fructicola, Phaffia rhodozyma, Pichia anomala, Pichia kudriavzevii, Pichia occidentalis, Pichia pastoris, Saccharomyces bulgari, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces exiguous, Saccharomyces uvarum, Scheffersomyces stipitis, Schizzosaccharomyces pombe, Schwanniomyces occidentalis, Sporobolomyces, Starmerella bombicolla, Tetrapisispora phaffii, Yarrowia lipolytica, Zygosaccharomyces bailii or Zygosaccharomyces rouxii. In one particular embodiment, the genetically modified yeast host cell is from the genus Saccharomyces and, in a further embodiment, from the species Saccharomyces cerevisiae.

The genetically modified host cell can be a bacteria. Suitable bacterial host cells that can be genetically modified as described herein can be a Gram-positive or a Gram-negative bacteria. The genetically modified bacterial host cell can be from the phylum Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Chlamydiae, Cholorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfobacteria, Thermotogae or Verrucomicrobia. In one particular embodiment, the genetically modified bacterial host cell is Escherichia coli.

As indicated above, the genetically modified host cells includes one or more heterologous nucleic acid molecule encoding one or more heterologous protein (e.g. , mutated Cys4p, mutated Yapl p, Glyl p, Gshl p and/or Gsh2p). The term "heterologous" when used in reference to a nucleic acid molecule (such as a promoter or a coding sequence) or a protein refers to a nucleic acid molecule or a protein that is not natively found in the host organism or cell. "Heterologous" also includes a native coding region, or portion thereof, that is removed or amplified from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g. , not in its natural location in the organism's genome. The heterologous nucleic acid molecule(s) is(are) purposively introduced into the host cell. An "heterologous" nucleic acid molecule or protein may be derived from any source, e.g. , eukaryotes, prokaryotes, viruses, etc. In an embodiment, the heterologous nucleic acid molecule may be derived from an eukaryote (such as, for example, a yeast from the same genus or from the same species as the genetically modified host cell). The term "heterologous" as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, a heterologous element could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g. , different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications). The term "heterologous" is also used synonymously herein with the term "exogenous".

(7) Mutated cystathionine beta-synthase (Cys4p)

Cysteine is known to be rate-limiting to GSH production. The cystathionine beta-synthase protein (also referred herein as Cys4p) catalyzes the first committed step in cysteine biosynthesis from homocysteine. Yeast strains exhibiting increased GSH production were shown to overexpress the transcripts encoding the Cys4p (Nisamedtinov et al. , 2010, Nisamedtinov et al. , 201 1 and Orumets et al. , 2012. The overexpression of the Cys4p has been shown to increase the GSH content in Cyberlindnera jadinii (formely Candida utilis) (Suzuki et al. , 201 1), Pichia pastoris (see CN 101220338 A and CN 101245363 A) and Saccharomyces cerevisiae (Suzuki et al. , 201 1). However, as shown in the present application, the overexpression of wild-type Cys4p in a yeast host cells leads only to a modest increase in total thiols (see results obtained for strain SIL005 on Figure 1 for example).

It has been reported that a gain-of-function allele of CYS4, CYS4.353, encodes a cystathionine β-synthase exempt of its regulatory domain, resulting in higher specific activity (Jhee et a/. , 2000), leading to an accelerated growth rate and cell division in S. cerevisiae (Blank et a/. , 2009). Surprisingly, as shown in the present application, when the gene CYS4.353 is expressed in a genetically-modified yeast host cell, a vigorous increase in total thiols is observed (see, for example, results obtained for strain SIL010 on Figure 1). The genetically modified host cell of the present disclosure (and especially the genetically modified yeast host cell of the present disclosure) can include a first heterologous nucleic acid molecule coding for a mutated Cys4p having an increased biological activity when compared to a wild-type Cys4p. Such genetically modified host cell is thus capable of expressing the mutated Cys4p and, when placed in the appropriate conditions, the genetically modified host cell expresses the mutated Cys4p which ultimately increases the total thiols (and GSH production) during fermentation. The first heterologous nucleic acid molecule can be integrated in one or more copies in the genetically modified host cell (at one or more neutral integration site(s)). The first heterologous nucleic acid molecule can replace one or more copies of the native nucleic acid molecule coding for the native Cys4p. In an embodiment, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies integrated copies of the first heterologous nucleic acid molecule (at one or more neutral integration sites) and in which at least one copy (or both copies) of the native nucleic acid molecule coding for the native Cys4p has been replaced by the first heterologous nucleic acid. When expressed in the genetically modified host cell, the mutated Cys4p has increased biological activity than the wild-type Cys4p. In the context of the present disclosure, the wild- type Cys4p refers to a protein having cystathionine β-synthase activity, i.e. capable of enzymatically converting homocysteine into cystathionine. The wild-type or native Cys4p is encoded by a yeast genome and comprises both a catalytic domain and a regulatory domain. The wild-type Cys4p include essential active-site residues threonine-81 , serine-82, threonine- 85, glutamine-157 and tyrosine- 158 (Aitken et a/. , 2004). Figure 7 provides an amino acid alignment of wild-type Cys4p obtained from various yeast species. In an embodiment, the mutated Cys4p possesses the essential active-site residues corresponding to threonine-81 , serine-82, threonine-85, glutamine-157 and tyrosine-158 as shown on Figure 7. ln an embodiment, the wild-type Cys4p has the amino acid sequence of any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 20 or 22. In another embodiment, the wild-type Cys4p is any one of the wild-type Cys4p shown on Figure 7. In still another embodiment, the wild-type Cys4p corresponds to the consensus sequence shown on Figure 7. In still another embodiment, the wild-type Cys4p of the present disclosure (which can be referred to as a variant) can share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity with any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 20 or 22 with the consensus sequence shown on Figure 7, provided that the wild-type Cys4p does exhibit cystathionine β-synthase activity. "Identity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5: 151 - 153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. As indicated above, the genetically modified host cell of the present disclosure (such as the genetically modified yeast host cell) can, in some embodiments, include a first nucleic acid molecule encoding for a mutated Cys4p. In the context of the present disclosure, a "mutated" Cys4p refers to a protein having increased cystathionine β-synthase activity with respect to the wild-type Cys4p. In addition, when compared to the wild-type Cys4p, the mutated Cys4p has one or more amino acid residue difference with the wild-type Cys4p. Since the mutated Cys4p exhibits cystathionine β-synthase activity, the mutated Cys4p however retains threonine at position 81 (or at a corresponding position), serine at position 82 (or at a corresponding position), threonine at position 85 (or at a corresponding position), glutamine at position 157 (or at a corresponding position) and tyrosine at position 158 (or at a corresponding position) in its catalytic site. ln an embodiment, the mutated Cys4p is a fragment of the wild-type Cys4p exhibiting an increased cystathionine β-synthase activity and/or stability with respect to the "wild-type" Cys4p. As used in the context of the present disclosure, the term "fragment" refers to a protein having at least one less amino acid residues that the wild-type protein. The deletion can occur either at the N-, at the C- or at both the N- and C-terminus of the wild-type Cys4p. In the context of the present disclosure, when the mutated Cys4p is a fragment of the wild- type Cys4p, it at least comprises the catalytic domain of the wild-type Cys4p. In some embodiments, the mutated Cys4p can have at least at least 300, 310, 320, 330, 340, 350 or more consecutive amino acid residues of the wild-type Cys4p. In a specific embodiment, the mutated Cys4p is obtained by deleting one or more amino acid residues from the carboxy terminus of the wild-type Cys4p, such as, for example, by deleting a part of or the entire regulatory domain of the wild-type Cys4p. The regulatory domain of the Cys4p is illustrated at Figure 7 and corresponds to positions 354 to 507 of SEQ ID NO: 1 . In an embodiment, the mutated Cys4p consists of the sequence defined by residues 1 to 353 of SEQ ID NO: 1 as well as corresponding sequences in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 22. In some specific embodiments, the mutated Cys4p consists of the amino acid sequence of SEQ ID NO: 2.

