BRPI0910599B1 - USE OF A CARBON SUBSTITUTE FOR YEAST PRODUCTION - Google Patents

Abstract

USE OF A CARBON SUBSTITUTE FOR YEAST PRODUCTION. The present invention relates to new strains of Saccharomyces that can be produced on a carbonaceous substrate that makes it possible to replace the use of sugar completely and/or partially and the derivative use for the production of yeast, in particular on an industrial scale. The invention also relates to a method for producing yeasts of the genus Saccharomyces on a carbonaceous substrate that makes it possible to replace the use of sugar completely or partially.

Description

TECHNICAL FIELD

The present invention concerns the production of yeast, in particular on an industrial scale. A subject of the present invention is the use of a carbonaceous substrate, making it possible to partially or totally replace the use of sugar for the production of yeast, especially on an industrial scale.

TECHNOLOGICAL BASIS

Yeast production involves the supply of different nutritional elements capable of guaranteeing its growth. These different nutritional elements in particular are carbon, nitrogen, phosphorus, sulfur, minerals and vitamins. Among the natural media that can be used for yeast production, molasses occupies a special position. In fact, molasses constitutes a relatively complete means capable of ensuring the supply of these nutritional elements. The average composition of molasses is as follows: 66 to 73% sugars, 15% to 23% organic compounds and 10% to 12% mineral compounds (as a percentage of total dry matter). The carbon source provided by molasses is essentially made up of sucrose or sugars resulting from its hydrolysis (glucose and fructose).

However, the growing scarcity of sugarcane and beet molasses, especially associated with their extensive use for the production of bioethanol, puts this type of production at risk. It is particularly necessary to find new carbonaceous substrates to replace the sucrose provided by molasses.

Yeast production can also be carried out in glucose or fructose syrups, for example, obtained from the hydrolysis of starch and in particular from cereals (corn, wheat, rice) or potatoes. However, these syrups are relatively expensive and do not represent an economically profitable solution to replace sucrose from molasses within the context of industrial yeast production.

Yeast of the genus Saccharomyces is capable of synthesizing glycerol under anaerobic conditions or in a situation of intense stress, for example, in the case of thermal or osmotic stress. Furthermore, Saccharomyces yeast is capable of degrading glycerol, but in a purely oxidative metabolism and in the absence of catabolic repression by sugars. Thus, Saccharomyces yeast, placed in the presence of glycerol and sugar in a batch culture, is not able to consume the glycerol, while the sugar remains in the medium.

Furthermore, in an oxidative metabolism, Saccharomyces yeast cultivated on a carbon substrate consisting exclusively of glycerol consumes glycerol very slowly, which is not compatible with yeast production on an industrial scale. (Lages and Lucas, 1997, Biochimica and Biophysica Acta).

Therefore, there is a real need for new strains for the production of yeast that make it possible to totally or partially replace sugar as a carbonaceous substrate, with yeast production yields compatible with economic and industrial exploitation and/or without loss in the quality of the yeast. yeast produced.

SUMMARY OF THE INVENTION

An object of the present invention is to provide new strains of Saccharomyces capable of being produced on a carbonaceous substrate, making it possible to totally and/or partially replace the use of sugar.

An object of the invention is also the use of these strains for the production of yeast, in particular on an industrial scale.

Another object of the invention refers to a method for the production of yeasts of the genus Saccharomyces on a carbonaceous substrate, making it possible to totally or partially replace the use of sugar, said method also making it possible to obtain a yield similar to that of production on a substrate of the type of sugar and/or making it possible to obtain yeast of similar quality.

An object of the present invention is a strain of Saccharomyces, characterized in that in the presence of a culture medium, consisting of a mixture of glycerol and sugar, under aerobic conditions, it consumes at least 80% of the glycerol of said mixture, said composite mixture by a proportion of glycerol of at least 20% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is also a strain, as defined above, characterized by consuming at least 85% of the glycerol in the mixture, preferably at least 90% of the glycerol in the mixture, more preferably at least 95% of the glycerol in the mixture, even more preferably at least 98% of the glycerol in the mixture.

An object of the present invention is also a strain, as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 30% as sucrose equivalent, preferably at least 40% as sucrose equivalent, more preferably at least 50% as sucrose equivalent. sucrose equivalent, even more preferably at least 60% as sucrose equivalent, even more preferably at least 70% as sucrose equivalent, even more preferably at least 80% as sucrose equivalent, even more preferably at least 90% as sucrose equivalent. sucrose.

The present invention relates in particular to a strain as defined above, characterized in that it is chosen from the species cerevisiae, boulardii, carlsbergensis and uvarum.

An object of the present invention is a strain as defined above, characterized by being chosen from a natural variant and/or a genetically modified strain.

A particular object of the present invention is a strain as defined above, characterized in that it is chosen from: - strain deposited in the Collection Nationale de Cultures de. Microorganismes (CNCM) on December 20, 2007 with CNCM number 1-3886, - strain deposited at CNCM on December 20, 2007 with CNCM number I-3887, - strain deposited at CNCM on December 20, 2007 with CNCM number I- 3888, - strain deposited at CNCM in CNCM on March 3, 2009 with number I- 4132 and - strain deposited at CNCM in CNCM on March 3, 2009 with number I- 4133.

A particular object of the present invention is a strain as defined above, characterized in that it is chosen from: strain deposited in the CNCM in CNCM on March 3, 2009 with number I- 4129, strain deposited in the CNCM in CNCM on March 3, 2009 with number I- 4130 and the strain deposited at CNCM in CNCM on March 3, 2009 with number I- 4131.

An object of the present invention is also the use of a strain as defined above for the production of yeast in the presence of a mixture of glycerol and sugar comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeasts from at least one strain of Saccharomyces, characterized in that said strain consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is also a method for producing yeast comprising the steps of: - introducing a strain of yeast of the genus Saccharomyces into a fermentor, and - multiplying said strain under aerobic conditions in a culture medium, comprising a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

A particular object of the present invention is a method, as defined above, characterized in that at least 80% of the glycerol in said mixture is consumed by said strain, preferably at least 85% of the glycerol in said mixture, more preferably at least 90% of the glycerol in said mixture, even more preferably at least 95% of the glycerol in said mixture, even more preferably at least 98% of the glycerol in said mixture.

An object of the present invention is a method as defined above, characterized in that the multiplication of said strain is carried out in a semi-continuous mode or in a continuous mode.

An object of the present invention is also a method as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 8% as sucrose equivalent, preferably at least 10% as sucrose equivalent, more preferably at least 12%. as sucrose equivalent, even more preferably at least 15% as sucrose equivalent, even more preferably at least 17% as sucrose equivalent, even more preferably at least 20% as sucrose equivalent.

A particular object of the present invention is a method as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 30% as sucrose equivalent.

An object of the present invention is a method as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 40%. as sucrose equivalent, preferably at least 50% as sucrose equivalent, more preferably at least 60% as sucrose equivalent, even more preferably at least 70% as sucrose equivalent, even more preferably at least 80% as sucrose equivalent. sucrose equivalent, even more preferably at least 90% sucrose equivalent.

An object of the present invention is a method as defined above, characterized in that said yeast strain is a strain as defined above.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Diagram of the expression cassette used to integrate the STL1 gene into the genome of yeast strains.

The STL1 gene is under the control of the pADH1 promoter and it is followed by the yeast CYC1 terminator. Two recombinogenic fragments (BUD5-A and BUD5-B) each of 500 nucleotides form the expression cassette, in order to ensure the insertion by homologous recombination of the cassette into the yeast genome, after transformation, at the BUD5 locus (YCR038c) located on chromosome III. The gene that confers resistance to KANMX geneticin is inserted downstream of the STL1 gene, formed by two loxP sites.

Figure 2: Expression level of the STL1 gene in strains genetically modified to express the STL1 gene, after culture in the presence of sugar.

The graph represents the relationship between the mRNA level of mutant strains of yeast expressing the STL1 gene and the mRNA level of the corresponding non-mutant strain (on the y axis). 1: relationship between the mRNA level of the strain deposited at CNCM with number I 3887 and that of the non-mutant strain NCYC 996; 2: relationship between the mRNA level of the strain deposited at CNCM with number I-3888 and that of the non-mutant strain NCYC 995; 3: relationship between the mRNA level of the strain deposited at CNCM with number I-3886 and that of the non-mutant strain I-3399.

Figure 3: Residual amount of glycerol, after culture in semi-continuous mode, of strain I-3887 compared to that of the non-mutant strain NCYC 996, as a function of time.

The mixture comprises 20% glycerol as sucrose equivalent. The y-axis on the left side indicates the amount of glycerol (in grams) and the y-axis on the right side indicates the amount of ethanol (in grams) in the yeast-free medium. The x-axis indicates time (in hours). The curve with triangles represents the residual amount of glycerol in the yeast-free medium during the culture of the non-mutant strain NCYC 996 and the curve with squares for the strain I-3887. The curve with circles represents the residual amount of ethanol in the yeast-free medium during the culture of the non-mutant strain NCYC 996 and the curve with diamonds for strain-3887.

Figure 4: Residual amount of glycerol, after culture in semi-continuous mode, of strain I-3888 compared to that of the non-mutant strain NCYC 995, as a function of time.

