WO1996041888A1

WO1996041888A1 - Yeast strains having a modified alcoholic sugar fermentation balance, uses thereof, and vectors for producing said strains

Info

Publication number: WO1996041888A1
Application number: PCT/FR1996/000854
Authority: WO
Inventors: Sylvie Dequin; Pierre Barre
Original assignee: Institut National De La Recherche Agronomique - Inra
Priority date: 1995-06-09
Filing date: 1996-06-06
Publication date: 1996-12-27
Also published as: AU6229796A; FR2735145A1; FR2735145B1

Abstract

Yeast strains having a modified alcoholic sugar fermentation balance, in particular as far as the production of glycerol and ethanol is concerned, and vectors or sequences for producing said strains, are disclosed. Said yeast strains are selected from Saccharomyces strains and contain at least one fragment of a gene coding for an NADH-dependent glycerol-3-phosphate dehydrogenase (GPDH), said fragment being modified by regulating sequences active in yeast comprising a strong promoter, for increasing the production of glycerol and reducing that of ethanol, in a sugar-rich medium during alcoholic fermentation.

Description

YEAST STRAINS HAVING A MODIFIED ALCOHOLIC FERMENTATION BALANCE FOR SUGARS AND THEIR APPLICATIONS, VECTORS FOR USE IN OBTAINING SUCH STRAINS.

The present invention relates to yeast strains having a modified alcoholic fermentation balance for sugars, in particular in this regard, concerns the production of glycerol and ethanol.

The present invention also relates to vectors or sequences which can be used for obtaining said strains.

Glycerol is an extremely important compound in industrial alcoholic fermentations carried out by yeasts, in particular because it is the main fermentation byproduct after ethanol and CO2 •

The production of glycerol, carried out in the cytoplasm, begins with the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, by a glyce¬ rol 3 phosphate dehydrogenase NADH-dependent (GPDH, EC 1.1.1.8). The enzyme is strongly inhibited by high concentrations of ethanol (NAGODA ITHANA et al. J. Am. Soc. Brew. Chem. 39, p. 179-183, 1977), solutions of high ionic strength as well as by different metabolites and cofactors [NADER et al., Biochem. Biophys. Acta, 571, p. 177-185, (1979)]. It is probably activated by one or more effectors [GANCEDO et al., European Biocem. , 5, p. 165-172, (1968)]. Its molecular weight is 42 kDa [NILSSON and ADLER, Biochem. Biophys. Acta, 1034, p. 180-185, (1990)]; the active form, on the other hand, has a molecular weight of 68 kDa, which would seem to show that the enzyme is active in the form of a di [ALBERTYN et al., FEBS, 308, p. 130-132, (1992)]. A gene, called GPD1, responsible for the synthesis of the enzyme has recently been cloned and characterized [LARSSON et al., Mol. Microbiol. 10, p. 1101-1111, (1993); ALBERTYN et al., Mol. Cell. Biol. 14, p. 4135- 4144, (1994)], but its disruption does not lead to a total loss of GPDH activity, this mistletoe leads to a maintenance of the production of glycerol, including in anaerobiosis, which supposes the existence of a second gene, GPD2, coding for an isoenzyme.

The second step consists in the dephosphorylation of glycerol-3-phosphate to glycerol by α-glycerophosphatase (glycerol 3-phosphatase or G3Pase, EC

3.1.3.21), stereospecific of L-glycerol-3-phosphate. This step is not considered to be limiting.

Whereas glycerol is produced in the cytoplasm, the enzymes involved in its assimilation are, on the other hand, localized in the itochondria: gly¬ cerol kinase coded by GUT1 and a GPDH flavin adenine dinucleotide (FAD) -dependent coded by GUT2.

The GPD1 gene [LARSSON et al., 1993 and ALBERTYN et al., 1994, cited above], obtained from the yeast Saccharomyces cerevisiae comprises 1176 bp and code for the above-mentioned GPDH (or GPDlp), protein of 391 amino acids. Glycerol is synthesized in yeast in response to two different environmental conditions:

- Osmotic shock:

Glycerol production is increased in response to this stress. The glycerol serves as a solute allowing the intracellular osmotic pressure to be adjusted as a function of that of the medium. During osmotic stress, the synthesis of GPDlp (GPDH expressed by the GPD1 gene) is significantly increased.

- Anaerobiosis and in particular the redox state of the cell:

The formation of glycerol during alcoholic fermentation is a means for the cell to balance its oxidation-reduction balance (imbalanced balance notably during cell growth), by providing a complementary electron acceptor system ( figure 1) . The study of osmosensitive mutants osgl-1, (LARSSON et al., 1993, cited above), shows that they are incapable of growing in an environment with a low potential of water and exhibit both a significant decrease in glycerol production and GPDH activity.

ALBERTYN et al., 1994, cited above, also sequenced the same GDPl gene and more particularly studied the regulation of the expression of GDPlp in S. cerevisiae by the HOG metabolic pathway (= High-Osmolari ty Glycerol response). The preeminent physiological effect, in yeasts having undergone an osmotic shock, is the intracellular increase in the production of compatible solutes to counterbalance the osmotic pressure (essentially production of glycerol). The GDPl gene and a high rate of glycerol production are essential for the growth of yeasts, in the presence of an osmotic shock.