The present disclosure also includes using fragments and/or variants of the mutated Cys4p, provided that such fragments and variants exhibit an increased cystathionine β-synthase activity and/or stability with respect to the wild-type Cys4p. Fragments of the mutated Cys4p refer to a mutated Cys4p having at least one less amino acid residues that the mutated Cys4p. The deletion can occur either at the N-, at the C-terminus or at both the N- and C- terminus of the wild-type protein. Variants of the mutated Cys4p may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue. A "variant" of the mutated Cys4p can be a conservative variant, or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the mutated Cys4p. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the mutated Cys4p. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the mutated Cys4p more hydrophobic or hydrophilic, without adversely affecting the biological activities of the mutated Cys4p. (ii) Mutated Yaplp

Yapl p is a transcription factor involved in S. cerevisiae oxidative stress response. It is a positive transcriptional regulator of the GSH1, GSH2, CYS3 and CYS4 genes. Yeast strains exhibiting increased GSH production where shown to overexpress the transcripts encoding the Yapl p (Nisamedtinov et al. , 2010 and Nisamedtinov et al. , 201 1). Wild-type Yapl p overexpression was also shown to enhance GSH accumulation (Orumets et al. , 2012).

The biological activity of the Yapl p is influenced by its subcellular localization: under oxidative stress, the wild-type Yapl p is localized in the nucleus and mediates its biological activity, while when the oxidative stress is reduced, the wild-type Yapl p is translocated in the cytoplasm, thus halting its biological activity (Kuge et al. , 1997). The reversible nuclear localization of the wild-type Yapl p is mediated by its C-terminal Cysteine-Rich Domain (CRD). The CRD is capable of forming disulfide bonds between specific cysteine residues which, in some circumstances, conceals the nuclear export sequence of the Yapl p, allowing it to remain active in the nucleus, despite an unchanged expression at the RNA level and even a lower protein level (Kuge et al. , 2001 and Kuge et al. , 1997).

The genetically modified host cell of the present disclosure can include an heterologous nucleic acid molecule encoding a mutated Yapl p. The mutated Yapl p has, when compared to the wild-type Yapl p, a decreased ability to be translocated from the nucleus to the cytoplasm. After its initial translation in the cytoplasm, the mutated Yapl p is thus substantially, and in some embodiments exclusively, located in the nucleus of the genetically modified host cell (such as, for example, in the nucleus of the genetically modified yeast host cell). The genetically modified host cell is capable of expressing the mutated Yapl p and, when placed in the appropriate conditions, the genetically modified host cell expresses the mutated Yapl p. The mutated Yap1 -encoding heterologous nucleic acid molecule can be integrated at one or more copies in the genetically modified host cell (at one or more neutral integration site(s)). The second heterologous nucleic acid molecule can replace one or both copies of the native nucleic acid molecule coding for the native Yapl p. In an embodiment, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies integrated copies of the heterologous nucleic acid molecule (at one or more neutral integration sites) and in which at least one copy (or both copies) of the native nucleic acid molecule coding for the native Yapl p has been replaced by the first heterologous nucleic acid.