The mixture consists of 20% glycerol as sucrose equivalent. The y-axis on the left side indicates the amount of glycerol (in grams) and the y-axis on the right side indicates the amount of ethanol (in grams) in the yeast-free medium. The x-axis indicates time (in hours). The curve with triangles represents the residual amount of glycerol in the yeast-free medium during the culture of the non-mutant strain NCYC 995 and the curve with squares for the strain I-3888. The curve with circles represents the residual amount of ethanol in the yeast-free medium during the culture of the non-mutant strain NCYC 995 and the curve with diamonds for strain-3888.

Figure 5: Yield and residual concentration of glycerol after chemostat cultivation of strain 1-3887 and strain NCYC 996, as a function of the proportion of glycerol in the mixture of glycerol and sugar in the growth medium.

The y-axis on the left side indicates the yield of the strain as a percentage of the yield of the NCYC 996 strain grown in the absence of glycerol. The y-axis on the right indicates the residual glycerol concentration in the yeast-free medium (in ppm). The x-axis indicates the proportion of glycerol in the mixture of glycerol and sugar as sucrose equivalent. The production yield is represented by squares and the residual glycerol concentration by circles. Results from the non-mutant strain NCYC 996 (control) are shown in white and those from the corresponding mutant strain 1-3887 in black.

Figure 6: Yield and residual concentration of glycerol after chemostat cultivation of strain 1-3888 and strain NCYC 995, as a function of the proportion of glycerol in the mixture of glycerol and sugar in the growth medium.

The y-axis on the left side indicates the yield of the strain as a percentage of the yield of the NCYC 995 strain grown in the absence of glycerol. The y-axis on the right indicates the residual glycerol concentration in the yeast-free medium (in ppm). The x-axis indicates the proportion of glycerol in the mixture of glycerol and sugar as sucrose equivalent. The production yield is represented by squares and the residual glycerol concentration by circles. Results from the non-mutant strain NCYC 995 (control) are shown in white and those from the corresponding mutant strain 1-3888 in black.

Figure 7: Percentage of glycerol consumed after chemostat cultivation of strain 1-3887 and strain NCYC 996, as a function of the proportion of glycerol in the mixture of glycerol and sugar in the growth medium.

The percentage of glycerol consumed (on the y-axis) is given as a function of the proportion of glycerol in the mixture of glycerol and sugar as sucrose equivalent (on the x-axis). The percentage of glycerol consumed by the non-mutant strain NCYC 996 (control) is represented in white and the corresponding percentage for the mutant strain 1-3887 is shown.

Figure 8: Percentage of glycerol consumed after chemostat cultivation of strain 1-3888 and strain NCYC 995, as a function of the proportion of glycerol in the mixture of glycerol and sugar in the growth medium.

The percentage of glycerol consumed (on the y-axis) is given as a function of the proportion of glycerol in the mixture of glycerol and sugar as sucrose equivalent (on the x-axis). The percentage of glycerol consumed by the non-mutant strain NCYC 995 (control) is represented in white and the corresponding percentage for the mutant strain 1-3888 is shown.

Figure 9: Percentage of glycerol consumed after semi-continuous culture of natural variants in the presence of a mixture of glycerol and sugar comprising 20% glycerol as sucrose equivalent.

The percentage of glycerol consumed (on the y-axis) is indicated by different natural variants tested (1 to 53). The percentage of glycerol consumed by the non-mutant strain from which the natural variants (denoted T) are obtained is also indicated.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention is a strain of Saccharomyces, characterized in that in the presence of a culture medium, comprising a mixture of glycerol and sugar, under aerobic conditions, it consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of at least 20% as sucrose equivalent, the sum of the percentages of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

Surprisingly and unexpectedly, the inventors obtained strains of Saccharomyces yeast capable of consuming at least 80% of the glycerol in a mixture of glycerol and sugar, simultaneously with their consumption of sugar, under aerobic conditions, in a continuous or semi-continuous culture.

The inventors have actually shown that this industrial standard baker's yeast, such as the strains deposited in the NCYC (National Collection of Yeast Cultures) with the numbers NCYC995 and NCYC996, consume glycerol only partially in the presence of a mixture of glycerol and sugar in the culture. semi-continuous and/or continuous culture (see example 2). At the end of the culture, the culture medium that is free of yeast then contains a large residual amount of glycerol that accumulated during the culture.

The strains according to the invention are characterized by the fact that, in the presence of a culture medium, comprising a mixture of glycerol and sugar, they consume at least 80% of the glycerol in said mixture, the percentage being given in weight/weight. In other words, the strain according to the invention consumes at least 80% of the total mass of glycerol present in the mixture of glycerol and sugar.

A particular object of the present invention is a strain of Saccharomyces, characterized in that in the presence of a culture medium comprised of a mixture of glycerol and sugar, under aerobic conditions, it consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of 20% as sucrose equivalent, the sum of the percentages of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

The determination of glycerol consumption can be carried out by measuring the residual amount of glycerol in the culture medium, after a semi-continuous culture, or by measuring the residual amount of glycerol in the drained medium in the case of a continuous culture.

In the case of a semi-continuous culture, the measurement of the residual amount of glycerol in the culture medium is carried out at the end of the culture.

By "end of culture" is meant the moment when the sugar supply is interrupted.

The duration of the culture is in particular less than or equal to the duration of a culture of the same strain in a mixture comprising 100% sugar as sucrose equivalent.

The culture duration is in particular less than 24 hours.

The culture duration is in particular less than 20 hours.

The duration of the culture is, for example, 22 hours.

In the case of a continuous culture, the residual amount of glycerol is measured at any time once steady state is reached.

Steady state is reached when concentrations in the fermenter are stable for at least 3 residence times, the residence time being the ratio 1 / [dilution rate].

Conventionally, culture conditions are such that the metabolism of the yeast strain is essentially respiratory and/or such that there is essentially no production of ethanol.

The word "essentially" means that these conditions are satisfied throughout the entire duration of the culture, but it is possible that from time to time these conditions are not satisfied in semi-continuous mode, for example during the first few hours of the culture, in particular for a duration comprised between 1/4 and 1/3 of the duration of the culture and, in particular during the first 5 hours of a culture in semi-continuous mode.

In a chemostat, the culture conditions are such that the metabolism of the yeast strain is respiratory and/or such that there is no production of ethanol when steady state is reached.

The expression "the metabolism of the yeast strain is essentially respiratory" means that the carbon is oxidized and not fermented, during the entire period of the culture, part of the carbon may be temporarily fermented.

The expression "essentially no ethanol production" means that the ethanol concentration at the end of the culture is less than 1%, preferably less than 0.1%, more preferably less than 0.01%, more preferably less than 0.001%.

A person skilled in the art knows how to adjust the parameters for culturing a given strain of yeast so that the culturing conditions are such that the metabolism of said strain is essentially respiratory and/or such that there is essentially no production of ethanol. .

The residual amount of glycerol is preferably measured in the culture medium that is free of yeast.

The residual amount of glycerol in the culture medium, for example, is measured by HPLC or GC chromatography, enzymatic assay or any other appropriate method.

The yeast strains according to the invention, therefore, are particularly useful, as they are capable of consuming glycerol continuously, during a production period compatible with industrial exploitation.

The expression "capable of consuming glycerol continuously" means that there is essentially no accumulation of glycerol during the entire culture period, but that from time to time accumulation becomes possible if the metabolism of the yeast is not essentially respiratory and/or if there is a alcohol production.

By the expression "mixture of glycerol and sugar", is meant a mixture consisting of glycerol and sugar in variable proportions, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

The mixture of glycerol and sugar constitutes the only source of sugar and glycerol in the culture medium.

The mixture of glycerol and sugar is preferably prepared before its introduction into the culture medium. Glycerol, also called glycerin or propan-1,2,3-triol, is a polyol. Pure glycerol is presented in the form of a viscous and transparent liquid and is soluble in water.

The glycerol according to the invention can be used in pure form and/or in the form of a product containing glycerol, and/or supplied in any form capable of producing glycerol.

The sugar is preferably a sugar that can be immediately assimilated by yeast, in particular a simple sugar of glucose, fructose or sucrose type and/or a mixture of these sugars.

Sugar can be supplied in the form of glucose syrups and/or fructose syrups and/or hydrolyzed starches and/or in the form of molasses and/or in the form of a substance capable of producing sugars that can be assimilated by the strain of yeast and/or in the form of a mixture thereof.

The sugar is preferably supplied in the form of molasses.

The mixture of glycerol and sugar constitutes at least 90% of the carbon source for yeast, preferably at least 95% of the carbon source, more preferably at least 98% of the carbon source. The carbon source in the culture medium, with the exception of the mixture of glycerol and sugar, comprises, for example, amino acids, lipids, organic acids naturally present in molasses or crude hydrolysates of agricultural origin.

The expression "the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent" means that when the proportion of glycerol expressed in sucrose equivalent is x%, the proportion of sugar in the mixture expressed in equivalent in sucrose it is (100-x)%.

The equivalence is determined here in relation to glycolytic metabolism, according to which one mole of sucrose is converted into one mole of fructose and one mole of glucose is converted into four moles of pyruvate (cf. Príncipes de la Biochimie, Lehninger, 1994).