To study the regulation of glycerol production, these Authors carried out studies of overexpression and deletion of the GPDl gene. Overexpression is achieved by subcloning the GPDl gene into a multicopy YEpIaclδl (= YEpGPDl) plasmid. The transformants (laboratory yeasts) carrying the multicopy plasmid YEpGPDl have a dependent GPDH-NADH enzymatic activity, greater by a factor of 5 to 7, compared to the enzymatic activity of the non-transformed cells; however, such transformants, although they overproduce the enzyme (GPDlp), do not have higher levels of glycerol, in a culture medium containing glucose, whereas when galactose is used in place of glucose, the same transformants produce about 3 to 5 times more glycerol than untransformed cells. However, the study of osmotic shock on these transformed cells, led the Authors of this article to specify that the cells overproducing GPDH do not appear to be more resistant to osmotic shock than wild cells, this undoubtedly being linked to the fact that they do not produce more gly¬ cerol during an alcoholic fermentation. Modulation of glycerol production

(increase or decrease) and of the glycerol / ethanol ratio, during the alcoholic fermentation, is of interest in the food industry: oenology, brewery, distillery, bakery, cider house. In oenology, glycerol is an important compound with regard to the organoleptic qualities of wine. It brings in particular a property of "softness", and a light sweet taste. The average concentration of glycerol in wines is 5 to 12 g / 1; the variability observed depends on different parameters, such as the composition of the medium and the growth conditions, and also the nature of the strain used.

Obtaining higher glycol contents has two major advantages: 1) an improvement in the organoleptic qualities of the product, and

2) a decrease in the ethanol content. In fact, the glycerol normally produced during alcoholic fermentation is the result of the functioning of a complementary electron acceptor system, intended to balance the general oxidation-reduction balance of glycolysis. The predominant role of glycerol in anaerobic conditions is therefore the reoxidation of NADH (Figure 1).

Glycerol production Amplification will on the one hand limit the carbon flux normally uti¬ Lisé to the production of pyruvate and ethanol, and on the other hand, cause an imbalance of the balance of redox ^1, using increased pool of NADH. This imbalance can be compensated by a limitation of the main pathway involved in the regeneration of the NAD (ethanol synthesis route) at the level of the acetaldehyde to ethanol conversion.

This is why the Applicant has set itself the goal of providing transformed yeast strains which better meet the needs of the practice than the transformed strains of the prior art, in particular in that they effectively allow the amplification of glycerol production with a concomitant modification of the glycerol / ethanol ratio. The present invention relates to yeast strains, characterized in that they are selected from Saccharomyces strains, in that they contain at least one fragment of a gene coding for a glycerol 3 phosphate dehydrogenase NADH- dependent (GPDH), which fragment is modified by active regulatory sequences in yeast containing a strong promoter, to increase the production of glycerol and reduce the production of ethanol, in a medium rich in sugars during alcoholic fermentation. For the purposes of the present invention, the term “medium rich in sugars” is understood to mean a medium containing at least 30 g / 1 of sugars (3%) (glucose, fructose, molasses, for example), preferably between 60 g / 1 ( 6%) and 250 g / 1 (25%). In accordance with the invention, said yeast strains produce, when sown in a fermentation medium comprising more than 30 g / l of sugars, approximately 2 to 5 times more glycerol with a concomitant reduction in the level of ethanol d '' at least 4 g / 1, compared to untransformed yeast cells and cultivated in a fermentation medium comprising less than 30 g / 1 of sugars.

In addition, the initial glycerol / glucose ratio is significantly different for the transformed strains (between 0.07 and 0.15) according to the invention compared to the untransformed strains (0.03-0.04). According to the invention, said gene coding for a GPDH is selected from the group consisting of the GPD1 gene, the GPD2 gene or any homologous gene capable of coding for a GPDH. Said Saccharomyces strains are preferably industrial strains, in particular oenological, distillery, brewery, bakery or cider maker strains.

According to a preferred embodiment of the invention, said yeasts belong to the species Saccharomyces cerevisiae.

By way of example, mention may be made of the strain ScV5M, derived from an industrial oenological strain.

Advantageously, the invention provides, for obtaining strains capable of increased production of glycerol and a reduction in the production of ethanol, on a medium rich in sugars, the following construction:

Said Saccharomyces strains are characterized in that they contain at least two copies of the gene coding for a NADH-dependent GPDH (GPDl or GPD2), under the control of sequences regulating the expression of said gene in yeast.

By "sequence regulating the expression of a gene" is meant promoter and terminator type sequences active in yeast. Promoters and terminators of different genes can be used, associated with different combinations. Preferably, promoters and terminators of a gene other than the GPD1 gene will be used. Mention will be made, by way of nonlimiting example, of the promoters and terminators, known per se, of the alcohol dehydrogenase I (ADHI), phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes.

The invention also comprises the expression cassette obtained by combining said regulatory sequences and the gene coding for a GPDH NADH- dependent (GPDl for example); this cassette can be carried by a plasmid, or integrated into the chromosomal DNA of the host yeast.

The invention also encompasses expression vectors, comprising an expression cassette as defined above, and which can be used for obtaining transformed yeast strains in accordance with the invention.

These vectors can be selected on the basis of the nature and the strength of the regulatory elements which enter the expression cassette. The promoters and terminators described above can be chosen, or any other sequence making it possible to control the expression of a gene in yeast. Another criterion for the choice of vectors resides in the number of copies thereof, which is conditioned by the choice of the origin of replication.

In accordance with the invention, the GPDl gene, associated with control sequences, is carried by a replicative plasmid with a high number of copies, having as origin of replication in yeast: part of the endogenous 2μ plasmid, an ARS chromosomal sequence or telomeric sequences.

Preferably, the vectors in accordance with the invention also comprise markers for selection in yeast, such as markers for autotrophy: URA3, LEU2, HIS3, TRP ^, ADE etc ... and / or antibiotic resistance markers (G318, hygro-mycin B, chloramphenicol, pleomycin), herbicides (sulfometuron methyl), copper ...

Advantageously, the vectors according to the invention are shuttle vectors, also having an origin of bacterial replication and a selection marker in a bacterium (for example, gene for resistance to an antibiotic). The plasmids according to the invention, carrying the gene coding for the yeast-dependent GPDH-NADH can be introduced into any yeast strain, by different transformation techniques. The most common transformation techniques include the protoplast technique, the permeabilization technique with lithium salts and electroporation.