When expressed in the genetically modified host cell (and especially in the genetically modified yeast host cell), the mutated Yapl p has a decreased ability to be translocated from the nucleus in the cytoplasm when compared to the ability of the wild-type Yapl p. In the context of the present disclosure, the wild-type Yapl p refers to a protein having basic leucine zipper transcription factor activity, i.e. a protein capable of modulating the expression of various genes. For example, one of the wild-type Yapl p's transcription factor activity is to increase the expression of the GSH1 and GSH2 genes respectively coding for the v- glutamylcysteine synthetase protein (Gshl p) and the glutathione synthetase 2 protein (Gsh2p). In an embodiment, the wild-type Yapl p is encoded by a eukaryotic genome, such as, for example, a yeast genome. Figure 8 provides an amino acid alignment of wild-type Yapl p from various yeast species. In an embodiment, the wild-type Yapl p has the amino acid sequence of any one of SEQ ID NO: 7, 9, 1 1 , 13, 15, 17, 18 19, 21 or 23. In another embodiment, the wild-type Yapl p is any one of the wild-type Yapl p shown on Figure 8. In still another embodiment, the wild-type Yapl p corresponds to the consensus sequence shown on Figure 8.

In still another embodiment, the wild-type Yapl p of the present disclosure can share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity with any one of SEQ ID NO: 7, 9, 1 1 , 13, 15, 17, 18 19, 21 or 23 or the consensus sequence shown on Figure 8, provided that the wild-type Yapl p is capable of being translocated from the nucleus to the cytoplasm when oxidative stress is reduced. "Identity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5: 151 - 153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

As indicated above, the genetically modified host cell of the present disclosure can, in some embodiments, include an heterologous nucleic acid molecule encoding for a mutated Yapl p. ln the context of the present disclosure, a "mutated" Yapl p refers to a protein having a reduced capacity of or lacking the ability of being translocated from the nucleus to the cytoplasm of the host cell, when compared to the ability of the "wild-type" Yapl p. In some embodiments, after its initial translation in the cytoplasm, the mutated Yapl p is constitutively expressed in the nucleus of the host cell. In addition, when compared to the wild-type Yapl p, the mutated Yapl p has one or more amino acid residue difference with the wild-type Yapl p.

In an embodiment, the mutated Yapl p is a fragment of the wild-type Yapl p exhibiting having a reduced capacity of or lacking the ability of being translocated from the nucleus to the cytoplasm of the yeast host cell when compared to the "wild-type" Yapl p. As used in the context of the present disclosure, the term "fragment" refers to a protein having at least one less amino acid residues that the wild-type protein. The deletion can occur either at the N-, at the C- or at both the N- and C-terminus of the wild-type protein. In the context of the present disclosure, when the mutated Yapl p is a fragment of the wild-type Yapl p, it at least comprises the basic region and the leucine zipper of the wild-type Yapl p. For example, the mutated Yapl p can be obtained by deleting (at least partially and in some embodiments entirely) the cysteine-rich domain from the wild-type Yapl p. The cysteine-rich domain of selected Yapl p is shown in Figure 8. In some embodiments, the mutated Yapl p can have at least at least 100, 200, 300, 400, 500, 600 or more consecutive amino acid residues of the wild-type Yapl p. In an embodiment, the mutated Yapl p can be obtained by substituting one or more of amino acid residues of the wild-type Yapl p. In an embodiment, the mutated Yapl p does include an amino acid substitution in the cysteine-rich domain of the wild-type Yapl p. The cysteine-rich domain of wild-type Yapl p is illustrated at Figure 8 and corresponds to residues 604 to 635 of SEQ ID NO: 3. The cysteine-rich domain of the wild-type Yapl p comprises three cysteine residues: a first cysteine residue corresponding to position 604 of SEQ ID NO: 3, a second cysteine residue corresponding to position 626 of SEQ ID NO: 3 and a third cysteine residue corresponding to position 635 of SEQ ID NO: 3. In an embodiment, the mutated Yapl p (in the domain corresponding to the cysteine-rich domain of the Yapl p) has the first cysteine residue (corresponding to residue at position 604 of SEQ ID NO: 3) and the third cysteine residue of the wild-type Yapl p (corresponding to residue at position 635 of SEQ ID NO: 3) and includes a substitution at the second cysteine residue (corresponding to residue at position 626 of SEQ ID NO: 3). The mutated Yapl p can be obtained by substituting the second cysteine residue of the cysteine-rich domain of the wild-type Yapl p by an hydrophilic amino acid residue, such as, for example aspartic acid, leucine, arginine, histidine, glutamic acid, serine, threonine, asparagine, glutamine, lysine, serine, tyrosine, methionine or tryptophan. In another embodiment, the mutated Yapl p can be obtained by substituting the second cysteine residue of the cysteine-rich domain of the wild-type Yapl p by an aspartic acid residue. In an embodiment, the mutated Yapl p comprises the amino acid sequence of SEQ ID NO: 4 or 5. In another embodiment, the mutated Yapl p comprises the amino acid sequence of any one of SEQ ID NO: 7, 9, 1 1 , 13, 15, 17, 18 19, 21 or 23 in which the second cysteine of the cysteine-rich domain (corresponding to residue at position 626 of SEQ ID NO: 3) has been substituted by an hydrophilic amino acid residue (as indicated above), preferably by an aspartic acid residue.