Therefore, one mole of glucose is converted into two moles of pyruvate and one mole of fructose is converted into two moles of pyruvate.

According to this same metabolism, one mole of glycerol is converted into one mole of pyruvate.

Thus, four moles of glycerol correspond to one mole of sucrose equivalent.

Two moles of glucose correspond to one mole of sucrose equivalent.

Two moles of fructose correspond to one mole of sucrose equivalent. 1.076 g of glycerol corresponds here to one gram of sucrose equivalent.

For example, one kilogram of a mixture finally containing 250 g/kg of sucrose equivalent and a proportion of 20% glycerol as sucrose equivalent therefore consists of 50 g of sucrose equivalent in the form of glycerol and 200 g of equivalent in sucrose in the form of sugar. Given raw molasses with a sugar content of 500 g/kg as sucrose equivalent, to prepare one kilogram of this mixture, 50 x 1.076 g = 54 g of pure glycerol are first mixed (which corresponds to 50 g of sucrose equivalent in the form of glycerol) and 400 g of said raw molasses (which corresponds to 200 g of sucrose equivalent in the form of sugar), then pure water is added to reach a final mass of the mixture of 1 kilogram.

The culture medium here is called fresh culture medium when it has not been placed in the presence of yeast strains

The culture medium here is called reaction culture medium that has been placed in the presence of yeast strains.

The yeast strain according to the invention is placed in the presence of the culture medium under aerobic conditions.

Preferably, the yeast strain according to the invention is placed in the presence of the culture medium in a fermentor suitable for the production of yeast. The volume of the fermenter can vary from a few milliliters to several cubic meters.

The fermentate is also called a reactor hereinafter.

The fermenter is preferably suitable for culturing under aerobic conditions.

The yeast strain is preferably placed in the presence of the culture medium within the context of a semi-continuous mode and/or continuous mode culture.

By the expression "semi-continuous mode culture" or "semi-continuous culture", or "semi-continuous mode" also called "fed batch", here is meant a culture in a fermenter that is progressively fed by the culture medium, but no volume of the medium is drained. In such a method, the culture volume is variable (and generally increasing) in the fermenter. The feeding rate in a culture in semi-continuous mode is constant or variable.

An example of a semi-continuous culture is a culture carried out under the conditions described in the reference book “Yeast Technology”, Chapter 6, 2nd edition, 1991, G. Reed and T. l/V. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442- 31892-8.

By the expression "continuous mode culture" or "permanent culture" or "continuous mode", here is meant a culture in a fermentor during which the fermenter is fed with fresh culture medium and in which part of the reaction medium is drained . In a continuous mode culture, the volume of the fermenter culture is variable or constant.

According to a particular incorporation of the culture by continuous mode, the feeding index and the drainage index are equal.

An example of a continuous culture is a culture carried out under the conditions described in the reference book “Yeast Technology, Chapter 6, 2nd edition, 1991, G. Reed and T. W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442- 31892-8 .

Chemostat culture is a particular form of continuous culture.

In a chemostat culture, different parameters such as dissolved oxygen level, nutrient concentrations, pH, cell density, are kept constant.

The culture medium comprises as constituents the mixture of glycerol and sugar and other elements necessary for the growth of yeast.

The other elements necessary for yeast growth are the following: at least one source of nitrogen, at least one source of sulfur, at least one source of phosphorus, at least one source of vitamins and/or at least one source of minerals.

These other elements are supplied in sufficient quantities so that they do not constitute a limiting factor for yeast growth and to maintain an essentially respiratory metabolism.

The nitrogen source, for example, is supplied in the form of ammonium sulfate, ammonium hydroxide, diammonium phosphate, ammonia, urea and/or a combination thereof.

The sulfur source, for example, is supplied in the form of ammonium sulfate, magnesium sulfate, sulfuric acid and/or a combination thereof.

The phosphorus source, for example, is supplied in the form of phosphoric acid, potassium phosphate, di-ammonium phosphate, mono-ammonium phosphate, or a combination thereof.

The source of vitamins, for example, is supplied in the form of molasses, yeast hydrolyzate, pure vitamin solution or a mixture of pure vitamins and/or a combination thereof.

The vitamin source supplies all vitamins to the yeast in amounts at least equivalent to those recommended in reference works. Several sources of vitamins can be combined.

The source of minerals, for example, is supplied in the form of molasses, a mixture of mineral salts and/or a combination thereof.

The mineral source supplies all macroelements and trace elements for the yeast in quantities at least equivalent to those recommended in reference works. Various mineral sources can be combined.

The same substances can supply several different elements.

The yeast strains according to the particular invention are chosen from the strains that make it possible to produce baker's yeast, brewer's yeast, winemaking yeast, yeast for the production of alcohol and/or yeast for the production of recombinant proteins, for example , for the production of therapeutic proteins.

The glycerol consumption of a strain of Saccharomyces can, for example, be measured under the conditions described in example 2, in order to determine whether said strain, in the presence of a culture medium, comprising a mixture of glycerol and sugar, under conditions aerobic, consumes at least 80% of glycerol in the mixture, said mixture comprising a proportion of 20% of glycerol as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is also a strain of Saccharomyces, characterized in that in the presence of a culture medium, comprising a mixture of glycerol and sugar, under aerobic conditions, it consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of 30% of glycerol as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is also a strain of Saccharomyces, characterized in that in the presence of a culture medium, comprising a mixture of glycerol and sugar, under aerobic conditions, it consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of 40% of glycerol as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

A particular object of the present invention is a strain, as defined above, characterized by consuming at least 85% of the glycerol in the mixture, preferably at least 90% of the glycerol in the mixture, more preferably at least 95% of the glycerol in the mixture, even more preferably at least 98% of the glycerol in the mixture.

A particular object of the present invention is a strain, as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 30% as sucrose equivalent, preferably at least 40% as sucrose equivalent, more preferably at least 50 % as sucrose equivalent, even more preferably at least 60% as sucrose equivalent, even more preferably at least 70% as sucrose equivalent, even more preferably at least 80% as sucrose equivalent, even more preferably at least 90% as equivalent in sucrose.

An object of the present invention is also a strain, as defined above, characterized in which the proportion of glycerol in said mixture is at most 95% as sucrose equivalent, preferably at most 90% as sucrose equivalent, more preferably at most 85%. as sucrose equivalent.

A particular object of the present invention is a strain, as defined above, characterized in which the proportion of glycerol in said mixture is from 20% to 90% as sucrose equivalent, preferably from 20% to 80% as sucrose equivalent, more preferably from 30% to 70% as sucrose equivalent, even more preferably from 30% to 60% as sucrose equivalent.

An object of the present invention is a strain as defined above, characterized in that it is chosen from the species cerevisiae, boulardii, carlsbergensis and uvarum.

A preferred strain according to the invention is a strain of Saccharomyces cerevisiae.

An object of the present invention is a strain as defined above, characterized in that it is chosen from a natural variant and/or a genetically modified strain.

The term "natural variant" here denotes a strain obtained by crossing and/or hybridization of yeast strains and/or obtained by spontaneous mutation and/or random mutagenesis, for example, after exposure to stress conditions, a UV treatment or to a treatment with mutagens.

The gene pool of a natural variant has not been modified through genetic engineering.

Two methods of random mutagenesis, for example, can be used to obtain natural variants according to the invention (see example 3 below).

The first method consists of a selection of natural variants based on their glycerol consumption and yield, immediately after exposure to UV radiation followed by the isolation of clones.

The second method comprises: - a first stage of exposure to UV radiation, - a second stage of culture in a chemostat in the presence of a mixture of sugar and glycerol, - a third stage of clone isolation, and - a fourth stage of selection of natural variants based on your glycerol consumption and your yield.

This second method makes it possible to enrich the culture with natural variants by consuming glycerol in the presence of sugar, before starting the selection stage.

A particular object of the invention is, therefore, the strain deposited with the CNCM on March 3, 2009 with the number CNCM 1-4129, the strain deposited with the CNCM on March 3, 2009 with the number CNCM 1-4130 and the strain deposited at CNCM on March 3, 2009 with number CNCM 1-4131.

These three strains were obtained by random mutagenesis following a UV treatment and selection based on their glycerol consumption and their yield (see example 3).

An object of the present invention is a natural variant as defined above, characterized by strain number CNCM 1-4129.

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM 1-4129 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM 1-4129 consumes more than 80% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% to 50%. %.

An object of the present invention is a natural variant, as defined above, characterized by strain number CNCM 1-4130.

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM 1-4130 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM 1-4130 consumes more than 80% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% and 50%. %.

An object of the present invention is a natural variant as defined above, characterized by strain number CNCM 1-4131.

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar with a proportion of glycerol of 20% as sucrose equivalent, the yeast strain CNCM 1-4131 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM 1-4131 consumes more than 80% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% to 50%. %.

By the expression “genetically modified strain” or GMO strain, we mean the gene pool of a strain that has been modified by genetic engineering.

A genetically modified strain may undergo a modification of its endogenous DNA, for example, a partial or total point mutation in a gene. The mutation may consist of an insertion, deletion and/or substitution of at least one nucleotide.

The mutation preferably induces a modification of the gene product after transcription and/or translation. The particular mutation may lead to a truncation of the gene product, a modification of at least one amino acid in the sequence of the gene product and/or a change of a reading frame during transcription.