Surprisingly, the Saccharo-myces yeasts, transformed by a plasmid with a high number of copies, in which the gene coding for a GPDH (GPD1 gene or GPD2 gene) is associated with regulatory sequences of said gene, have an increased production of glycerol and a reduction in the production of ethanol on a medium rich in sugars, that is to say comprising at least 30 g of sugars / l; by way of example, transformed yeasts in accordance with the invention allow the multicopy expression of the GPD1 gene coding for a GPDH, under the control of the strong ADHI promoter (clone SIM7, for example) and thus a significantly increased production of glycerol during alcoholic fermentation in a medium containing 30 g / l of sugars or more, whereas the multicopy expression of this gene, in a medium containing only 20 g / l of sugars, as described in ALBERTYN et al. (1994), that is to say under the control of its own promoter, in laboratory yeast had hitherto failed to observe an increase in the production of glycerol.

The method for constructing strains according to the invention overexpressing GPDH and consequently overproducing glycerol, on a medium rich in sugars (alcoholic fermentation) comprises the following steps: construction of an expression cassette comprising the coding gene for a GPDH (GPD1 gene or GPD2 gene) and strong regulatory elements; - introduction of this cassette in multiple copies in a yeast.

The glycerol overproducing yeast strains in accordance with the invention find numerous applications in the food industry. They can be used wherever alcoholic fermentation must be accompanied by both a decrease in the etha¬ nol rate and a decrease in the sugar rate. This is particularly interesting in the field of the production of fermented drinks with a low ethanol content, whether wines or more recent products such as "wine-based cocktails". In the latter category, for example, the sugar content must not exceed 80 g / l, and the final degree of alcohol 4 ° C, which presupposes the use of musts with a high sugar content of 150 g / 1 maximum, which is rarely the case. Other models such as sparkling grapes are also affected.

More generally, there has been, for a few years, a confirmed trend of consumption for products less rich in sugar but also in ethanol, while retaining the sweet taste.

However, the physical processes capable of being implemented for this purpose: reverse osmosis, evaporation under vacuum, present in this context, limits, insofar as these techniques, under development, are expensive and often affect the quality products. For these reasons, the yeast strains, in accordance with the invention, capable of degrading more sugar by producing less alcohol in favor of glycerol (sweet flavor) have indisputable advantages. In addition, the glycerol overproductive yeasts, in accordance with the invention, have the advantage, when implemented, of not requiring any particular investment. By getting rid of traditional metabolisms We can obtain original products within the fermented drinks sector.

The bakery sector is also affected; but in this case, it is the increase in intracellular production of glycerol which is sought after; such yeasts have, in fact, greater osmotolerance and increased resistance to freezing and drying.

The present invention also relates to an alcoholic fermentation process with a balance of fermen¬ tation of modified sugars, characterized in that to increase the production of glycerol and reduce the production of ethanol, said process comprises:

(1) the transformation of a Saccharomyces yeast strain by a vector overexpressing the protein

GPDH; and

(2) seeding the fermentation medium, containing at least 30 g / 1 of sugars, with the transformed yeast strain (transformant), obtained in (1). In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention, as well than in the appended drawings, in which: FIG. 1 represents the oxidation-reduction balance during alcoholic fermentation, FIG. 2 represents the sequence of oligonucleotides 01 and 02 and their respective positions in the GPD1 gene,

FIG. 3 illustrates the cloning of the GPD1 gene in the plasmid pGEM-T,

- Figure 4 represents the cloning of the GPDl gene in the plasmid pVTlOO-U, - Figure 5 represents the evolution of the cell population according to the progress of reaction in V5p (control strain ScV5M transformed with the plasmid pVTlOO-U) (O) and SIM7 (strain ScV5M transformed with the plasmid pVTlOO-U-GPDl) (•),

- Figure 6 represents the production of glycerol according to the progress of reaction in V5p

(O) and SIM7 (•), FIG. 7 represents the evolution of the specific GPDH activity in V5p (O) and SIM7 (•) as a function of the reaction progress, - FIG. 8 represents the immunodetection of the GPDlp protein in SIM7. The protein fractions are obtained at different moments of the reaction progress. Each protein deposit contains the same amount of protein (10 μg), - Figure 9 represents the evolution of the cell population in V5p (O) and SIM7 (•), as a function of time (μ (V5p) = μ (SIM7) = 0.54 gen / h),

- Figure 10 shows the evolution of the remaining glucose concentration in the medium in V5p (O) and SIM7 (•), as a function of time (fermentation on YNB + 5 g / 1 of casamino acids pH 3.4, 28 ° C with [glucose] = 100 g / 1),

FIG. 11 represents the evolution of the production of acetaldehyde as a function of the progress of the reaction in V5p (O) and SIM7 (•),

FIG. 12 represents the oligonucleotides used to isolate the TEF1-ZEO gene-CYC1 terminator promoter cassette, from the vector pVT332,

FIG. 13 represents the recombinant plasmid pVTlOOU-ZEO-GPDl,

FIG. 14 illustrates the evolution of the production of the metabolites produced respectively by the K ^ pVTlOOU-ZEO strains (control strains, also referred to below as K ^ pVTU) (S) and the transformed strains mées ./PVT100U-ZEO-GPD1 (also called K _x - GPD1 below) (O).

It should be understood, however, that these examples are given by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation. EXAMPLE 1: CLONING OF GPDl.