The present disclosure also includes using fragments and/or variants of the mutated Yapl p, provided that such fragments and variants exhibit a reduced ability of or lacking the ability of being translocated from the nucleus to the cytoplasm of the host cell when compared to the wild-type Yapl p. Fragments of the mutated Yapl p refer to a mutated Yapl p having at least one less amino acid residues that the mutated Yap1 p. The deletion can occur either at the N- , at the C- or at both the N- and C-terminus of the wild-type protein. Variants of the mutated Yapl p may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue. A "variant" of the mutated Yapl p can be a conservative variant, or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the mutated Yapl p. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the mutated Yapl p. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the mutated Yapl p more hydrophobic or hydrophilic, without adversely affecting the biological activities of the mutated Yapl p.

(Hi) Heterologous threonine aldolase (Glylp)

Glycine is required for the production of GSH. The L-threonine aldolase protein (also referred to as Gly1 p) catalyzes the removal of acetaldehyde from threonine to generate glycine.

The genetically modified host cell of the present disclosure (and especially the genetically modified yeast host cell of the present disclosure) can include an heterologous nucleic acid molecule coding for an heterologous Glyl p. Such genetically modified host cell is thus capable of expressing the heterologous Glyl p and, when placed in the appropriate conditions, the genetically modified host cell expresses the heterologous Glyl p which can favor an increase in total thiols (and GSH production), optionally in combination with the expression of the heterologous Gshl p and/or the mutated Cys4p. The Glyl p-encoding heterologous nucleic acid molecule can be integrated in one or more copies in the genetically modified host cell (at one or more neutral integration site(s)). In an embodiment, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies (e.g. , at least two, three or four) integrated copies of the Glyl p- encoding heterologous nucleic acid molecule (at one or more neutral integration sites).

In an embodiment, the heterologous Glyl p has the amino acid sequence of any one of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31 or 32. In another embodiment, the heterologous Glyl p is any one of the heterologous Glyl p shown on Figure 9. In still another embodiment, the heterologous Glyl p corresponds to the consensus sequence shown on Figure 9.

The present disclosure also concerns variants of the heterologous Glyl p may be one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue. A "variant" of the heterologous Glyl p can be a conservative variant, or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the heterologous Glyl p. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the heterologous Glyl p. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the heterologous Glyl p more hydrophobic or hydrophilic, without adversely affecting the biological activities of the heterologous Glyl p.

In still another embodiment, the variant of the heterologous Glyl p of the present disclosure can share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity with any one of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31 or 32 with the consensus sequence shown on Figure 9, provided that the heterologous Glyl p exhibits threonine aldolase activity. "Identity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151 -153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

In an embodiment, the heterologous Glyl p is a fragment of the wild-type Glyl p exhibiting an threonine aldolase activity. As used in the context of the present disclosure, the term "fragment" refers to a protein having at least one less amino acid residues that the wild-type protein. The deletion can occur either at the N-, at the C- or at both the N- and C-terminus of the wild-type heterologous Glyl p. In the context of the present disclosure, when the heterologous Glyl p is a fragment of the wild-type Glyl p, it at least comprises the catalytic domain of the wild-type Glyl p. In some embodiments, the fragment of the heterologous Glyl p can have at least at least 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 or more consecutive amino acid residues of the wild-type Glyl p.

(iv) Additional heterologous nucleic acid molecules

The genetically modified host cell comprises at least one of the first heterologous nucleic acid molecule encoding for the mutated Cys4p, the second heterologous nucleic acid molecule encoding for the mutated Yapl p and/or the third heterologous nucleic acid molecule encoding for the heterologous Glyl p. In a further embodiment, the genetically modified host cell comprises the first heterologous nucleic acid molecule (coding for a mutated Cys4p) and at least one of the second nucleic acid molecule (coding for a mutated Yapl p) or the third heterologous nucleic acid molecule (coding for a threonine aldolase protein (Glyl p)). In an embodiment, the genetically modified host cell comprises both the first heterologous nucleic acid molecule coding for the mutated Cys4p and the second heterologous nucleic acid molecule coding for the mutated Yapl p. In a further embodiment, the genetically modified host cell comprises both the second heterologous nucleic acid molecule coding for the mutated Cys4p and the third heterologous nucleic acid molecule coding for the heterologous Glyl p. In another embodiment, the genetically modified host cell comprises both the second heterologous nucleic acid molecule coding for the mutated Yapl p and the third heterologous nucleic acid molecule coding for the heterologous Glyl p. In yet another embodiment, the genetically modified host cell comprises the first heterologous nucleic acid molecule encoding for the mutated Cys4p, the second heterologous nucleic acid molecule encoding for the mutated Yapl p and the third heterologous nucleic acid molecule encoding for the heterologous Glyl p. ln the embodiments in which more than one heterologous nucleic acid molecules are present in the genetically modified host cell (such as the genetically modified yeast host cell), each of the heterologous nucleic acid molecules can be integrated (in one or more copies) at neutral integration site(s) and/or replace one or both copies of the nucleic acid molecule coding for the native (wild-type) nucleic acid molecules (for example the native nucleic acid molecule coding for Cys4p or Yapl p). For example, the first, second and third heterologous nucleic acid molecule can both be integrated at one or more copies in the genetically modified host cell (at one or more neutral integration site(s)). In such embodiment, the neutral integration sites can be the same or different. In another example, the first heterologous nucleic acid molecule can replace one or more copies of the native nucleic acid molecule coding for the native Cys4p and one or more copies of the second heterologous nucleic acid molecule can be integrated at one or more neutral integration site(s). In still another example, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies integrated copies of the first and the second heterologous nucleic acid molecules (at one or more neutral integration sites) and in which at least one copy (or both copies) of the native nucleic acid molecule coding for the native Cys4p has been replaced by the first heterologous nucleic acid. In still another example, the first heterologous nucleic acid molecule can be integrated at one or more copies in the genetically modified host cell (at one or more neutral integration site(s)) while the second nucleic acid molecule can replace one or both copies of the nucleic acid molecule coding for the native wild-type Yapl p. The first heterologous nucleic acid molecule can replace one or more copies of the native nucleic acid molecule coding for the native Cys4p and the second heterologous nucleic acid can replace one or more copies of the native nucleic acid molecule coding for the native Yapl p. In still another example, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies integrated copies of the first heterologous nucleic acid molecule (at one or more neutral integration sites) and in which at least one copy (or both copies) of the native nucleic acid molecule coding for the native Cys4p has been replaced by the first heterologous nucleic acid and at least one copy (or both copies) of the native nucleic acid molecule coding for the native Yapl p has been replaced by the second heterologous nucleic acid molecule. In still a further example, the genetically modified host cell (and especially the genetically modified yeast host cell) can have one or more copies integrated copies of the first and of the second heterologous nucleic acid molecule (at one or more neutral integration sites) and in which at least one copy (or both copies) of the native nucleic acid molecule coding for the native Cys4p has been replaced by the first heterologous nucleic acid and at least one copy (or both copies) of the native nucleic acid molecule coding for the native Yapl p has been replaced by the second heterologous nucleic acid molecule. The genetically modified host cell can include further heterologous nucleic acid molecules encoding additional genes for favoring the production and accumulation of glutathione. For example, the genetically modified host cell can include a fourth nucleic acid molecule coding for the Gshl p and/or a fifth nucleic acid molecule coding for the Gsh2p. In yet another example, the genetically modified host cell can include both the fourth nucleic acid molecule coding for the Gshl p and the fifth nucleic acid molecule coding for the Gsh2p. Each of the heterologous nucleic acid molecules of the recombinant host cell can be present in one or more copies in the genetically modified host cell. For example, each of the heterologous nucleic acid molecules can be present in one, two, three or four copies in the genetically modified host cell. The number of copies of each of the heterologous nucleic acid molecule is independently selected.