A genetically modified strain may comprise exogenous DNA, whether or not integrated into at least one of the yeast chromosomes and/or into at least one yeast plasmid.

A preferred genetically modified strain comprises an exogenous DNA integrated into at least one of the yeast's chromosomes.

The exogenous DNA can be a DNA fragment, - for example a gene fragment, a complete or partial expression cassette -, and/or a plasmid.

When the genetically modified strain comprises an integrated exogenous DNA, it is a single integration or multicopy integration.

An object of the present invention is a strain as defined above, characterized by being a genetically modified variety at the level of at least one gene involved in at least one glycerol metabolic pathway.

An object of the present invention is a strain as defined above, characterized by being a genetically modified variety at the level of at least one gene involved in glycerol catabolism or in the regulation of glycerol catabolism.

A more specific object of the present invention is a strain, as defined above, characterized by being a genetically modified strain at the level of at least one gene chosen from the STL1, GUP1, GUP2, GUT1, GUT2, FPS1 and/or ADR1 and/or or a gene involved in glucose repression and/or a combination thereof.

A genetically modified strain according to the invention presents in particular an increase in the expression of a gene and/or a reduction in the expression of a gene and/or a constitutive expression and/or a differential expression of a gene, and/or the expression of a gene absent in the original yeast strain.

A more specific object of the present invention is a strain, as defined above, characterized by being a genetically modified strain at the level of at least one gene chosen from the STL1, GUP1, GUP2, GUT1, GUT2 genes and/or a combination thereof.

Modification of strains according to the invention is carried out using techniques well known to those skilled in the art (see Molecular Cloning, 3rd edition, Sambrook and Russell).

A genetically modified yeast strain according to the invention preferably consumes more than 90% glycerol in the mixture of glycerol and sugar.

The STL1 gene encodes the glycerol permease which in particular allows glycerol to enter the cell.

A strain according to the invention genetically modified at the level of the STL1 gene expresses the STL1 gene in the presence of sugar.

A preferred strain genetically modified at the level of the STL1 gene according to the invention overexpresses the STL1 gene in the presence of sugar compared to the corresponding non-mutated strain.

The STL1 gene in a genetically modified strain according to the invention is, for example, placed under the control of a constitutive promoter or a glucose-inducible promoter.

A preferred strain genetically modified at the level of the STL1 gene according to the invention has a transcription rate of the STL1 gene multiplied by at least 100 with respect to the corresponding non-mutant strain, preferably multiplied by at least 200, more preferably at least by 250. the transcription rate being calculated after quantitative PCR testing on cDNA obtained by reverse transcription.

The GUP1 gene encodes a membrane protein involved in the active transport of glycerol in the cell.

A strain genetically modified according to the invention at the level of the GUP1 gene expresses the GUP1 gene in the presence of sugar.

The GUP2 gene encodes a membrane protein involved in the active transport of glycerol in the cell.

A strain according to the invention genetically modified at the level of the GUP2 GUP1 gene expresses the GUP2 GUP1 gene in the presence of sugar.

The FPS1 or YLL043W gene encodes a membrane protein that allows in particular the export of glycerol.

A strain according to the invention genetically modified at the level of the FPS1 gene does not express the FPS1 gene, for example, followed by deletion of the gene.

The ADR1 or YDR216W gene encodes a transcription activator involved in the expression of genes involved in the ethanol, glycerol and fatty acid utilization pathways. Such genes in particular are regulated by glucose repression.

A strain according to the invention genetically modified at the level of the ADR1 gene expresses the ADR1 gene in the presence of sugar.

The GUT1 or YHL032C gene encodes a glycerol kinase that converts glycerol to glycerol-3-phosphate.

A strain according to the invention genetically modified at the level of the GUT1 gene expresses the GUT1 gene in the presence of sugar.

The GUT2 or YIL155C gene encodes glycerol-3-phosphate dehydrogenase that converts glycerol-3-phosphate into DHAP (dihydroxyacetone phosphate).

A strain according to the invention genetically modified at the level of the GUT2 gene expresses the GUT2 gene in the presence of sugar.

A strain according to the invention, for example, is modified by conversion with an expression cassette allowing expression of a gene as defined above, in particular in the presence of sugar.

The expression cassette comprises a promoter, a gene of interest and a terminator. The terminator is an artificial sequence or a yeast termination sequence.

The promoter is a constitutive or inducible promoter.

A preferred promoter according to the invention is the pADH1 glucose-inducible promoter.

An object of the present invention is a strain as defined above, characterized by being the strain deposited with the CNCM on December 20, 2007, with CNCM number I-3886.

The yeast strain number CNCM I-3886 was obtained by integrating the expression cassette represented in Figure 2 into the genome of the strain deposited at CNCM with number CNCM I-3399.

The yeast strain number CNCM I-3886 strongly expresses the STL1 gene in the presence of sugar (mRNA level more than 270 times higher than the non-mutant strain I-3399).

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM I-3886 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM I-3886 consumes more than 90% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% to 80%.

An object of the present invention is a strain, as defined above, characterized by being the strain deposited with the CNCM on December 20, 2007, with CNCM number I-3887.

The yeast strain number CNCM I-3887 was obtained by integrating the expression cassette represented in Figure 2 into the genome of the strain deposited at NCYC (National Collection of Yeast Cultures) with number NCYC 996.

Yeast strain number CNCM I-3887 strongly expresses the STL1 gene in the presence of sugar (mRNA level more than 270 times higher than that of the non-mutant strain NCYC 996).

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM I-3887 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM I-3887 consumes more than 90% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% and 80%. %.

An object of the present invention is a strain, as defined above, characterized by being a strain deposited with the CNCM on December 20, 2007, with CNCM number I-3888.

The yeast strain number CNCM I-3888 was obtained through the integration of the expression cassette represented in Figure 2 into the genome of the strain deposited at NCYC (National Collection of Yeast Cultures) with number NCYC 995.

The yeast strain number CNCM 1-3888 therefore strongly expresses the STL1 gene in the presence of sugar (mRNA level more than 300 times higher than the non-mutant strain NCYC 995).

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM I-3888 consumes more than 95% of the glycerol in the mixture.

In continuous culture, in a culture medium containing a mixture of glycerol and sugar, the yeast strain CNCM I-3888 consumes more than 90% of the glycerol in the mixture, when the proportion of glycerol in said mixture varies between 20% to 80%. %.

An object of the present invention is a strain as defined above, characterized by being the strain deposited in the CNCM on March 3, 2009, with the number CNCM 1-4132.

The yeast strain number CNCM I-3886 was obtained through the integration of the expression cassette represented in Figure 2 into the genome of the strain deposited at CNCM with the number CNCM I-3399.

The yeast strain number CNCM 1-4132 therefore strongly expresses the STL1 gene in the presence of sugar (mRNA level more than 200 times higher than the non-mutant strain I-4072).

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of 20% glycerol as sucrose equivalent, the yeast strain CNCM 1-4132 consumes more than 80% of the glycerol in the mixture.

An object of the present invention is a strain as defined above, characterized by being the strain deposited at the CNCM on March 3, 2009, with the number CNCM 1-4133.

The yeast strain CNCM 1-4133 was obtained through the integration of the expression cassette represented in Figure 2 into the genome of the strain deposited at CNCM on September 4, 2008, with the number 1-4071.

Yeast strain number CNCM 1-4133 strongly expresses the STL1 gene in the presence of sugar (mRNA level more than 200 times higher than non-mutant strain 1-4071).

In semi-continuous culture, in a culture medium containing a mixture of glycerol and sugar, with a proportion of glycerol of 20% as sucrose equivalent, the yeast strain CNCM 1-4133 consumes more than 80% of the glycerol in the mixture.

An object of the present invention is also a strain of Saccharomyces derived from strain CNCM I-3886, strain CNCM I-3887, strain CNCM I-3888, strain CNCM 1-4132, strain CNCM 1-4133 and strain CNCM 1 -4129, strain CNCM I-4130 or strain CNCM 1-4131, characterized in that in the presence of a culture medium, comprising a mixture of glycerol and sugar, consume at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of at least 20% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is also a strain of Saccharomyces derived from strain CNCM I-3886, strain CNCM I-3887, strain CNCM I-3888, strain CNCM 1-4132, strain CNCM 1-4133, strain CNCM 1-4129, strain CNCM I-4130 or strain CNCM 1-4131, characterized in that in the presence of a culture medium comprising a mixture of glycerol and sugar, consuming at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of 20% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

By the expression “derived strain”, we mean a strain derived by any conversion, such as, for example, by one or more crossings and/or by mutation and/or by genetic transformation.

An object of the present invention is also the use of a yeast strain as defined above, for the production of yeast, in the presence of a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol of at least 5% as equivalent in sucrose, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

An object of the present invention is the use of a yeast strain as defined above, for the industrial production of yeast, in the presence of a mixture of glycerol and sugar.

An object of the present invention is the use of a yeast strain as defined above, for the production of yeast, the production being carried out in accordance with a standard system well known to those skilled in the art, but carried out under aerobic conditions and using a mixture of glycerol and sugar as a carbon source.