The cloning of GPDl was carried out by PCR amplification, from total DNA of the strain ScV5M, using the two oligonucleotides 01 and 02 (FIG. 2), chosen from the sequence of GPDl [LARSSON and al., (1993)]. This strain was deposited on June 18, 1992 with the National Collection of Cultures of ^' Micro¬ organisms, held by the INSTITUT PASTEUR, under the number 1-2222. It is a haploid strain, ura-, MAT a, derived from an oenological strain

1) Extraction of Enomicrue DNA from ScV5M The strain ScV5M was cultured in YEPD medium, the composition of which is as follows: yeast extract 10 g / 1, peptone 20 g / 1, glucose 20 g / 1. 10 ml of YEPD medium are seeded with ScV5M and incubated overnight at 28 ° C. 50 ml of the same medium are inoculated at an OD (660 nm) of 0.05, and incubated at 28 ° C, until an OD (660 nm) of 4 to 6 is reached. The cells are recovered by centrifugation for 10 minutes at 1000 g and the DNA extracted according to the method described by VAN SOLIGEN and VAN DER PLAAT, J. Bacteriol., 130, p. 946-947, (1977), with some modifications: The cell pellet is taken up in a buffer

SCE pH 7 (1.2 M sorbitol, 0.1 M Na3 citrate, 60 mM EDTA), with 5 μl / ml of β-mercaptoethanol, and incubated for 15 minutes at room temperature.

- 5 mg / ml of zymolyase 20000 are added to the cell suspension, which is placed at 37 ° C for 45 minutes, until a protoplast level greater than 90% is obtained.

- The protoplasts are recovered by centrifugation for 5 minutes at 350 g, then taken up in 1 ml of lysis buffer (0.2 M Tris-HCl pH 8, 0.1 M EDTA, 1.5% sarkosyl) supplemented with 100 μl of proteinase K (20 mg / ml). The solution is incubated for 1 hour at 50 ° C.

- The suspension is centrifuged for 30 minutes at 15,000 g, and the lysate recovered. The DNA is precipitated with 0.05 volume of 3M sodium acetate pH 4.8 and 2 volumes of ethanol. The precipitate is recovered using a glass rod and dissolved in 500 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8). The concentration of DNA in solution is determined by measuring the OD at 260 nm. 2) Amplification of GPDl by PCR

From the GPDl sequence [LARSSON et al., Mol. Microbiol., 10, p. 1101-1111, (1993)] two oligonucleotides 01 and 02 were synthesized. 01 is chosen before the translation initiation codon. 02 is located after the stop codon. At the 5 ′ ends of each oligonucleotide, additional bases have been added, so as to introduce a Xhol restriction site for 01 and BamHI for 02. The sequence of the oligonucleotides is indicated in FIG. 2. The two primers were used to amplify GPDl, from the total DNA isolated from ScV5M, under the following conditions:

Primer 01 5 μl (20 pmoles)

Primer 02 5 μl (20 pmol) TaQ 10X buffer (PROMEGA) 10 μl

TaQ (PROMEGA, 5 U / μl) 0.5 μl dNTP (2 mM) 10 μl (100 ng)

MgCl. (25 mM) 6 μl

H ₂ 0 qs 100 μl Amplification conditions: 30 seconds 94 ° C, 30 seconds 39 ° C, 1 minute 72 ° C for 30 cycles, on Perkin-Elmer Cetus model 9600 amplification.

The amplification product is analyzed on 0.8% agarose gel. A single band of about 1300 base pairs is observed, in agreement with the theoretical size of 1260 bp.

3) Identification of the amplified fragment

In order to verify that the cloned fragment corresponds to GPD1, the PCR product was subcloned into the vector PGEM-T (PROMEGA), in order to partially determine its sequence. For this, the amplified product was precipitated by adding to the amplification mixture 0.25 volumes of ammonium acetate ION and 2.5 volumes of ethanol. After 15 minutes at -20 ° C, the suspension is centrifuged for 15 minutes at 15000g. The supernatant is removed and the pellet washed under the same conditions with 1% ethanol.

After drying, the DNA is taken up in 50 μl of TE buffer.

100 ng of amplified DNA are ligated to 50 ng of vector pGEM-T in a final reaction mixture of

10 μl, in the presence of 2 μl of 5X buffer (GIBCO BRL) and 1 unit of T4 DNA ligase (GIBCO BRL), overnight at 14 ° C. The E. coli DH5α (GIBCO BRL) strain of genotype:

F-; endAl; hsdR17 (K-, m_K-); supE44; Thi-1; λ-; recAl; gyrA96; relAl, was used. The protocols used for the preparation of competent bacteria and those for transformation are described by HANAHAN, DNA cloning, vol. 1, DM Glover (ed) IRL Press, p. 109-135, (1985). The ligation mixture was used to transform the competent DH5α bacteria. The colonies obtained were selected on LB dishes (bacterotryptone 10 g / 1, yeast extract 5 g / 1, NaCl 10 g / 1) plus ampicillin (50 μg / ml). The DNA of the clones obtained is then extracted and analyzed by enzymatic digestion [SAMBROOK et al., Molecular Cloning: A laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor HN.Y. , (1989)]. A clone having the 1260 bp insert was selected. The plasmid obtained, called pGEMT-GPDl is represented in FIG. 3. The insert was sequenced by the enzymatic method of chain termination [SANGER et al ,, Proc. Natl. Acad. Sci. USA, 74, p. 5463-5467, (1977)] using the A370 automatic sequencer controlled by the A373 software (APPLIED BIOSYSTEMS). The primers coupled to the fluorescent ddNTPs which have been used are T7 and M13 reverse. The sequence reactions were carried out using the “PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit” (APPLIED BIOSYSTEMS) on the Perkin-Elmer Cetus model 9600 thermal cycler, in accordance with the supplier's instructions.

- The sequences obtained were analyzed using DNA Strider 1.0 software [MARCK, (1988)], and homology searches were carried out on the FASTA program with the EMBL database from Heidelberg and GENE BANK. The sequence of at least 300 bases of each strand of the cloned fragment was thus produced. A 100% identity was found with the published GPD1 sequence [LARSSON et al., Mol. Microbiol. 10, p. 1101-1111, (1993)], which confirms that the amplified fragment corresponds to the GPD1 gene.

