HIGH YIELDS

DESCRIPTION

The present invention relates to a process for producing glutathione. In particular, the invention relates to a fermentation and a chemical and/or mechanical treatment for producing extracellular glutathione (referred to hereinafter as GSH for the sake of brevity) which enables GSH to be obtained with high yields and at relatively low cost.

GSH, γ-L-glutamyl-L-cysteinylglycine, is a known tripeptide which is present in small quantities in all cells of living organisms. Its many functions are important in various biomedical sectors, including enzymology, pharmacology, toxicology, endocrinology and microbiology.

GSH is one of the most abundant antioxidants present in living organisms.

It is efficient in reducing damage caused by free radicals and other oxidising agents.

GSH is also used as a detoxicant (for example, in paracetamol or heavy metal poisoning) and as a diet integrator for the treatment of, inter alia, diabetes, infertility, cancer, cataracts and human immunodeficiency virus (HIV).

GSH is produced industrially by fermentation processes in which the product is extracted from microbial cells. In order to obtain high GSH yields, it is possible to work A) on increasing the content of intracellular GSH (by means of mutagenesis or suitable cultivation strategies), B) on increasing cell density (by optimising the production process) and C) on optimising the extraction procedure. Methods for increasing the production of GSH by micro-organisms are described in various patents and/or scientific studies.

Li Y. et al. in "Glutathione: a review on biotechnological production", Appl. Microbiol. Biotechnol., 2004, vol. 66, 233-242 summarises the technological advances and results achieved in the field of GSH production over the last thirty years. In particular, it describes both the various culturing and extraction strategies, and the mutagenesis techniques adopted by the various research groups.

WO2004/003217 describes a process for the production of GSH which provides for the cultivation, under suitable conditions promoting GSH production, of a yeast strain which has one or more gene mutations resulting in GSH secretion in the culture medium which is significantly increased compared with the non-mutated parent strain. Up to 20 pmol/l of extracellular GSH are obtained with that process.

Liao X.Y. et al., in "Improved glutathione production by gene expression in Escherichia coli", Lett. Appl. Microbiol., 2006, vol. 43, 211-214, describes how to increase GSH production in Escherichia coli using various genetic constructs containing the GSH biosynthesis genes, gshl and gshll, to the extent that 34.8 mg/g by wet weight of cells are obtained.

Lin J. et al., in "Enhancement of glutathione production in a coupled system of adenosine-deaminase-deficient recombinant Escherichia coli and Saccharomyces cerevisiae", Enzyme and Microbiol. Technology, 2009, 44, 269-273, describes a high-efficiency coupled system for the regeneration of ATP, an energy metabolite necessary for the enzymatic production of GSH. That system, constructed with E. coli and S. cerevisiae, which are both deficient in the adenosine deaminase gene, enables the yield to be improved to up to 2.94 times that obtained with the control. The GSH concentration achieved after 6 hours is 7.82 mM.

The use of engineered micro-organisms overcomes the problem of the low production yields, with the disadvantage that the mutations introduced are often not stable.

EP1391517 describes a method for producing GSH comprising a) the production of a biomass by the pre-culturing under aerobic conditions of a GSH-producer yeast strain in which the GSH content per unit of biomass is > 1.2% w/w; b) the cultivation under aerobic conditions of the resultant biomass until a biomass having a density > 50 g/l is obtained; c) the activation of the biomass obtained in order to trigger GSH production and, finally, d) the recovery of the cultivated biomass, the extraction of the GSH by breaking the cells and the purification thereof.

The GSH _. production yield obtained by the method mentioned above is low when compared with the amount of biomass used. In addition, the extraction of GSH takes place by breaking the cells.

Zhang T. et al., in "Optimization of the medium for glutathione production in Saccharomyces cerevisiae", Process Biochem, 2007, vol. 42, 454-458, describes how to optimise the culture medium in order to increase GSH production yields. The optimised culture medium gives a yield of intracellular GSH equal to 74.6 mg/l.

Udeh K.O. et al., in "High-glutathione containing yeast Saccharomyces cerevisiae: optimization of production", Acta Microbiol Pol, 1997, vol. 46, 105-114, describes how to optimise the composition of the culture medium for the production of GSH using a Saccharomyces cerevisiae strain having a high glutathione content. With the method described, up to 17 mg of GSH/g of yeast dry weight are obtained.