In an embodiment, the genetically modified host cell comprises at least two and preferably at least four copies of the first heterologous nucleic acid molecule encoding the mutated Cys4p (integrated at a neutral position) and wherein at least one and preferably two copies of the native nucleic acid molecule encoding the wild-type Yapl p has been replaced by the second heterologous nucleic acid molecule encoding the mutated Yap1. In another embodiment, the genetically modified host cell comprises at least two and preferably at least four copies of the first heterologous nucleic acid molecule encoding the mutated Cys4p (integrated at a neutral position) and at least two and preferably at least four copies of the second heterologous nucleic acid molecule coding for the heterologous Glyl p. In such embodiments, it may be necessary to include at least two and preferably four copies of each of the third heterologous nucleic acid molecule coding for the Gshl p and the fourth heterologous nucleic acid molecule coding for the Gsh2p.

In an embodiment, the genetically modified host cell (and particularly the genetically modified yeast host cell) bears (in an integrated form) the second, the third, the fourth and the fifth heterologous nucleic acid molecules. Both copies of the native nucleic acid molecule coding for the native wild-type Yapl p are respectively replaced by the second heterologous nucleic acid molecule. In addition, the second nucleic acid molecule is integrated in multiple copies (preferably four) in the genome of the genetically modified host cell. Further, both the third and the fourth nucleic acid molecules are each integrated in multiple copies (preferably four) in the genome of the genetically modified host cell.

In a further embodiment, the genetically modified host cell (and particularly the genetically modified yeast host cell) bears (in an integrated form) the first, the second, the third, the fourth and the fifth heterologous nucleic acid molecules. Both copies of each of the native nucleic acid molecule coding for the native wild-type Cys4p and the native wild-type Yapl p are respectively replaced by the first and the second heterologous nucleic acid molecule. In addition, the second nucleic acid molecule is integrated in multiple copies (preferably four) in the genome of the genetically modified host cell. Further, both the third and the fourth nucleic acid molecules are each integrated in multiple copies (preferably four) in the genome of the genetically modified host cell.

In an embodiment, the genetically modified host cell does not include an heterologous nucleic acid molecule coding for a wild-type or mutated cystathionine γ-lyase protein (Cys3p), but nevertheless does express a native cystathionine γ-lyase protein (Cys3p).

Process for making genetically modified host cells

The genetically modified host cell can be prepared by using conventional molecular biology tools and teaches. In the context of the present disclosure, the process for making the genetically modified host cell comprises introducing a first heterologous nucleic acid molecule coding for the mutated Cys4p, a second heterologous nucleic acid molecule coding for the mutated Yapl p and/or a third heterologous nucleic acid molecule coding for the heterologous Glyl p. Optionally, the process can also include introducing a fourth heterologous nucleic acid molecule coding for the Gshl p and/or a fifth heterologous nucleic acid molecule coding for the Gsh2p. In an embodiment, the process does not include (e.g. , excludes) the introduction of an heterologous nucleic acid molecule coding for a wild-type or mutated cystathionine γ- lyase protein (Cys3p), but nevertheless does express a native cystathionine γ-lyase protein (Cys3p). Each of the heterologous nucleic acid molecule can be independently integrated or independently replicating in the genetically modified host cell. The term "integrated" as used herein refers to genetic elements that are placed, through molecular biology techniques, into the genome of a host cell. For example, genetic elements can be placed into the chromosomes of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies in the host cell's genome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the host cell's genome. In such embodiment, the heterologous nucleic acid molecule can be stable and self- replicating.