EXAMPLE 2: OVEREXPRESSION OF GPD1 IN THE YEAST S. CEREVISIAE SCV5M.

In order to obtain a strong and constitutive expression of GPDl in yeast, the coding region of GPDl was placed under the control of regulatory elements (promoter and terminator) of yeast, in a yeast / E. coli shuttle vector.

1) Introduction of GPDl onto the multitemplar plasmid PVTIOO-U The expression plasmid pVTlOO-U was used (FIG. 4). This plasmid contains the origin of repli- yeast cation 2 μ, the selection marker URA3 and the strong regulatory elements ADH (promoter and terminator of alcohol dehydrogenase I), as well as the bacterial elements (origin of replication and gene for resistance to ampicillin ). This plasmid has been described by [VERNET et al., Gene, 52, p. 225-233, (1987)].

The amplification product obtained as described in Example 1, 2) was digested with Xhol and BamHI. 100 ng of this digested fragment were ligated to 50 ng of vector pVTlOO-U digested with Xhol and BamHI and dephosphorylated. After transformation of bacteria and selection of the recombinant clones as described in Example 1, 3), several recombinant clones were obtained. The map of the recombinant plasmid obtained, called pVTlOO-U-GPDl, is shown in Figure 4.

2) Yeast transformation

The yeast strain Saccharomyces cerevisiae

ScV5M was transformed by the vector pVTlOO-U-GPDl and by the vector pVTlOO-U (control). The transformation method used is that of lithium acetate described by ITO et al. (J.

Bacteriol., 1983, 153, 163-168)).

The selective medium used is YNB (Yeast nitrogen base 7 g / 1 DIFCO, glucose 20 g / 1). The absence of uracil makes it possible to maintain a selection pressure for the plasmids. Several recombinant clones were obtained. The SIM7 clone was studied subsequently.

3) Consequences of GPDl overexpression a) Obtaining anti-GPDlo polyclonal antibodies

Anti-GPDlp polyclonal antibodies were used as a tool in this study. They were produced by injection into a rabbit of a purified preparation of the GPDlp protein. The purification of this protein was carried out from the oenological strain S267: yeast ICV (88, 117.0), as described below: - Preparation of the raw extract

10 liters of YEPD medium are inoculated with S267, from a 12-hour preculture on the same medium. The culture is collected at the end of the exponential phase and the cells are recovered by centrifugation, 5 minutes at 4000 g. The pellet is then washed with 0.9% NaCl and the cells are taken up in one volume (v / w) of buffer at pH 7.0 (10 mM Tris-H ₂ S0 ₄ , 1 mM EDTA, 1 mM DTT, 0.02% NaN ₃ , 1 mM PMSF, 0.7% protamine sulfate). 30 ml of cell suspension are added,

15 g of glass beads (diameter 0.5 mm) and grinding is carried out using a type B device. BRAUN for 4 minutes at 4 ° C. The supernatant is recovered after centrifugation at 20,000 g, 20 minutes and stored at 4 ° C. - Fractional precipitation with ammonium sulphate

The crude extract is subjected to fractional precipitation with ammonium sulphate (40% saturation). The supernatant obtained is subjected to precipitation at 60% saturation. The pellet containing the active protein is resuspended in 50 ml of buffer A.

- Anion exchanging chromatography The crude extract is dialyzed for 48 h against a tam¬ pon A, then deposited at 4 ° C and at the rate of 20 ml / h on a column (1 = 10 cm, diameter = 1.6 cm ) of DEAE-Sepharose CL6B (PHARMACIA), previously balanced with buffer B (Buffer A without PMSF or potassium sulfate). After removal of the non-retained proteins by passage of three volumes of equilibration buffer, a linear gradient of ris-H2SO4 pH 7, between 0 and IM was applied. The enzyme is eluted for a concentration of Tris-H Sθ4 of 300 mM.

- Molecular exclusion chromatography After concentration, the enzymatic fraction obtained was deposited on a column (1 = 40 cm, diameter = 1.6 cm) of Sephadex-GlOO (fractionation domain 10000 at 120,000 Da). The elution is carried out with buffer B, at a rate of 5 ml / h. The fractions (5 ml) are recovered and the activity of the enzyme measured. The enzyme is eluted between 35 and 50 ml of buffer. The fractions between 36 and 48 ml of elution volume are kept.

- Affinity chromatography

The enzymatic fraction obtained was deposited on a column containing a Blue Sepharose CL-6B resin on which is fixed an analog of NAD +, blue Cibacron F3G-A. The column (1 = 5 cm, diameter = 1.6 cm) is equilibrated at 4 ° C with buffer B and the sample is deposited at the rate of 6 ml / h. The column is then washed with 10 column volumes (buffer B). The elution of the fixed enzyme was obtained by using a linear mixed gradient of Tris-H2SO4 (0 to 1.5 M) and NADH (0 to 3 mM). 1 ml fractions are collected and the activity of the enzyme measured. The enzyme is released from 1 M ris-H2SO4 and 2 mM NADH.

- Electrophoresis under denaturing conditions The purified extract was subjected to electrophoresis under denaturing conditions (SDS-PAGE), and the protein band (43 kDa) corresponding to GPDlp was cut and reduced to powder. The antibodies were produced by the company APPLIGENE, by injection into a rabbit. The serum is collected after two months. The specificity of the antibodies was tested by Western Blot. The nylon nitrocellulose membranes obtained after transfer of the proteins from the gel are revealed as described by SAMBROOK et al. [Molecular Cloning: A laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor NY, (1989)], using anti-rabbit goat immunoglobulins coupled with alkaline phosphatase as secondary reagent and the 5-bromo complex -4-chloro-3-indoyl phosphate / nitro blue tetrazolium as chromogenic substrate. b) Culture and dosing conditions:

Middle

Y B [Yeast Ni trogen Base wi thout amino acid (DIFCO) 6.7 g / 1, glucose 100 g / 1, casamino acid (DIFCO) 5 g / 1, pH 3.4]. The medium used does not contain ura¬ cil, which ensures a selection pressure for the plasmids.