An object of the present invention is also the use of a mixture of glycerol and sugar for the production of yeasts from at least one strain of Saccharomyces, characterized in that said strain consumes at least 80% of the glycerol in said mixture, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

Such use is of particular interest for the production of yeast on an industrial scale.

The expression at least one strain of Saccharomyces means that yeast production can be carried out from several different yeast strains, from the same species or from different species.

Preferably, yeast production is carried out from a single yeast strain.

The mixture of glycerol and sugar is as defined above.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that the production of yeast is carried out in a continuous or semi-continuous manner.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that the production of yeast is carried out under aerobic conditions.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that the production of yeast is carried out for a period preferably less than 48 hours, more preferably less than 36 hours, even more preferably less than 24 hours.

Advantageously, the production period is between 10 hours and 24 hours.

The production period is, for example, 22 hours.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that the production yield is similar to that obtained with a standard strain of yeast produced in the absence of glycerol.

For example, the yield of yeast production according to the invention is greater than 85%, preferably greater than 90%, more preferably greater than 95% of the yield of a standard strain produced in the absence of glycerol.

Standard strain means, for example, baker's yeast deposited at NCYC under number NCYC 995.

By “yield” or “production yield” or “biomass production yield”, we mean the ratio of the mass of yeast produced to the mass of sucrose equivalent used.

The mass of sucrose equivalent used is made up of the mass of sucrose equivalent consumed and that not consumed.

The culture medium is as defined above.

According to another embodiment, an object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that the culture medium comprises another carbon source that can be assimilated by the yeast strain. Another carbon source that can be assimilated by the yeast strain is, for example, ethanol. In such incorporation the mixture of glycerol and sugar constitutes at least 70% of the carbon source for the yeast, preferably at least 80% of the carbon source for the yeast, more preferably at least 90% of the carbon source for the yeast.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast as defined above, characterized in that not all of the culture medium is placed in the presence of the yeast strain from the beginning of the culture.

The culture medium is preferably placed in the presence of the yeast strain in installments over time, at a constant or variable rate, the different constituent elements of the culture medium being supplied at the same time or separately.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast, as defined above, characterized in that the mixture of glycerol and sugar is introduced into the culture medium in installments over time.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast, as defined above, characterized in that the mixture of glycerol and sugar is introduced into the culture medium separately from the other components of the culture medium.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast, as defined above, characterized in that the mixture of glycerol and sugar is prepared before its introduction into the culture medium and/or is formed in the culture medium, when glycerol and sugar are supplied separately.

An object of the present invention is the use of a mixture of glycerol and sugar for the production of yeast, as defined above, characterized in that the strain is not placed in the culture, in the presence of all the glycerol and/or all the sugar in the mixture. from the beginning of culture.

A particular object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeast from at least one strain of Saccharomyces, characterized in that said strain consumes at least 85% of the glycerol in the mixture, preferably at least 90% of the glycerol in the mixture, more preferably at least 95% of the glycerol in the mixture, even more preferably at least 98% of the glycerol in the mixture.

An object of the present invention is also the use as defined above of a mixture of glycerol and sugar for the production of yeast, characterized in that said mixture comprises a proportion of glycerol of at least 8% as sucrose equivalent, preferably at least 10%. as sucrose equivalent, more preferably at least 12% as sucrose equivalent, even more preferably at least 15% as sucrose equivalent, still more preferably at least 17% as sucrose equivalent, still more preferably at least 20% as sucrose equivalent .

A particular object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeast, characterized in that the proportion of glycerol in said mixture comprises from 8% to 30% as sucrose equivalent, preferably 10%. to 20% as sucrose equivalent, more preferably from 12% to 17% as sucrose equivalent.

An object of the present invention is also the use as defined above of a mixture of glycerol and sugar for the production of yeast, characterized in that said mixture comprises a proportion of glycerol of at least 30% as sucrose equivalent, preferably at least 40%. as sucrose equivalent, more preferably at least 50% as sucrose equivalent, even more preferably at least 60% as sucrose equivalent, still more preferably at least 70% as sucrose equivalent, still more preferably at least 80% as sucrose equivalent. sucrose, even more preferably at least 90% as sucrose equivalent.

An object of the present invention is also the use as defined above of a mixture of glycerol and sugar for the production of yeast, in that the proportion of glycerol in said mixture is at most 95% as sucrose equivalent, preferably at most 90% as equivalent. in sucrose, more preferably at most 85% as sucrose equivalent.

A particular object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeast, characterized in that the proportion of glycerol in said mixture comprises from 30% to 90% as sucrose equivalent, preferably 40%. to 80% as sucrose equivalent, more preferably from 50% to 70% as sucrose equivalent.

An object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeast of a strain, characterized in that said strain is chosen from the species cerevisiae, boulardii, carlsbergensis and uvarum.

An object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeasts from at least one strain of Saccharomyces, characterized in that said strain is chosen from a natural variant and/or from a genetically modified strain.

An object of the present invention is the use as defined above of a mixture of glycerol and sugar for the production of yeasts from at least one strain of Saccharomyces, characterized in that said strain is chosen from: - strain deposited with the CNCM on December 20, 2007 with CNCM number I- 3886, - strain deposited at CNCM on December 20, 2007 with CNCM number I- 3887, - strain deposited at CNCM on December 20, 2007 with CNCM number I- 3888, - strain deposited at CNCM on March 3, 2009 with number 1-4132, - strain deposited with CNCM on March 3, 2009 with number 1-4133, - strain deposited with CNCM on March 3, 2009 with number 1-4129, - strain deposited with the CNCM on March 3, 2009 with number 1-4130 or - strain deposited with the CNCM on March 3, 2009 with number 1-4131, or a mixture thereof.

An object of the present invention is also yeasts obtained by using as defined above a mixture of sugar and glycerol.

An object of the present invention is also a method for producing yeast comprising the steps of: introducing a strain of yeast of the genus Saccharomyces into a fermentor, and multiplying said strain under aerobic conditions in a culture medium comprising a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as sucrose equivalent.

The mixture of glycerol and sugar is as defined above.

The culture medium is as defined above.

An object of the present invention is a method for producing yeast as defined above, characterized in that the multiplication of said strain is carried out according to a semi-continuous mode or according to a continuous mode, the continuous or semi-continuous modes being as defined above.

The duration of the culture of the method for producing yeast according to the invention is preferably less than 48 hours, more preferably less than 36 hours, even more preferably less than 24 hours.

Advantageously, the duration of the culture of the method for producing yeast according to the invention is between 10 and 24 hours.

The production period, for example, is 22 hours.

The pH is preferably adjusted to a value between 4.0 and 6.5, for example, by the simultaneous supply of acids or bases.

The temperature is preferably adjusted to a value between 30°C and 34°C, for example, by heating or cooling the fermenter.

Advantageously, the yeast production method according to the invention produces yields similar to those obtained with standard strains of yeast produced in the absence of glycerol.

For example, the yield of yeast production according to the invention is greater than 85%, preferably greater than 90%, more preferably greater than 95% of the yield of a standard strain produced in the absence of glycerol.

By standard strain, for example, baker's yeast deposited at NCYC with number NCYC 995 can be mentioned.

By "yield" is meant the ratio between the mass of yeast produced and the mass of sucrose equivalent used.

The mass of sucrose equivalent used is made up of the mass of sucrose equivalent consumed and not consumed.

Yeasts obtained by culturing strains according to the invention in the presence of a mixture of glycerol and sugar and/or yeasts obtained according to the yeast production method of the invention have similar qualities to yeasts used in a standard way and produced in a sugar-type carbon substrate (in the absence of glycerol).

The term “quality” means both functional quality and preservation qualities.

For example, said yeasts have similar qualities in terms of resistance to drying, in terms of production yield, in terms of fermentation power to baker's yeast, in terms of alcohol production and/or resistance to strong concentrations of alcohol to alcohol yeasts are used in a standard way and produced on a carbonaceous sugar-type substrate (in the absence of glycerol).

The yeasts obtained by culturing strains according to the invention in the presence of. a mixture of glycerol and sugar and/or yeast obtained in accordance with the yeast production method of the invention has an intracellular glycerol concentration preferably less than 1.5% by dry mass, more preferably less than 1%.

This low concentration of intracellular glycerol reflects the absence of glycerol accumulation in the medium and, therefore, the fact that it was metabolized.

In a particular embodiment, the culture medium comprises another carbon source that can be assimilated by the yeast strain. Another carbon source that can be assimilated by the yeast strain is, for example, ethanol. In such an incorporation, the mixture of glycerol and sugar constitutes at least 70% of the carbon source for the yeast, preferably at least 80% of the carbon source for the yeast, more preferably at least 90% of the carbon source for the yeast. .

In a preferred embodiment, not all of the culture medium is placed in the presence of the yeast strain from the beginning of the culture. The culture medium is preferably placed in the presence of the yeast strain in installments over time, at a constant or variable rate, making it possible for the different constituent elements of the culture medium to be supplied at the same time or separately.

In a preferred embodiment according to the invention, the mixture of glycerol and sugar is introduced into the culture medium in installments over time.

In a preferred embodiment according to the invention, the mixture of glycerol and sugar is introduced into the culture medium separately from the other components of the culture medium.