Xiong Z.Q. et al., in "Efficient extraction of intracellular reduced glutathione from fermentation broth of Saccharomyces cerevisiae by ethanol", Bioresour. Technol., 2009, vol. 100, 1011-1014, describes how to extract GSH from the culture medium, without breaking the cells, using ethanol. Extraction with ethanol has many advantages, principally the lesser consumption of energy, the non-destruction of the cells and a lesser protein concentration in the culture broth, all factors which can reduce the complexity and costs of the purification process.

However, that method, if compared with conventional extraction techniques (for example, extraction with boiling water) does not have significant advantages with respect to the GSH production yield.

In recent years, GSH extraction techniques have been substantially optimised, reducing the consumption of solvent, shortening the extraction times and increasing the yields and quality of the extracts.

EP1500944 describes how to obtain high yields of extracellular GSH by the cultivation of the Saccharomyces cerevisiae yeast in a bioreactor which simulates a condition of reduced gravity. With that approach, up to 7% w/dw of extracellular GSH is obtained. The advantage of that method consists both in the high production yields and in the fact that the GSH produced is extracellular and therefore recoverable from the culture medium without breaking the cells.

Although the methods described above offer an increase in GSH production and/or optimisation of the extraction and purification process, they are sometimes complex, in particular, for example, difficult to use industrially, or still produce relatively low yields.

The object of the present invention is therefore to provide a method for the production of GSH which overcomes the limitations explained above with reference to the mentioned prior art.

A further object of the present invention is to provide a simple and efficient method for obtaining high yields of extracellular GSH, avoiding the introduction of gene mutations and/or the breakage of the cells used for the production thereof.

That object is achieved by a method for the production of GSH by means of a fermentation process in accordance with the claims which follow.

In particular, the process for the production of extracellular GSH according to the invention comprises the following stages: cultivating a microorganism in a suitable culture medium, providing the culture with at least one oxidising agent for inducing the production of GSH by that microorganism, administering to the culture at least one modifying agent suitable for promoting the opening of at least one cellular ion channel in the microorganism in order to promote the discharge of GSH.

With the process of the invention, high GSH yields are obtained and the amount of GSH released in the culture medium is > 8% (% by weight of GSH/yeast dry weight).

In addition, with the method of the invention it is possible to recover the GSH from the culture medium without having to destroy the producer micro-organism or even having to purify the GSH from other compounds released as a result of the breakage of that micro-organism, which breakage occurs in numerous methods of the prior art in order to recover the GSH produced therein.

With the process of the invention it is possible to isolate the GSH from the culture medium in a simple and economical manner.

In addition, the production of GSH with the process of the invention can potentially be carried out continuously.

In order to perform the process of the invention, wild-type or also mutated micro-organisms may be used.

In a preferred embodiment, the invention provides for the use of at least one yeast strain as the producer micro-organism.

According to a more preferred embodiment of the invention, it is proposed to use the Saccharomyces cerevisiae yeast strain, identified as L5267 (Lesaffre private collection). The morphological, sexual and nutritional characteristics of the strain used are in conformity with the typical characteristics of the species of yeast used, as described, for example, by J. A. Barnet et al. in "YEAST: Characteristics and identification", (Third ed. Cambridge University, 2000).

According to a further preferred embodiment, the use of Candida utilis yeast strains is provided for.

In order to perform the process of the invention, haploid or also polyploid yeasts may be used.

In other versions of the invention, it may be proposed to use, as the GSH- producer micro-organism, aquatic algae strains (e.g. marine diatoms) for the decontamination of water polluted by metals (such as those described in the article by Kawakami S.K. et al. BioMetals, 2006,19,51-60).

In other versions, it may be proposed to use, as producer organism, microbial cells which are in plankton form or which are organised in a biofilm.

According to a different preferred embodiment, a bacterial strain, even more preferably comprising Escherichia coli, is used as the producer microorganism. It is known from the literature that that strain is capable of producing GSH.

In order to carry out the process of the invention, the culture medium may be any liquid or solid culture medium suitable for ensuring cell vitality.

According to the invention, modifying agents are used to induce directly and/or indirectly the opening of at least some of the ion channels of the micro-organism in order to promote the discharge of GSH.

Preferably, in the process of the invention, modifying agents suitable for causing the opening of the chlorine channels of the micro-organism are used.