Culture conditions

10 ml of medium are inoculated with the yeast strains. The growth is carried out in an Erlenmeyer flask at 28 ° C., without stirring. The cultures are carried out anaerobically, in 250 ml fermenters filled with 200 ml of medium, placed at 28 ° C. The fermenters are seeded from the preculture, at a rate of 10 ^ cells / ml. The growth is followed by sampling an aliquot of culture medium and estimating the number of cells on a device of the Coulter counter (ZBI) type.

The metabolites are assayed in the supernatant, after centrifugation at 2000 g for 3 minutes, samples are taken.

Dose of metabolites:

Glucose

Glucose was measured by the reducing sugar assay method (DNS). The assay is carried out by adding, to a volume of the solution to be assayed, a volume of reagent (300 g of sodium and potassium tartrate, 16 g of NaOH and 10 g of dinitro 3,5 salicylic acid). The mixture is placed at 100 ° C for 5 minutes. The reaction is stopped in a 4 ° C bath. 10 volumes of water are added. The optical density is determined at 540 nm and the glucose concentration is determined by comparison of the values determined for a standard range of 0 to 2 g of glucose or by HPLC as specified below. Glucose. Glvcerol, ethanol, Acetaldehyde: The glycerol and acetaldehyde were assayed enzymatically using the corresponding assay kits, marketed by BOEHRINGER. All these metabolites, except acetaldehyde, were also assayed in parallel by HPLC according to the following conditions: Aminex HPX 87H Biorad column, mobile phase sulfuric acid 8 mN, flow rate 0.6 ml / min, at 45 ° C, refractometer detection. Measurement of enzyme activities

The cells were recovered from culture, by centrifugation for 3 minutes at 2000 g, and washed with a 50 mM H2PO4 buffer, pH 6.5. 100 mg yeast

(wet weight) are then ground by adding 1 g of glass beads (diameter = 0.5 mm) and 0.5 ml of 20 mM imidazole buffer pH 7.0 containing 1 mM DTT. The tube is shaken for 1 minute with a vortex and placed for 1 minute in ice.

The operation is repeated 5 times. After centrifugation for 1 minute at 5000 g, the supernatant is recovered and constitutes the acellular extract. Proteins are measured using a BCA Protein Assay Reagent kit (PIERCE). The glycerol-3-phosphate dehydrogenase activity (EC 1.1.1.8) is determined by the method described by GANCEDO (Eur. J.

Biochem., 1968, 5, 165-172). c) Results

- Fermentation in YNB medium, described above, was carried out with the two strains transformed by pVTlOO-U and pVTlOO-U-GPDl (SIM7).

The strain ScV5M transformed with pVTlOO-U (control) and SIM7 were tested for their capacity to produce glycerol during fermentations under conditions close to oenological conditions.

The growth and production of glycerol in the culture medium were monitored during the fermentation (Figures 5 and 6). The amount of glycerol produced is expressed as a function of the progress of reaction (1-S / SO) where S represents the glucose concentration at the moment, t and SO the initial concentration. The SIM7 strain overexpressed for the GPD1 gene produces a quantity of glycerol much greater than the control strain transformed by the plasmid alone pVTlOO-U. The final production reaches 14 g / 1 for the SIM7 strain against 4 g / 1 for the control strain (Figure 6).

A very significant increase in GPDH activity is observed in the SIM7 strain compared to the control strain (FIG. 7, in which the values represent the average of 3 determinations, the vertical bars illustrate the standard deviations). The GPDH activity increases very rapidly, and this, from the start of growth, to reach a maximum at the entry into stationary phase (Figures 5 and 7). This maximum is of the order of 180 mU per milligram of protein, or almost 2.5 times the activity observed in the control strain (Figure 7). This activity then decreases, the levels reached then being comparable to those of the control strain. In the control strain, the GPDH activity is relatively low at the start of fermentation and reaches a maximum of approximately 75 mU / mg of protein at a reaction progress of 0.6. The cells are then in stationary growth phase (FIG. 5). In order to confirm that the increase in activity in SIM7 is linked to an overproduction of the protein coded by GPDl, the production of this protein was monitored in the two strains by immunodetection using polyclonal antibodies raised against the protein GPD1. The results are shown for the SIM7 strain (Figure 8).

Different samples were taken during the fermentation. The proteins of a crude extract prepared as described above were separated by SDS-PAGE electrophoresis and transferred to a nylon-nitrocellulose membrane. The polyclonal antibodies obtained are then fixed on the membrane and revealed by anti-rabbit anti-bodies coupled to alkaline phosphatase [SAMBROOK et al., 1989, cited above]. The GPDlp protein is detected for the strain overexpressing GPDl during the exponential growth phase and remains produced at a comparable level until the end of fermentation (Figure 8). On the other hand, the GPDlp protein is not detected in the control strain except for the points situated between 0.4 and 0.6 of reaction progress, which correspond to the peak of GPDH activity observed (results not shown).

A close correlation is observed between the overproduction of glycerol (Figure 6) and the overproduction of GPDlp (Figure 8). On the other hand, the drop in GPDH activity observed during the second part of the fermentation, while the protein is still overproduced, is in contradiction with the importance of the production of glycerol also observed during this second half of the fermentation. This drop in activity in vi tro may reflect a partial inactivation of the enzyme in vivo. However, a slight instability of the protein can be excluded during this late phase, since under the experimental conditions used, the immunological response cannot be precisely quantified.