The mixture of glycerol and sugar can be prepared before its introduction into the culture medium and/or be formed in the culture medium when glycerol and sugar are supplied separately.

In a preferred embodiment according to the invention, the strain is not placed in the culture, in the presence of all the glycerol and/or all the sugar in the mixture from the beginning of the culture.

An object of the present invention is also a method for producing yeast as defined above which comprises the steps of: introducing a strain of yeast of the genus Saccharomyces into a fermentor, optionally multiplying said strain under aerobic conditions in a culture medium without glycerol, multiplication of said strain under aerobic conditions, according to semi-continuous or continuous mode, in culture medium, comprising a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent , the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as equivalent in sucrose, optionally multiplying said strain under aerobic conditions in a culture medium without glycerol.

An object of the present invention is a method for producing yeast comprising the steps of: introducing a strain of yeast of the genus Saccharomyces into a fermentor, optionally multiplying said strain under aerobic conditions in a culture medium without glycerol, multiplying the said strain under aerobic conditions, in culture medium, comprising a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol of at least 5% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100% as equivalent in sucrose, optionally multiplying said strain under aerobic conditions in a culture medium without glycerol, and separating by centrifugation the baker's yeast then produced from this culture medium, obtaining a “cream yeast” liquid containing approximately between 14 and 25% dry mass, or even more dry mass if the yeast cream is mixed with osmotic products.

An object of the present invention is also the method for producing yeast as defined above comprising the optional subsequent steps of: filtering the cream yeast liquid thus obtained, generally in a rotary vacuum filter, obtaining a fresh dehydrated yeast containing approximately 26 to 35% dry dough, mix this fresh dehydrated yeast to obtain a very homogeneous dough. extrusion of the yeast thus obtained, obtaining a pressed yeast in the form of chunks of fresh yeast or crushed fresh yeast, sold with approximately 30% dry mass, or if the yeast is intended to be dried, in the form of vermicelli, drying controlled way, in a stream of hot air, for example, by fluidizing yeast particles obtained by extrusion, and packaging.

A particular object of the present invention is a method for producing yeast as defined above, characterized in that at least 80% of the glycerol of said mixture is consumed by said strain, preferably at least 85% of the glycerol of said mixture, more preferably at least 90% of the glycerol of said mixture, even more preferably at least 95% of the glycerol of said mixture, even more preferably at least 98% of the glycerol of said mixture.

An object of the present invention is also a method for producing yeast, as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 8% as sucrose equivalent, preferably at least 10% as sucrose equivalent, more preferably at least 12% as a sucrose equivalent, even more preferably at least 15% as a sucrose equivalent, even more preferably at least 17% as a sucrose equivalent, even more preferably at least 20% as a sucrose equivalent.

A particular object of the present invention is a method for producing yeast, as defined above, characterized in that the proportion of glycerol in said mixture comprises from 8% to 30% as sucrose equivalent, preferably from 10% to 20% as sucrose equivalent. , more preferably 12% to 17% as sucrose equivalent.

A particular object of the present invention is a method for producing yeast, as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 30% as sucrose equivalent.

The most particular object of the present invention is a method for producing yeast, as defined above, characterized in that said mixture comprises a proportion of glycerol of at least 40% as sucrose equivalent, preferably at least 50% as sucrose equivalent, more preferably at least 60% as sucrose equivalent, even more preferably at least 70% as sucrose equivalent, even more preferably at least 80% as sucrose equivalent, even more preferably at least 90% as sucrose equivalent.

An object of the present invention is also a method for producing yeast, as defined above, characterized in that the proportion of glycerol in said mixture is at most 95% as sucrose equivalent, preferably at most 90% as sucrose equivalent, more preferably in maximum 85% as sucrose equivalent.

A particular object of the present invention is a method for producing yeast, as defined above, characterized in that the proportion of glycerol in said mixture comprises 30% to 90% as sucrose equivalent, preferably 40% to 80% as sucrose equivalent, more preferably 50% to 70% as sucrose equivalent.

An object of the present invention is a method for producing yeast, as defined above, characterized in that said yeast strain is chosen from the species cerevisiae, boulardii, carlsbergensis and uvarum.

An object of the present invention is a method for producing yeast as defined above, characterized in that said yeast strain is chosen from a natural variant and/or a genetically modified strain.

An object of the present invention is a method for the production of yeast, as defined above, characterized in that said yeast strain is chosen from: - strain deposited with the CNCM on December 20, 2007 with CNCM number I-3886, - strain deposited at the CNCM on December 20, 2007 with the number CNCM I- 3887, - strain deposited at the CNCM on December 20, 2007 with the number CNCM I- 3888, - strain deposited at the CNCM on March 3, 2009 with the number 1-4132, - strain deposited at CNCM on March 3, 2009 with number 1-4133, - strain deposited at CNCM on March 3, 2009 with number 1-4129, - strain deposited at CNCM on March 3, 2009 2009 with number 1-4130 or - strain deposited with the CNCM on March 3, 2009 with number 1-4131, or a derived mixture.

An object of the present invention is a method, as defined above, characterized in that said yeast strain is a strain as defined above.

An object of the present invention is also yeasts that can be obtained by implementing the yeast production method as defined above.

In a particular embodiment of the invention, residual glycerol is recycled for use in a mixture of glycerol and sugar.

According to another embodiment, the mixture consisting of glycerol and sugar can contain a proportion of glycerol in the range of up to 99.9% as a sucrose equivalent, or practically up to 100% as a sucrose equivalent.

The following examples illustrate the invention without limiting it.

EXAMPLES Example 1: Obtaining genetically modified yeast strains according to the invention Material and methods

The STL1 gene (YDR536w) encodes active glycerol permease. This gene was amplified from the genomic DNA of an industrial yeast and sequenced.

The sequence of the amplified STL1 gene, composed of 1710 nucleotides, was aligned with the nucleotide sequence of the YDR536w ORF from yeast S288c (SGD database). The alignment demonstrated the presence of 7 different nucleotides in the entire sequence, which corresponds to an identity percentage of 99.6%.

The translation of the nucleotide sequence of the STL1 gene from industrial yeast leads to a protein of 570 amino acids. The alignment of the protein sequence of the STL1 gene from industrial yeast with the protein sequence of the YDR536w ORF from S288c yeast demonstrated the presence of 3 different amino acids, which corresponds to a percentage of identity of 99.5%.

The construction of genetically modified industrial yeast strains is based on a molecular tool suitable for the industrial yeasts constructed by the applicant.

This tool in particular allows: - the cloning of a gene of interest, here the STL1 gene, between the ADH1 promoter and the yeast CYC1 terminator, which allows the expression of this gene in yeast, - the presence on both sides of the cassette of expression of 2 recombinogenic fragments (BUD5-A and BUD5-B, each of 500 nucleotides), which allow the insertion by homologous recombination of the cassette into the yeast genome, after transformation, in the BUD5 locus (YCR038c) located on chromosome III, - the presence of the KANMX selection marker, framed by loxP 2 sites, which allows the selection of yeast clones, having integrated the expression cassette containing the STL1 gene into their genome; these same clones are then transformed with a plasmid containing the Cre gene coding for a recombinase placed under the control of the Gal1 promoter; After inducing the expression of this gene by culture in the medium containing galactose, the action of the recombinase will induce the loss of the DNA fragment containing the KANMX selection marker.

The expression cassette is shown in Figure 1.

The 5 starter strains used are as follows: the Saccharomyces cerevisiae strain deposited at CNCM under number I-3399, the Saccharomyces cerevisiae strain deposited at NCYC under number NCYC996, the Saccharomyces cerevisiae strain deposited at NCYC under number NCYC995, the strain of Saccharomyces cerevisiae deposited at the CNCM with the number I-4072, and the strain of Saccharomyces cerevisiae deposited at the CNCM with the number 1-4071.

Each of the initial strains is transformed with the expression cassette. Geneticin-resistant clones are selected. Then, for each selected clone, the integration of the expression cassette into the yeast genome at the correct locus is verified by PCR. The selected clones are then transformed with a plasmid containing the Cre gene encoding by a recombinase placed under the control of the Gal1 promoter. After induction of expression of this Cre gene by culture in medium containing galactose, the action of recombinase makes it possible to induce the loss of the DNA fragment containing the KANMX selection marker. The loss of said KANMX selection marker at the site of integration in the chromosome is then verified by PCR, the presence of the expression cassette containing the STL1 gene in the yeast genome at the correct locus by PCR, as well as the loss of resistance to geneticin. Loss of the Cre plasmid is verified by loss of resistance to nourseothricin.

Finally, the transcription of the STL1 gene in the presence of sugar in the genetically modified strain is verified by measuring the level of mRNA originating from the transcription of the STL1 gene placed under the control of the ADH1 promoter. For this purpose, the modified strain is grown in the presence of sugar (YPG laboratory culture medium containing 2% glucose). The mRNA is extracted from said strains and the level of mRNA corresponding to the transcription of the STL1 gene is measured by quantitative PCR. The mRNA level of the mutant strain is then expressed as a function of the mRNA level of the corresponding non-mutant strain (set at 1).