In an embodiment, at least one heterologous nucleic acid molecule is integrated in the genome of the genetically modified host cell. In still another embodiment, all heterologous nucleic acid molecules are integrated in the genome of the genetically modified host cell. Each of the heterologous nucleic acid molecule can either be integrated at a neutral integration site (the same or different sites) or designed to specifically replace the corresponding native nucleic acid molecule encoding the corresponding wild-type protein.

In an embodiment, it is contemplated that one or more heterologous nucleic acid molecules intended to be introduced in the genetically modified host cell be codon optimized, at least partially or entirely, prior to its introduction in the intended recipient host cell.

In an embodiment, when present, the first heterologous nucleic acid molecule coding for the mutated Cys4p is integrated at a neutral integration site. When present in multiple copies (two, three or four copies for example), each of the first heterologous nucleic acid molecules can be integrated at the same or at different neutral integration sites. Alternatively or in combination, the first heterologous nucleic acid molecule can replace one or both copies of the nucleic acid molecule coding for the wild-type Cys4p.

In yet another embodiment, when present, the second heterologous nucleic acid molecule coding for the mutated Yapl p can replace one or preferably both copies of the nucleic acid molecule coding for the wild-type Yapl p in the yeast host cell. Alternatively or in combination, the second heterologous nucleic acid molecule can be integrated at a neutral integration site (which can be the same or different than the integration site(s) for the first heterologous nucleic acid molecule). When present in multiple copies (two, three or four for example), each of the second heterologous nucleic acid molecules can be integrated at the same or different integration sites (which can be the same or different than the integration site(s) for the first heterologous nucleic acid molecule).

In still a further embodiment, when present, the third heterologous nucleic acid molecule (coding for the Glyl p) can be integrated at the same or different neutral integration sites (which can be the same or different than the integration site(s) for the first or second heterologous nucleic acid molecules). In an embodiment, when present, the fourth heterologous nucleic acid molecule (coding for the Gshl p) and the fifth heterologous nucleic acid molecule (coding for the Gsh2p) can be integrated at the same or different neutral integration sites (which can be the same or different than the integration site(s) for the first or second heterologous nucleic acid molecules). In still another embodiment, the process can include integrating one or more copies of the first heterologous nucleic acid molecule coding for the mutated Cys4p at one or more neutral integration sites and replacing one or preferably both copies of the nucleic acid molecule coding for the wild-type Yapl p in the host cell (a yeast host cell for example) by the second heterologous nucleic acid molecule coding for the mutated Yapl p. In such embodiment, the process can also include integrating one or more copies of the third heterologous nucleic acid molecule coding for the threonine aldolase (Glyl p), the fourth heterologous nucleic acid molecule coding for the Gshl p and the fifth nucleic acid molecule coding for the Gsh2p at the same or different neutral integration sites.

The heterologous nucleic acid molecules can be introduced in the host cell using a vector. A "vector," e.g. , a "plasmid", "cosmid" or "YAC" (yeast artificial chromosome) refers to an extra chromosomal element and is usually in the form of a circular double-stranded DNA molecule. Such vectors may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double- stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. When the heterologous nucleic acid molecule is intended to be integrated at a neutral integration site, it may be necessary to include a promoter on the heterologous nucleic acid molecule. In such embodiment, the promoter and the nucleic acid molecule coding for the protein of interest are operatively linked to one another. In the context of the present disclosure, the expressions "operatively linked" or "operatively associated" refers to fact that the promoter is physically associated to the nucleotide acid molecule coding for the heterologous protein in a manner that allows, under certain conditions, for expression of the heterologous protein from the nucleic acid molecule. In an embodiment, the promoter can be located upstream (5') of the nucleic acid sequence coding for the heterologous protein. In the context of the present disclosure, one or more than one promoter can be included in the heterologous nucleic acid molecule. When more than one promoter is included in the heterologous nucleic acid molecule, each of the promoters is operatively linked to the nucleic acid sequence coding for the heterologous protein.

When the heterologous nucleic acid molecule is intended to be integrated at a neutral integration site, it may be necessary to include a terminator on the heterologous nucleic acid molecule. In such embodiment, the terminator and the nucleic acid molecule coding for the protein of interest are operatively linked to one another. In the context of the present disclosure, the expressions "operatively linked" or "operatively associated" refers to fact that the terminator is physically associated to the nucleotide acid molecule coding for the heterologous protein in a manner that allows, under certain conditions, for marking the end of the coding sequence of the heterologous protein. In an embodiment, the terminator can be located upstream (3') of the nucleic acid sequence coding for the heterologous protein. In the context of the present disclosure, one or more than one terminator can be included in the heterologous nucleic acid molecule. When more than one terminator is included in the heterologous nucleic acid molecule, each of the terminators is operatively linked to the nucleic acid sequence coding for the heterologous protein.