The growth of the SIM7 strain and of the control strain was compared (FIG. 9). Despite a difference in the final biomass reached, the growth rate is exactly the same for the two strains. It is 0.54 generations per hour. On the other hand, all the glucose present in the culture medium (100 g / l initially) is degraded by the two strains (FIG. 10).

The glycerol production pathway is therefore significantly amplified, which must result in an increased use of NADH. In order to see whether the cell, in order to maintain its redox balance, corrects this imbalance by reducing the production tion of ethanol, itself a consumer of NADH, the production of ethanol at the end of fermentation was measured in the two strains and the results are illustrated in Table I. Table I: Ethanol production at SIM7 and

ScV5M-pVT100-U during alcoholic fermentation on YNB medium with 100 g / 1 of glucose.

Ethanol strain g / 1 glycerol / glucose

ScV5M-pVT100-U 46.8 0.04

ScV5M-pTVl00-U-GPD1 37.5 0.13

A significant decrease (of the order of 10 g / l) in the amount of ethanol produced by the SIM7 strain is effectively observed.

This decrease in ethanol production, resulting from the lower availability of NADH necessary for the transformation of acetaldehyde into ethanol by intervention of alcohol dehydrogenase I, the evolution of the production of acetaldehyde was followed, during fermentation (Figure 11). In agreement with the decrease in ethanol production, an accumulation of acetaldehyde is observed in the strain overexpressed for GPDl. The final concentration of acetaldehyde at the end of fermentation is around 150 mg / 1 and therefore remains very moderate.

The overexpression of GPDl therefore results in a medium rich in sugars, by a very significant increase in the production of glycerol, counterbalanced by a reduction in the production of ethanol.

- Fermentation on synthetic must The production of glycerol is proportional to the initial amount of glucose present in the culture medium. This was shown by testing the production of glycerol by the strains ScV5M-pVTl00-U and ScV5M-pVT100-U-GPD1, on synthetic MS medium in the presence of 2 glucose concentrations. Synthetic must is a medium that mimics the composition of natural musts, the composition has been described by BELY M. et al. (Journal of Fermentation and Bioengineering, 1990, 70, 246-252). The nitrogen composition of this medium was modified as follows: 80 mg / 1 of NH ₄ C1 and 120 mg / 1 of α-amino nitrogen were used. Two glucose concentrations 100 g / 1 and 200 g / 1 were tested. The fermentations were carried out in 1 1 fermenters. Furthermore, the other culture conditions and the monitoring of fermenta¬ tion were carried out as described above. The glycerol production results are summarized in Table II:

TABLE II: Production of glycerol in SIM7 and ScV5M-pVTl00-U during alcoholic fermentation on MS medium with 100 and 200 g / 1 of glucose

Glycerol strain g / 1

MS glucose 100 g / 1 MS glucose 200 g / 1

ScV5M-pVTl00-U not determined 6.6

ScV5M-pVTl00-U-GPD1 19 27.9

In the case of fermentation in a medium containing 200 g / l of glucose, the glycerol product / initial glucose ratio is significantly different for the strain ScV5M-pVT100-U-GPDl (0.14) compared to the ratio obtained with the strain ScV5M-pVTl00-U (0.03) control.

A very significant increase in the production of glycerol is observed in the presence of 200 g / l of glucose compared to a fermentation carried out on 100 g / l of glucose. In order to determine the consequences of this significant production of glycerol on the ethanol content, the production of ethanol at the end of fermentation was measured on this medium and the results are illustrated in Table III. Table III: Production of ethanol in SIM7 and ScV5M-pVT100-U during alcoholic fermentation on MS medium with 200 g / 1 of glucose.

Ethanol strain g / 1

ScV5M-pVT100-U 86.7

Scv5M-pVT100-U-GPDl 74

The very large production (27.9 g / l) of glycerol on MS medium 200 g / l of glucose is accompanied by a decrease in the production of ethanol by approximately 13 g / l.

EXAMPLE 3 Overexpression of GPD1 in the oenological yeast S. cerevisiae Kl (Languedoc) under oenological conditions.

1) Introduction of the positive selection marker ZEO on the multicopy plasmid pVTlOO-U-GPDl:

Since the industrial oenological strains are prototrophic, a dominant positive marker has been introduced into the vector pVTlOO-U-GPDl, in order to be able to select transformants of the strain Kl.

The cassette “TEF1 promoter-ZEO gene-terminator CYC1” was isolated from the vector pUT 332 (WENZEL TJ et al., Yeast, 1992, 8, 667-668), by PCR using the oligonucleotides described in FIG. 12. These primers make it possible to introduce Hpal sites at the ends of the amplified fragment. _.

The amplification was carried out as follows:

Oligonucleotide (1) 5 μl (20 pmol)

Oligonucleotide (2) 5 μl (20 pmol)

Buffer TaQ 10X 10 μl dNTP 2 mM 10 μl MgCl ₂ 25 mM 6 μl pUT332 (50 ng / μl) 1 μl TaQ 0.5 μl

Water 52.5 μl

30 cycles (30 sec 94 ° C, 30 sec 45 ° C, 1 min

72 ° C) The fragment of 1175 bp amplified was analyzed on 0.8% agarose gel, then digested with Hpal, and purified on agarose gel with low melting point. The agarose strip containing the fragment was cut and used directly in the ligation mixture. Thirty ng of fragment thus obtained were ligated to 50 ng of plasmid pVTlOO-U-GPDl digested with Hpal and dephosphorylated, overnight at 20 ° C, in the presence of 2 units of T4 DNA ligase. After transformation of bacteria and selection of the recombinant clones, several clones having the expected structure were obtained. The map of the recombinant plasmid obtained, called pVTlOOU-ZEO-GPDl, is shown in FIG. 13.