Results

The strains genetically modified to express the STL1 gene thus obtained were deposited at the CNCM with the following numbers: strain number I-3886, originating from the initial strain deposited at the CNCM with number I-3399, strain number I-3887, originating from the initial strain deposited at NCYC under number NCYC996, strain number I-3888, originating from the initial strain of Saccharomyces cerevisiae deposited at NCYC under number NCYC995, strain number 1-4132, originating from the initial strain deposited at CNCM under number I-4072, and strain number 1-4133, originating from the initial strain deposited at the CNCM with number 1-4071.

The transcription rates indicated in Figure 2 show a large increase (270- to 300-fold) in transcription of the STL1 gene in three genetically modified strains I-3886, I-3887 and I-3888 grown in the presence of sugar, compared to gene transcription rate in the corresponding non-mutant control strain grown in the same medium.

Regarding the transcription rate of strains 1-4132 and 1-4133 grown in the presence of sugar, a large increase (more than 200 times) in the transcription of the STL1 gene is also demonstrated, compared to the transcription rate of the gene in the strain no corresponding control mutant grown in the same medium.

EXAMPLE 2: Production of yeast from genetically modified strains according to the invention in a mixture of glycerol and sugar Material and methods (i) Semi-continuous Mode

The production of fresh yeast is carried out in the manner described in the work "Yeast Technology, 2nd edition, 1991, G. Reed and T.W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.

The fermenter used is seeded with a certain initial volume containing water, some nitrogen and phosphorus, nutritional elements such as vitamins and minerals.

Before seeding the fermenter with a certain amount of live yeast strains, some molasses is added to start the culture. The pH and temperature are adjusted respectively to values between 3.5 and 7.0 for pH (with simultaneous supplies of acids or bases as necessary) and between 30 and 34°C for temperature (with heating or cooling of the fermenter). as necessary). The so-called "fed batch" culture in semi-continuous mode is started by supplying molasses or a mixture of molasses and glycerol comprising a proportion of 20% glycerol as sucrose equivalent. The flow rate of the molasses or the mixture of glycerol and molasses determines the rate of yeast growth. This variable flow rate is fixed over time so that the sucrose equivalent supplied over time can be completely respired (oxidized) by the yeast. Under such conditions, ethanol production by yeast over time is zero, or limited to the first hours of testing. In order to meet these conditions, culture is, for example, carried out at an average growth rate of 0.16 - 1 h for strains I-3886, I-3887, I-3888 and the corresponding non-mutant strains, and at an average growth rate of 0.13 -1 h for strains 1-4132 and 1-4133 and the corresponding non-mutant strains.

The medium is sufficiently aerated and mixed so as not to be limiting in terms of oxygen.

In addition to molasses or a mixture of glycerol and molasses, a continuous source of nitrogen in the form of urea and phosphorus in the form of phosphoric acid makes it possible to meet the yeast's needs for the synthesis of proteins and other constituents of the cell.

The supply of molasses or the mixture of glycerol and molasses is maintained for 22 hours in order to obtain the desired amount of yeast, taking into account the equipment used.

During the last hours of culture, the supply of molasses or the mixture of glycerol and molasses is progressively reduced in order to reduce the level of yeast germination in the fermenter.

According to this method of use, the supply of the carbon substrate (e.g. the mixture of glycerol and molasses comprising a proportion of 20% glycerol as sugar equivalent) makes it possible to multiply the initial biomass by 40 in 22 hours of culture. , maintaining a metabolism that is mainly respiratory and ensuring a germination rate when harvested of less than 5-10%. The total amount of carbonaceous substrate dumped is a function of the oxygen and calorie transport capacity of the fermentation tank.

The final concentration of the biomass is between 5 and 7% and the nitrogen content of this biomass is between 6 and 9%.

During culture and at the end of culture, the amount of glycerol in the culture medium is measured by HPLC and is compared with the amount of glycerol supplied.

At the end of the culture, the yeast is harvested by centrifugation and the lv mass is measured.

The yeast produced (in g DM) is the difference between the harvested yeast (in g DM) and the yeast in the inoculum (in g DM) (DM meaning Dry Mass).

The percentage of yeast is then obtained by taking the relationship between "yeast produced (g DM)" / "carbon substrate introduced as sucrose equivalent (g)" and multiplying by 100.

The percentage of glycerol consumed is calculated by taking the relationship “amount of glycerol measured at the end of culture (g)” “amount of glycerol supplied during culture (g)” and multiplying by 100.

(ii) In chemostat

The fermenter is fed by a carbon source consisting of a mixture of glycerol and sugar, a nitrogen source, a sulfur source and a phosphorus source. Vitamins and minerals are supplied in excess.

Mixtures of glycerol and glucose comprising 0% to 80% glycerol as sucrose equivalent are tested.

The fermenter is placed on a scale that controls a draft pump to maintain a constant volume in the fermenter, controlling its weight. The culture is limited by the carbon substrate (glucose and glycerol), the other constituents being supplied in excess. The medium is sufficiently aerated and mixed so that it is never limiting in terms of oxygen.

The medium is supplied continuously at a constant flow rate, making it possible to obtain a dilution rate (= flow rate/volume) of 0.15 tr1 and therefore at steady state a constant growth rate of 0.15 h1.

The concentration of sucrose equivalent in the feeding medium is equal to 19 g/kg.

The yield, as well as the residual amount of glycerol, are measured in the culture at steady state.

Steady state is reached when concentrations in the fermenter are stable for at least 3 residence times, the residence time being the relationship 1/[dilution rate].

The biomass production yield in supplied sugar is the ratio between the biomass produced and the sugar supplied. At steady state, the biomass produced per unit time is equal to the draft rate multiplied by the fermenter biomass concentration (determined by filtering a known volume of wort containing the yeast and weighing the yeast cake that has been previously washed and dried).

When determining the yield, a sample of the must containing the yeast is taken and immediately filtered to remove the biomass. The filtrate is analyzed by HPLC to test the residual amount of glycerol.

Results (i) Semi-continuous mode

The non-mutant strains 1-3399, NCYC 996, NCYC 995, 1-4072 and 1-4071 and the corresponding mutant strains I-3886, I-3887, I-3888, 1-4132 and 1-4133 respectively, were produced from according to the procedure described above, with pure molasses or a mixture of glycerol and molasses as carbon source, comprising a proportion of 20% glycerol as sucrose equivalent.

Figures 3 and 4 and Tables 1,2, 3, 4 and 5 illustrate the results obtained with the non-mutant strains and the corresponding mutant strains.

The production yield is determined for each non-mutant strain in the presence of pure molasses (without glycerol). In tables 1, 2, 3, 4 and 5, the values indicated in the "yield" line correspond to the percentage of yield in relation to the yield of the non-mutant strain produced in pure molasses.

Table 1 indicates the results of tests performed with the non-mutant strain NCYC 996 and the corresponding mutant strain I-3887.

Table 2 indicates the results of tests performed with the non-mutant strain I-3399 and the corresponding mutant strain I-3886.

Table 3 indicates the results of tests performed with the non-mutant strain NCYC 995 e. the corresponding mutant strain I-3888.

Table 4 indicates the results of tests performed with the non-mutant strain I-4072 and the corresponding mutant strain 1-4132.

Table 5 indicates the results of tests performed with the non-mutant strain I-4071 and the corresponding mutant strain 1-4133.

Analyzing these tables, it is noted that during production using a mixture of glycerol and molasses (comprising a proportion of 20% glycerol as sucrose equivalent) as a carbon source, the production yield obtained with non-mutant strains is significantly lower. than the production yield obtained from pure molasses. This reduction in yield must be linked to the non-use of a significant part of the sucrose equivalent supplied in the form of glycerol, the glycerol accumulating in the culture medium as indicated in the results in the line “glycerol supply consumed”.

These same tables show the results of tests carried out with the mutant strains, when the carbon source supplied is a mixture of molasses and glycerol. The measurements carried out indicate that the consumption of glycerol supplied is practically total for the three mutant strains I-3887, I-3886 and I-3888 (glycerol consumption greater than 99%), which results in production yields at least equivalent to those obtained with non-mutant strains produced in pure molasses (Tables 1 to 3).

With regard to the mutant strains 1-4132 and 1-4133, the consumption of glycerol supplied is greater than 80% and the yield is greater than 90% of the yield obtained with the non-mutant strain in the absence of glycerol (Tables 4 and 5 ).

This difference in glycerol consumption by non-mutant strains and the corresponding mutant strains is illustrated in Figures 3 and 4, which show the accumulation of glycerol over time during production tests in a mixture of glycerol and molasses comprising a proportion of 20% glycerol as sucrose equivalent.

The curve with triangles in Figure 3 represents the residual amount of glycerol in the yeast-free medium during the culture of the non-mutant strain NCYC 996 and the curve with squares that of the strain I-3887.

The curve with triangles in Figure 4 represents the residual amount of glycerol in the yeast-free medium during the culture of the non-mutant strain NCYC 996 and the curve with squares that of the mutant strain I-3888.