More preferably, modifying agents suitable for causing the opening of the bromine and/or potassium and/or sodium and/or calcium channels may be used.

The modifying agents used in the invention to open one or more ion channels of the cells present in the culture medium may be of the chemical and/or mechanical type.

In a preferred embodiment of the invention, a modifying agent such as to cause the disorganisation/depolarisation/depolymerisation of components of the cytoskeleton of the micro-organism is provided. The treatment produces changes in the cell cytoskeleton such as to cause directly and/or indirectly the opening of at least one of the ion channels with the consequent discharge of GSH from the micro-organism, after its production therein has been stimulated.

In some preferred embodiments, it is proposed to provide modifying agents suitable for acting directly or indirectly on the actin of the micro-organism. The modifying agent may be a chemical compound (for example of the class of the cytochalasins) which, by acting directly on the actin, alters the monomer/polymer balance, depolymerising the actin fibres, causing the opening of at least some of the cellular ion channels and consequently the discharge of GSH . It is in fact known that cytochalasins are also activators of cellular ion channels.

In a preferred version, it is proposed to use dihydrocytochalasin B (DHCB), a saturated derivative of cytochalasin B, which shortens the actin filaments with the advantage of not interfering with the transport of the sugars.

In other versions of the invention, chemical agents that are indirect actin disorganisers/depolarisers/depolymerisers may be used.

Those compounds, mediating a broad spectrum of cell responses which involve the cytoskeleton, alter indirectly the monomer/polymer balance of actin, thus causing the opening of the ion channels and, consequently, the discharge of GSH from the cells.

Examples of indirect actin depolarisers/depolymerisers are inhibitors of Rho- kinase (ROCK). Rho-kinase is a target serine-threonine kinase of Rho. It is an important regulator of the cytoskeleton which mediates the effects of RhoA (Ras homolog gene family, member A) on the stabilisation of actin fibres, the contraction of the smooth musculature, cell adhesion, formation of protrusions on the membrane and cell motility.

Of the various compounds of the above-mentioned type, there may be used, for example, the chemical agent Y-27632, (R)-(+)-tfans-4-(l- aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate.

In a further different preferred embodiment of the process of the invention, in order to promote the discharge of GSH from the micro-organism, a mechanical agent, such as, for example, hypergravity, may be used.

A variation in the external/internal cell pressure such as to modify the osmotic balance in fact alters the structure of the cytoskeleton, causing the opening of at least some of the ion channels and promoting the discharge of GSH. In order to obtain hypergravity, it is possible, for example, to subject the micro-organism to centrifugation.

In the process of the invention, in order to promote the production of GSH in the producer micro-organism, it is proposed to provide a stress-inducing agent, such as, for example, oxygen or another oxidising agent.

Preferably, an amount of oxygen such as to generate moderate oxidative stress without damaging the cells is provided. More preferably, the amount of oxygen is > 4%. The oxidative stress is generated, for example, by bubbling molecular oxygen through the culture medium or by surface gaseous exchange.

The process of the invention can be carried out in batch, fed-batch or continuously.

The process of the invention permits a great increase in the production of GSH on the part of the micro-organism and the discharge thereof in order to render it available and separable from the culture medium without breaking the cells.

Finally, the process of the invention may also comprise a stage in which it is proposed to extract and collect the GSH produced in order to render it available for subsequent use.

The stage of extracting the GSH from the culture medium may be carried out, for example, by precipitating the corresponding salt in the presence of sulphuric acid (H ₂ S0 ₄ ) in which the above-mentioned salt, after salification of the sulphuric acid, becomes insoluble.

The procedure of the invention provides a simple, efficient and potentially more economical method for producing extracellular GSH, enabling the production of GSH to be increased and at the same time enabling the GSH to be recovered from the culture medium in a simple manner.

The following examples illustrate the features and advantages of the invention which are described in more detail by way of non-limiting example with reference to the appended drawings in which :

- Figure 1 is a graph showing the extracellular GSH produced by centrifugation (hypergravity achieved equal to lOg) in accordance with the method of the present invention compared with a control culture and a further culture to which NPPB has been added (Example 1);

- Figure 2 is a graph showing the extracellular GSH produced by the addition of DHCB in accordance with the method of the present invention compared with a control culture and a further culture to which NPPB has been added (Example 2); - Figure 3 is a graph showing the extracellular GSH produced by the addition of Y-27632 in accordance with the method of the present invention compared with a control culture and a further culture to which NPPB has been added (Example 3).