2) Yeast transformation:

The oenological yeast strain S. cerevisiae Kl was transformed by the vector pVTlOOU-ZEO-GPDl and by the vector pVTlOOU-ZEO (control), by the method of transformation with lithium acetate described by ITO et al. (1983). The selective medium used is YEPD (yeast extract 10 g / 1, bacto peptone 20 g / 1, glucose 20 g / 1) containing 40 μg / ml of phleomycin.

3) Consequences of GPD1 overexpression under oenological fermentation conditions:

10 ml of synthetic must (described in Example 2) are inoculated with the yeast strains. Growth is carried out in a tube for 24 h at 28 ° C. The cultures are carried out anaerobically, in 220 ml fermenters, filled with 200 ml of the same medium and placed at 28 ° C. with stirring using a magnetic bar. The fermenters are seeded from the preculture at the rate of 10 ⁶ cells / ml. Growth is followed by sampling of aliquots of culture medium and estimation of the number of cells on a Coulter counter device (ZBI). The metabolites are assayed in the supernatant, after centrifugation at 2000 g for 3 minutes.

* Dose of metabolites:

Glucose, glycerol, ethanol, acetate, succinate, pyruvate, acetoin and butane-diol: are assayed by HPLC according to the protocol described in Example 2. The acetaldehyde is assayed by the enzymatic route (Boehringer). The butane diol extraction method used is that previously described (HAGENAUER-HENER U. et al., Dtsch. Lebensm. Rundsch., 1990, 86, 273-276).

* Results: Fermentation on synthetic wort was carried out with the strains Kl / pVTlOOU-ZEO (K ^ pVTU) and Kl / pVTlOOU-ZEO-GPD1 (K.-GPD1). The various metabolites were dosed after complete consumption of the sugar. The results presented in FIG. 14 show a production of glycerol of 14 g / l for the strain overexpressed for GPDl instead of 6.9 g / l for the control strain. This overproduction of glycerol is accompanied by a decrease in the production of ethanol by approximately 8 g / 1 (80 g / 1 against 88.4 g / 1). This deviation of the carbon flow towards the glycerin leads to an increase of a factor of approximately 2 in the final concentration of acetaldehyde, pyruvate and acetate. Larger variations are observed in the succinate (1.8 g / 1 against 0.5 g / 1) and 2.3 butane-diol (1.1 g / 1 against 0.25 g / 1). In the case of fermentation in a medium containing 200 g / l of glucose, the glycerol product / initial glucose ratio is significantly different for the strain K ^ GPDl (0.07) compared to the ratio obtained with the control strain K.- pVTlOO-U-ZEO (0.03). These results are explained by the imbalance of the redox balance caused by the overexpression of GPDl. NADH, preferably reduced to NAD by the GPDH route, becomes limiting for the conversion of acetaldehyde to ethanol. This results in a decrease in the amount of ethanol formed, as well as an accumulation of acetaldehyde, acetate and pyruvate. However, the variations in these metabolites at the end of fermentation remain minor. The accumulation of succinate and butanediol could be a means of detoxifying the cell compared to acetaldehyde. The routes by which these two compounds are accumulated have not been studied. However, in the hypothesis where the succinate is formed by the oxidative pathway of the Krebs cycle, its formation could allow the cell to regenerate a little NADH. On the contrary, the formation of 2,3 butane diol (acetoin reduction product) from acetaldehyde, which is not advantageous at the redox level, would rather be a means of detoxification. In terms of the predictable effects of observed metabolic changes, it should be noted that the concentrations of all these metabolites remain within a range compatible with concentrations which can be observed in fermented beverages and in particular in wine including for succinate ( 0.5-2 g / 1, RADLER F., Wine microbiology and biotechnology; Harwood Académie Publishers, 1992, Ed G. Fleet. Chur, Switzerland) and 2.3 butane-diol (0.3-1.3 g / 1, RIBEREAU-GAYON et al., Treaty of Enology, 1972). In addition, high amounts of succinate could be advantageous in wine, through the acid character which it could bring. As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which can come to the mind of the technician in the matter, without deviate from the framework or the scope of this invention.

Claims

1 °) Yeast strains, characterized in that they are selected from Saccharomyces strains, in that they contain at least one fragment of a gene coding for a glycerol 3 phosphate dehydrogenase NADH-dependent (GPDH), which fragment is modified by active regulatory sequences in yeast comprising a strong promoter, to increase the production of glycerol and reduce the production of ethanol, in a medium rich in sugars during alcoholic fermentation.

2 °) Yeast strains according to claim

1, characterized in that they are selected from industrial Saccharomyces strains, in particular oenological, distillery, brewery, bakery or cider house strains.

3) yeast strains according to claim 1 or claim 2, characterized in that they belong to the species Saccharomyces cerevisiae. 4 °) Yeast strains according to any one of claims 1 to 3, characterized in that they contain at least two copies of the gene coding for a NADH-dependent GPDH.

5) Expression cassette, characterized in that it comprises a gene coding for a yeast-dependent GPDH NADH- associated with active regulatory sequences in yeast.

6 °) Expression vectors, comprising an expression cassette according to claim 5, and usable for obtaining the yeast strains transformed according to any one of claims 1 to 4.

7 °) Vectors according to claim 6, charac¬ terized in that they are shuttle vectors, also having an origin of bacterial replication and a selection marker in a bacterium. 8 °) Fermented drinks with a reduced ethanol content, characterized in that they are obtained by alcoholic fermentation, in the presence of yeast strains according to any one of claims 1 to 4. 9 °) Use of a yeast strain according to any one of claims 1 to 4, for the preparation of bread.

10 °) Alcoholic fermentation process with modified sugar fermentation balance, characterized in that to increase the production of glycerol and reduce the production of ethanol, said process comprises:

(1) transformation of a Saccharomyces yeast strain with a vector overexpressing the GPDH protein; and (2) seeding the fermentation medium, containing at least 30 g / 1 of sugars, with the transformed yeast strain (transformant), obtained in (1).