The difference in consumption between the non-mutant strains and the corresponding mutant strains is significant after the first five hours of culture. During the first five hours of culture, the respiratory capacity of non-mutant strains and mutant strains is saturated by the supplied sugar ("Crabtree" effect), this phenomenon being accompanied by the production of ethanol. When this phenomenon disappears, that is, when the yeasts have the respiratory capacity to oxidize all the sugar and glycerol supplied, the mutant strains virtually consume all the glycerol supplied simultaneously with the sugar, while the non-mutant strains accumulate the glycerol. Table 1 Table 2 Table 3 Table 4 Table 5

(ii) Continuous mode

In Figures 5 and 6, each point corresponds to a steady state obtained with a feed medium in which the proportion of glycerol in the mixture of glycerol and sugar is 0%, 20%, 50% or 80% as sugar equivalent.

With a feeding medium whose only carbon source is glucose (absence of glycerol), the biomass production yield of the mutant strain (CNCM-3887) and that of the non-mutant strain (NCYC 996) are identical. Therefore, the mutation of the STL1 gene does not modify the biomass production yield in glucose.

The replacement of 20%, 50% or 80% of glucose as sucrose equivalent 15 by glycerol in the mixture of glycerol and sugar in the feed is accompanied, in the case of the non-mutant strain, by an increasing accumulation of glycerol in the reaction medium ( Figures 7 and 8). This amount of glycerol that accumulates in the reaction medium leads to a marked reduction in the biomass production yield in spilled sugar equivalent (Figures 5 and 6). Thus, at a substitution level of 50%, the production yield of non-mutant strains decreased by 25% relative to the yield of the same strain produced in a medium containing only glucose.

The glycerol consumption of the non-mutant strains is less than 57% at a 20% substitution level, less than 38% at a 50% substitution level, and less than 22% at an 80% substitution level (Figures 7 and 8).

In contrast, the mutant strains consume almost all of the supplied glycerol, simultaneously with sugar, as shown by the very low concentration of residual glycerol measured in the reaction medium. Thus, the mutant strains consume more than 95% of the glycerol in the mixture, up to a replacement level of 80% (Figures 7 and 8). Consequently, the production yield in sugar equivalent supplied is not affected, or only slightly affected, by the level of substitution of glycerol for sugar. The production yield of the mutant strain is always higher than that of the corresponding non-mutant strain in the presence of a glycerol-containing mixture (Figures 5 and 6).

Furthermore, in the presence of a mixture of glycerol and sugar comprising 20% or 50% glycerol as sucrose equivalent, the production yield of the mutant strains is greater than 90% in relation to the non-mutant strain grown in the absence of glycerol, and is greater than 85% in the presence of a mixture of glycerol and sugar comprising 80% glycerol as sucrose equivalent (Figures 5 and 6).

Example 3: Obtaining natural variants according to the invention Material and methods (i) Direct screening

A population of cells from a standard baker's yeast strain is subjected to UV radiation of 1200 to 2000 J/cm2, leading to a loss of viability of 99.9%

The natural variants obtained are isolated in a non-selective medium (YPG), then screened based on their yield and glycerol consumption in the presence of a mixture of glycerol and sugar.

Sorting is carried out after culture in semi-continuous mode, in the presence of a mixture of glycerol and sugar comprising 20% glycerol as sucrose equivalent, in 1 liter and 7 liter fermenters.

The experimental conditions of the semi-continuous culture are as described in Example 2 for a period of 22 hours, with an average growth rate of 0.16 Ir1.

(ii) Screening after enrichment in guimyostats

A population of cells from the same initial strain as before is subjected to increasing levels of UV radiation (from 400 to 4400 J/cm2) leading to viability losses ranging from 80 to 99.98%.

The natural variants subjected to these different levels of radiation are then combined and part is used to sow a culture in a chemostat.

The carbon source of the culture is constituted by a mixture of glycerol and glucose comprising 50% glycerol as sucrose equivalent.

The culture is carried out until approximately 220 cells are generated.

Glycerol consumption is measured during culture.

Culture samples are regularly taken to screen for natural variants.

The natural variants from the different samples are isolated in a non-selective medium (YPG), then triaged based on their yield and their glycerol consumption in the presence of a mixture of glycerol and sugar.

Sorting is carried out after culture in semi-continuous mode, in the presence of a mixture of glycerol and sugar comprising 20% glycerol as sucrose equivalent, in 1 liter and 7 liter fermenters.

The experimental conditions of the semi-continuous culture are as described in Example 2 for a period of 22 hours, with an average growth rate of 0.16.

Results

Natural variants from direct screening or screening after enrichment in chemostats present very varied glycerol consumption and yield profiles.

An example of a result obtained on 53 natural variants from screening after enrichment in chemostats is shown in Figure 9. The glycerol consumption of the initial strain (T) is also indicated.

A number of natural variants show a glycerol consumption greater than 80% in the presence of a mixture of glycerol and sugar comprising 20% glycerol as sucrose equivalent and a yield greater than or equal to 85% of the initial strain.

These include the natural variants deposited in the CNCM with the numbers I- 4129 (natural variant no. 4), 1-4130 (natural variant no. 2O) and 1-4131 (natural variant no. 2) which were obtained by screening after enrichment in corresponding sampling chemostat to obtain approximately 145 generations of cells in a chemostat.

It is noted that the analysis of glycerol consumption during culture in the chemostat reveals that during the first 100 generations, residual glycerol decreases regularly, which suggests an adaptation of the culture to the mixture of glycerol and sugar.

Furthermore, the level of mRNA corresponding to the transcription of the STL1 gene is measured by quantitative RT-PCR, as indicated in Example 1, in different natural variants according to the invention. The mRNA level obtained is then compared with that of the initial strain that has not been subjected to UV treatment.

Depending on the natural variants, an mRNA level similar to that of the initial strain or multiplied by a factor of 2 to a factor of 7 is observed.

The ability to consume glycerol in the presence of a mixture of glycerol and sugar of the strains according to the invention is therefore not necessarily linked to an overexpression of the STL1 gene.

Example 4: Production of yeast of the natural variants according to the invention in a mixture of glycerol and sugar Material and methods

Cultures in semi-continuous mode are carried out as in Example 2, for a period of 22 hours, with an average growth rate of 0.16 h1.

Results

The natural variants 1-4129, 1-4130, and 1-4131, as well as the initial strain of yeast from which they originate, are produced according to the procedure described above, with pure molasses or a mixture of glycerol and molasses comprising a proportion of 20% or 40% glycerol as sucrose equivalent as carbon source.

The production yield for the initial strain is determined in the presence of pure molasses (no glycerol). In tables 6 and 7, the values shown in the "yield" line correspond to the percentage of yield in relation to the yield of the initial strain produced in pure molasses.

Tables 6 and 7 indicate the results of the tests carried out with the natural variants and the initial strain (T) in the presence of a mixture of glycerol and sugar comprising 20% and 40% glycerol respectively. Table 6 Table 7

The measurements carried out indicate that the consumption of glycerol supplied is practically total for the three natural variants in the presence of a mixture of glycerol and sugar comprising 20% glycerol as sucrose equivalent 15 (glycerol consumption greater than 99%), while consumption of the initial strain is less than 40% (Table 6).

In the presence of a mixture of glycerol and sugar comprising 40% glycerol as sucrose equivalent, the measurements carried out indicate that the consumption of the glycerol supplied is practically total for the natural variant 1-4129, greater than 85% for the natural variant 1 -4130 and greater than 90% for the I-4131 variant.

Claims (5)

1. A STRAIN OF Saccharomyces cerevisiae characterized by being selected from the group consisting of: - the strain deposited at the CNCM on December 20, 2007 with the CNCM number I-3886, - the strain deposited at the CNCM on December 20, 2007 with the CNCM number I-3887, - the strain deposited with the CNCM on December 20, 2007 with the CNCM number I-3888, - the strain deposited with the CNCM on March 3, 2009 with the number I 4132 and - the strain deposited with the CNCM on March 3, 2009 with number I 4133. 2. USE OF A STRAIN OF Saccharomyces cerevisiae according to claim 1, for the production of yeast in the presence of a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol between 5% and 90% as sucrose equivalent, characterized in that the sum of the proportions of glycerol and sugar in said mixture is equal to 100% as sucrose equivalent, wherein the sugar is in the form of glucose syrup, fructose syrup, hydrolyzed starches, molasses, or any combination thereof, and in which said strain of Saccharomyces cerevisiae consumes glycerol simultaneously with sugar. 3. The USE according to claim 2, characterized in that the mixture of glycerol and sugar comprises 20% glycerol as sucrose equivalent, and said strain of Saccharomyces cerevisiae consumes at least 80% glycerol in said mixture. 4. A METHOD FOR PRODUCING YEAST comprising the steps of: - introducing a strain of Saccharomyces according to claim 1 into a fermentor, and - multiplying said strain under aerobic conditions in accordance with a semi-continuous mode or in accordance with a continuous in a culture medium, comprising a mixture of glycerol and sugar, said mixture comprising a proportion of glycerol between 5% and 90% as sucrose equivalent, the sum of the proportions of glycerol and sugar in said mixture being equal to 100 % as sucrose equivalent characterized by said strain of Saccharomyces cerevisiae consuming glycerol simultaneously with sugar, and - wherein the sugar is in the form of glucose syrup, fructose syrup, hydrolyzed starches, molasses, or any combination thereof. 5. The METHOD according to claim 4, characterized in that the mixture of glycerol and sugar comprises 20% glycerol as sucrose equivalent, and said strain of Saccharomyces cerevisiae consumes at least 80% glycerol in said mixture.