Process for making glutathione

The present disclosure also relates to processes for making glutathione based on the use of the genetically modified host cells described herein or obtained by the process describes herein. Generally, the process comprises fermenting a substrate with the genetically modified host cell so as to obtain a fermented mixture comprising glutathione. As used in the context of the present disclosure, the term "substrate" refers to a source of carbon for the host cell that can be used during fermentation. The substrate is preferably in a liquid form and can optionally be supplemented with carbohydrates (glucose for example), a sugar alcohol (glycerol for example), vitamins, minerals, and/and amino acids (cysteine and/or glycine for example). When the genetically modified host cell is a genetically modified yeast host cell, such substrate can be, without limitation, a chemically defined medium or a non-chemically define medium such as molasses (obtained from sugar beet or sugar cane), etc. The fermentation is conducted under circumstances allowing the expression of the heterologous nucleic acid molecule(s) and the accumulation of glutathione. The process includes fermenting the substrate with the genetically modified host cell to obtain a fermented mixture comprising GSH. The term "fermented mixture" refers to the fermented substrate, the genetically modified host cells and the metabolites produced during the fermentation by the genetically modified host cells. The fermented mixture can be further processed by various downstream operations. For example, an extract of the fermented can be obtained, and such extract can be optionally dehydrated or dried. In the context of the present disclosure, in the extract of the fermented mixture, the genetically modified host cells can be inactivated and whole (i.e., the genetically modified host cell is relatively intact) or can be further fractionated (i.e., the genetically modified host cell's is intentionally ruptured). When the genetically modified host cell is a yeast, a yeast extract can be produced from the fermented mixture. For example, a yeast extract can be obtained by hydrolyzing or autolyzing the fermented mixture (chemically, thermally and/or enzymatically) and subsequently separating (isolating) the insoluble from the soluble fraction (corresponding to the yeast extract). The yeast extract consists of the hydrolyzed genetically modified host cells. The fermented mixture can be hydrolyzed to provide a hydrolysate which can be optionally dehydrated or dried. When the genetically modified yeast host cell is a yeast, a yeast hydrolysate can be provided from the fermented mixture. A yeast hydrolysate can be obtained by allowing the lysis of the of the genetically modified yeast cells with their own enzymes. The yeast hydrolysate usually contains both a soluble and a non-soluble fraction. In some embodiments, the process further comprising purifying the genetically modified host cells (such as, for example, the genetically modified yeast host cells) from the fermented mixture. The purified genetically modified host cells can be processed (divided into aliquots, diluted, frozen, filtrated and/or lyophilized) so as to maintain their cellular integrity and allow their subsequent proliferation. Alternatively, the genetically modified host cells (such as, for example, the genetically modified yeast host cells) can be inactivated (e.g. , killed) prior to being processed (divided into aliquots, diluted, frozen, filtrated and/or lyophilized). Optionally, the glutathione can be purified (at least partially and, in some embodiments, completely).

The fermented mixtures or the various products that can be obtained thereof can be advantageously used in many applications, such as, for example, in pharmaceutical applications, in cosmetic applications as well as in animal and human food applications (nutrition, bread making and wine making for example). Glutathione and glutathione- comprising products can be formulated for oral administration, for topical administration or for parenteral administration.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - GENETICALLY MODIFIED SACCHAROMYCES CEREVISIAE HOST CELLS

Gene cassettes (including native promoters and terminators) were amplified by polymerase chain reaction and integrated at the indicated target sites (Table 1) by homologous recombination.

Table 1 - Description of the various strains of Example I

Strain designation Genotype

Parental strain Wild type (not genetically modified)

SIL005 AYLR296W::CYS4 (2 copies)

SIL016 AYLR296W::CYS4 -CYS3 (2 copies of each)

SIL064 ACYS4::CYS4.353

SIL010 AYLR296W::CYS4.353 (2 copies)

AYLR296W::CYS4.353 (2 copies)

SIL019

AFCY1 -GSH1-GSH2 (2 copies each)

AYLR296W::CYS4.353-CYS3 (2 copies of each)

SIL020

AFCY1 -GSH1-GSH2 (2 copies each)

SIL022 AYAP1:: YAP1 ^C626D Strain designation Genotype

AYAP1::YAP1 ^C626D

SIL024

AFCY1::GSH1-GSH2 (2 copies each)

AYLR296W::CYS4.353 (2 copies)

SIL030

AYAP1::YAP1 ^C626D

AYLR296W::CYS4.353 (2 copies)

SIL078 ACYS4::CYS4.353

AYAP1:.YAP1 ^C626D

AYLR296W::CYS4.353 (2 copies)

SIL043 AYAP1::YAP1 ^C626D

AFCY1-GSH1-GSH2 (2 copies each)

AYLR296W::CYS4.353 (2 copies)

SIL070 AYAP1::YAP1 ^C626D

AFCY1::GSH1-GSH2-GSH1-GSH2 (4 copies each)

AYLR296W::CYS4.353 (2 copies)

AYAP1::YAP1 ^C626D

SIL073

AFCY1::GSH1-GSH2-YAP1 ^C626D -GSH1-GSH2 (4

copies of GSH1 &GSH2, 2 copies of YAPI ^C626D )

AYLR296W::CYS4.353 (2 copies)

AYAP1::YAP1 ^C626D

SIL085 ACYS4::CYS4.353

AFCY1::GSH1-GSH2-YAP1 ^C626D -GSH1-GSH2 (4

copies of GSH1 &GSH2, 2 copies of YAPI ^C626D )

AYAP1::YAP1 ^C626D

SIL068 AFCY1::GSH1-GSH2-YAP1 ^C626D -GSH1-GSH2-

_YAP1 C626D _{(4 co p jes Qf eacn)}

AYAP1::YAP1 ^C626D

ACYS4::CYS4.353 (2 copies)

SIL090

AFCY1::GSH1-GSH2-YAP1 ^C626D -GSH1-GSH2-

_YAP1 C626D _{(4 co p jes Qf eacn)}

ACYS4::CYS4.353

AFCY1::pPMA1-GSH1- pPMA1-GSH1 (4 copies)

SIL061

AAPT2::pPMA1-GSH1- pPMA1-GSH1 (4 copies)

ASTR2::pGPM1-GSH2 (2 copies)

ACYS4::CYS4.353

AFCY1::pPMA1-GSH1- pPMA1-GSH1 (4 copies)