EXAMPLE 1

A Saccharomyces cerevisiae strain, in particular the polyploid homothallic strain L5267 (Lesaffre private collection), was used.

The yeast was suspended at a concentration of 2.5% dry weight in an aqueous culture medium having the following composition : 3.5 g/l of KH ₂ P0 ₄ , 0.37 g/l of KOH, 7 g/l of (NH ₄ )2S0 ₄ , 0.5 g/l of MgS0 ₄ , 10 g/l of Na ₃ C ₃ l-l ₅ 0(COO)3, 4 g/l of cysteine, 4 g/l of glycine, 40 g/l of glucose.

The yeast was cultivated at 28 °C for 24 hours (fermentation time).

Hypergravity obtained by subjecting the cells to centrifugation of 10 g using MidiCAR (Medium Sized Centrifuge for Acceleration Research, DESC, Amsterdam, The Netherlands) was used as the mechanical actin- disorganising/depolarising/depolymerising agent.

The culture medium is kept oxidised throughout the entire fermentation period in a saturated oxygen environment.

The yeast is metabolically active throughout the entire fermentation period, as confirmed by the consumption of extracellular glucose and by the concomitant production of ethanol and glycerol. The content of intracellular trehalose is almost constant.

When fermentation is complete (after 24 hours) the yeast cells are separated from the culture medium and the concentration of intra- and extracellular glutathione produced is measured by high performance liquid chromatography (HPLC).

The GSH concentration is determined in accordance with the method reported by Lakritz et al. Anal. Biochem. 1997, 247, 63-68. In particular, the samples are analysed by reverse phase HPLC using a 5 pm Purospher® RP-18 endcapped chromatography column (Merck) and a spectrophotometric detector (λ=210 nm). The solvent used contains NaH ₂ P0 ₄ 25mM. The pH, obtained by using trifluoroacetic acid 1M, is 3.5. The flow, optimised, is 0.6 ml/min.

The identification and measurement of the GSH concentration are effected using a standard GSH solution (Sigma).

Alternatively, the GSH is identified by NMR spectroscopy and its concentration is determined by integrating the relative peaks with respect to an internal standard at known concentration (benzene d6, C ₅ D ₆ ).

The process of the invention permits the production of high yields of extracellular GSH. In particular, with the process described, «8% of extracellular GSH is obtained (% by weight of GSH on the yeast dry weight). The concentration of intracellular GSH remains constant in the range of 1- 1.4%.

The addition to the culture medium of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which inhibits the opening of the chlorine channel, at a concentration of 10 ^"5 M, inhibits the discharge of GSH mentioned above. The concentration of intracellular GSH remains constant in the range of 1- 1.4%.

The appended Figure 1 shows a histogram featuring three different concentrations of extracellular GSH produced in three different experiments: - as described in Example 1 above;

- as described in Example 1 without subjecting the micro-organism to centrifugation;

- as described in Example 1 and also adding NPPB to the culture medium as described above.

As will be appreciated, centrifugation substantially increases the production of extracellular GSH compared with the situation in a similar process in which centrifugation does not take place, while the introduction of NPPB, blocking the opening of the cellular ion channels (in particular the chlorine channel), prevents the discharge of GSH, causing the substantial absence of extracellular GSH even when centrifugation is applied.

EXAMPLE 2

A Saccharomyces cerevisiae strain, in particular a polyploid homothallic strain, the same strain as that used in Example 1, was utilised.

A yeast suspension was generated until a concentration equal to 2.5% dry weight was obtained in the culture medium.

The culture medium selected has the same composition as the culture medium used in Example 1.

The strain was cultivated at 28 °C for 24 hours.

Throughout the entire fermentation period, the yeast cells are treated with dihydrocytochalasin B (DHCB), a saturated derivative of cytochalasin B, which depolymerises actin and blocks monomer addition at the growth end of the polymer, and does not interfere with the transport of the sugars.

The dihydrocytochalasin B is added to the culture medium at a concentration of 3 Μ. The culture medium is kept oxidised throughout the entire fermentation period in a saturated oxygen environment.

The yeast is metabolically active throughout the entire fermentation period, as confirmed by the consumption of extracellular glucose and the concomitant production of ethanol and glycerol.