SIL143 AAPT2::pPMA1-GSH1- pPMA1-GSH1 (4 copies)

ASTR2::pGPM1-GSH2 (2 copies)

AYOL085C::pGPD1-GLY1- pGPD1-GLY1 (4 copies)

Determination of thiol content. Five milliliter-cultures of strains containing these constructs, alone or combined, were grown for 18 hours in defined medium in shake flasks at 32°C, 200 rpm. Measurement of total thiols using Ellman's reagent was then performed. The strain yielded 1.2 times more thiols upon addition of two copies of CYS4, but the effect was significantly more pronounced when using CYS4.353, with a 1.7-fold thiols content increase. Constitutive expression of Yapl p increased the total thiols content of 1.3-fold, and of 1.8-fold when combined with CYS4.353. Additional strains have been designed which express more copies of a constitutively active Yapl p, more copies of CYS4.353, as well as extra copies of GSH1 and GSH2, three genes positively regulated by Yapl p. GSH1 and GSH2 were added at neutral integrations sites. All genes were under the control of their native promoters and terminators. The resulting total thiols (apparent GSH) increase compared to the parental strain is shown in Figure 1 and indicated in table 2.

Table 2. Total thiols increase of various constructs when compared to the parental strain.

Inactive dry yeast was produced from the strain SIL068 after growth in industrial 20L fermenters, with no addition (commercial cream), with the addition of cysteine only (commercial cream +Cys) or with the addition of both cysteine and glycine (commercial cream +Cys+Gly). To precisely measure the free cysteine, y-GC and true GSH distribution, the thiols were quantified using HPLC, and results are displayed in Figure 2. Interestingly, SIL068 grown in industrial conditions in molasses displays a true GSH content of 1 .6% (total thiols of 1 .9%). Upon addition of cysteine and glycine, the true GSH could be increased up to 4.8% (total thiols of 6.3%).

Additional strains were constructed which contained additional copies of CYS3, along with extra copies of CYS4 (SIL016) or CYS4.353 (SIL020) (Table 1). Such strains did not display increased apparent GSH content compared to those overexpressing solely CYS4 or CYS4.353 (Figure 1 , Table 2).

Inactive dry yeast (IDY) was produced from commercial creams propagated with cysteine or with a cysteine and glycine addition. The IDY was tested for its bread dough relaxant effect in baguettes using a standard no time dough recipe. The relaxing effect is assessed by measuring the baguettes (the higher the dough relaxing effect is, the longer the baguette is).

The SIL068 IDY1 (obtained by making an inactive dry yeast with the SIL068 strain propagated in cysteine and glycine) used for the test was measured at 4.92% true GSH, for a total thiols of 6%. The SIL068 IDY2 (obtained by making an inactive dry yeast with the SIL068 strain propagated in cysteine only) was measured at 1 .9% true GSH, for a total thiols of 5.7%. The amount of IDY used was thus adjusted accordingly to reflect the amount of total thiols from the Fermaid-SR™ commercial product, which specific lot was measured at 17% total thiols. As seen on Figure 3 and in Table 3 below, both SIL068 IDYs have dough relaxing effect proportional to their total thiols content. The dough relaxation performance of the sample seems linked to its apparent GSH content.

Table 3. Measurement of the length of baguettes supplemented with the commercial Fermaid-SR™ additive, the SIL068 IDY1 or the SIL068 IDY2. Results correspond to the mean baguette length for four baguettes (generated from the same dough).

In order to further increase the GSH content in the recombinant host cell, the role of GLY1 was further investigated. Genetically-modified yeast strain SIL061 contains 8 extra copies of GSH1 under the control of a constitutive promoter, 2 extra copies of GSH2 under the control of a constitutive promoter, and 2 copies of CYS4.353 under the control of the endogenous CYS4 promoter. SIL061 was further engineered to contain 4 additional copies of GLY1 , under the control of a constitutive promoter (SIL143). When grown in minimal media with the addition of 1 mM of cysteine, the extra copies of GLY1 resulted in the conversion of more than 60 % of the intermediate γ-GC to GSH (Figure 4).

EXAMPLE II - GENETICALLY MODIFIED PI CHI A PASTORIS HOST CELLS

To determine if the genetic modifications of Example I could be applied to other genus, genetically-modified P. pastoris host cells have been made. Gene cassettes (including native promoters and terminators) were amplified by polymerase chain reaction and integrated at the indicated target sites (Table 2) by homologous recombination.

Table 2 - Description of the various Pichia pastoris strains of Example II

Five milliliter-cultures of P. pastoris, wild-type or genetically modified, were grown for 18 hours in defined medium in shake flasks at 32°C, 200 rpm. Thiols were derivatized using Ellman's reagent and quantified by HPLC, as described in Example I.

The strain with wild-type CYS4 (SIL148) does not show an improvement in total thiol content compared to the wild-type strain. However, as shown in Figure 5, the strain expressing CYS4.340 (SIL 150) has a 1 .58-fold increase in true GSH content. This demonstrates that the mutated CYS4 (coding for Cys4p without its regulatory domain) has a positive effect on the GSH production in the genus Komagataella.

The strain SIL151 , containing an extra copy of YAP1 , shows a 1 .39-fold increase in GSH content compared to the wild-type X-33. The integration of an extra copy of the mutated YAP1 , YAP1 .C414D, in strain SIL153, increased GSH production by 2.07-fold over of X-33, thus significantly more than wild-type YAP1 . Strain SIL153 displayed a 1 .48-fold increase in true GSH content over strain SIL151. This demonstrates that the mutated YAP1 has a positive effect on the GSH production in the genus Komagataella.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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