The dihydrocytochalasin B brings about both the depolymerisation of actin and the activation of the ion channels, promoting the discharge of the GSH produced.

When fermentation is complete, the yeast is separated from the culture medium and the concentration of intra- and extracellular GSH produced is measured by HPLC analysis as indicated in Example 1.

Alternatively, the GSH is identified by NMR spectroscopy and its concentration is determined as indicated in Example 1.

The results are confirmed using the Glutathione Assay kit (Sigma).

The process of the invention enables high yields of extracellular GSH to be obtained.

In particular, ^■ « 8% of extracellular GSH (% by weight of GSH on yeast dry weight) is obtained with the process described.

The concentration of intracellular GSH remains constant in the range 1- 1.4%.

The addition to the culture medium of NPPB, which inhibits the opening of the chlorine channel, at a concentration of 10 ^"5 M (as in Example 1) inhibits the discharge of GSH mentioned above, as shown in Figure 2. The concentration of intracellular GSH remains constant in the range of 1-1.4%. The appended Figure 2 shows a histogram featuring three different concentrations of extracellular GSH produced in three different experiments:

- as described in Example 2 above;

- as described in Example 2 without the addition of DHCB;

- as described in Example 2 and also adding NPPB to the culture medium (not DHCB only) as described above.

As will be appreciated, the addition of DHCB substantially increases the production of extracellular GSH compared with the situation in a similar process in which that addition is not made, while the introduction of NPPB, blocking the opening of the cellular ion channels (in particular the chlorine channel), prevents the discharge of GSH, causing the substantial absence of extracellular GSH even when DHCB is present.

EXAMPLE 3

A Saccharomyces cerevisiae strain, in particular a polyploid homothallic strain, the same strain as that used in Examples 1 and 2, was utilised.

A yeast suspension was generated until a yeast concentration equal to 2.5% dry weight is obtained in the culture medium having the same composition as that used in Examples 1 and 2.

The strain was cultivated at 28 °C for 24 hours.

Throughout the entire fermentation period, the yeast cells are subjected to the action of the compound ( ?)-(+)-trans-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a very potent and selective non-toxic ROCK inhibitor capable of permeating the cells, which brings about actin depolymerisation.

Y-27632 is added to the culture medium at a concentration of 10 ^"4 M.

The culture medium is kept oxidised throughout the entire fermentation period (24 hours) in a saturated oxygen environment.

The yeast is metabolically active throughout the entire fermentation period, as confirmed by the consumption of extracellular glucose and the concomitant production of ethanol and glycerol.

When fermentation is complete, the yeast is separated from the culture medium and the concentration of intra- and extracellular GSH produced is measured by HPLC analysis, as in Examples 1 and 2.

Alternatively, the GSH is identified by NMR spectroscopy and its concentration is determined as indicated in Examples 1 and 2.

The results are confirmed using the Glutathione Assay kit (Sigma).

The process of the invention enables high yields of extracellular GSH to be obtained. In particular, ~ 12.5% of extracellular GSH (% by weight of GSH on yeast dry weight) is obtained with the process described. The concentration of intracellular GSH remains constant in the range of 1-1.4%. The addition to the culture medium of NPPB, at a concentration of 10 ^"5 M (as in Examples 1 and 2), inhibits the discharge of GSH mentioned above. The concentration of intracellular GSH remains constant in the range of 1- 1.4%.

The appended Figure 3 shows a histogram featuring three different concentrations of extracellular GSH produced in three different experiments:

- as described in Example 3 above;

- as described in Example 3 without the addition of Y-27632;

- as described in Example 3 and also adding NPPB to the culture medium (not Y-27632 only) as described above.

As will be appreciated, the addition of Y-27632 substantially increases the production of extracellular GSH compared with a similar process in which that addition is not made, while the introduction of NPPB, blocking the opening of the cellular ion channels (in particular the. chlorine channel), prevents the discharge of GSH, causing the substantial absence of extracellular GSH even when Y-27632 is present.

The data shown in all three Examples demonstrate that, by treating the cells in accordance with the process of the invention, production yields of extracellular GSH which are much higher than those obtainable with the techniques already known are obtained, without having to resort to mutations and/or breakage of the cells.

In addition, with the process of the invention, it is possible to obtain extracellular GSH without destroying the cells of the culture.