Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
  • C12G1/00—Preparation of wine or sparkling wine

Definitions

• the present invention relates to the field of yeasts intended for use in the food industry, and in particular relates to yeasts intended for use in the biotransformation of grape juice into wine.
• Grape juice suitable for winemaking is commonly referred to as grape must.
• the present invention relates more particularly, but not exclusively, to a process for the selection of sourdoughs of the genus Saccharomycese ⁇ in particular of the species Saccharomyces cere visiae in order to better control the pH of the product of the fermentation of grape juice into wine and this by selecting their ability to degrade (or produce) organic acids and in particular malic acid and succinic acid.
• the content of all the organic acids in wine contributes significantly to its final acidity and therefore to the final pH, which is an important parameter of its organoleptic quality.
• Grape juice naturally contains many organic substances, including organic acids such as malic acid and tartaric acid.
• the acidity of grape juice and wines prepared from this juice is closely dependent on the content of these two acids.
• the malic acid content in the grape decreases during its ripening to reach values of 2 to 7 g / L at the time of maturity.
• the yeasts can partially degrade the malic acid and produce other organic acids such as succinic acid and pyruvic acid which will in turn modify the acidity.
• the malic acid content is mainly controlled by the malolactic fermentation carried out by lactic bacteria in particular by Oenococcus oeni.This fermentation takes place after alcoholic fermentation, and can be initiated using industrial leaven inoculated into the wine or in the fermenting must.
• Patent EP 2 183364 describes the use of the species Saccharomyces cerevisiae with a selection within the genetic variability of the species. oenological characteristics linked to the production of volatile phenols.
• the species Saccharomyces cerevisiae exhibits a particularly attractive natural genetic variability, but so far no candidate allowing a significant reduction of malic acid has been suitably selected and no strain capable of consuming almost all of the malic acid, with at the very least a significant reduction in malic acid of at least 50% could not be properly identified.
• this species can in certain cases produce small amounts of malic acid as described in Yeramian et al. J. Agric. Food Chem., Vol. 55, No. 3, 2007 912-919. However, the level of malic acid production of these strains remains less than or equal to 0.7 g / L.
• the present invention relates to a yeast strain of the species Saccharomyces cerevisiae comprising at least one peptide sequence polymorphism in at least one of the protein sequences encoded by the genes selected from the eleven genes YLL041C (SDH2), YGL008C (PMA 1), YDL054C (MCH1), YKL029C (MAE 1), YDL021W (GPM2), YOR380W (RDR1), YOR090C (PTC5), YBR218C , YGL037C (PNC1), YBL036C and YDL237W (AiM6), which are located on chromosomes (chr) 2, 4, 7, 11, 12 and 15.
• a polymorphism of a peptide sequence is preferably linked to a single sequence polymorphism (known by the English acronym “SNP”, meaning “single nucleotide polymorphism”) or a succession of several polymorphisms. unique in sequence to the nucleic acid sequence of the gene encoding this protein.
• SNP single nucleotide polymorphism
• the genetic and / or peptide sequences are those identified in the Saccharomyces GENOME DATABASE (SGD) database for the S288C strain and correspond, more particularly, to the sequences detailed in the “sequence listing” part of the present description. .
• the yeast strain according to the invention comprises at least one, preferably from one to six, peptide sequence polymorphisms at the level of the amino acids selected from the following positions in the reference genome: the acid l ' amino acid (Lys158) of the protein encoded by the YLL041C gene, the amino acid (Pro74) of the protein encoded by the YGL008C gene, the amino acid (Asp200) of the protein encoded by the YGL008C gene, the amino acid (Ser63) of the protein encoded by the YDL054C gene, the amino acid (Ile605) at the level of the protein encoded by the YKL029C gene, the amino acid (Met419) at the level of the protein encoded by the YOR090C gene, the amino acid (Glu722) of the protein encoded by the YBR218C gene and the amino acid (Glu 165) of the protein encoded by the YDL237W gene.
• said strain comprises the peptide sequence polypeptide sequence poly
• the yeast strain of the species Saccharomyces cerevisiae comprises at least one peptide sequence polymorphism in the sequence of each of the proteins encoded by the two genes YLL041C and YGL008C located on chromosomes 7 and 12
• Such a selection of individuals makes it possible to obtain an average content of malic acid consumed, which is taken over the entire population, and which is greater than 51%, being expressed as a percentage by mass calculated relative to the total mass of malic acid present at the beginning.
• said strain comprises two sequence polymorphisms peptide at the level of two amino acids selected from the following positions in the reference genome: the amino acid (Lys 158) of the protein encoded by the YLL041C gene, the amino acid (Pro74) of the protein encoded by the YGL008C gene , the amino acid (Asp200) of the protein encoded by the gene and YGL008C and the amino acid (Ile605) at the level of the protein encoded by the YKL029C gene.
• the yeast strain of the Saccharomyces cerevisiae species according to the invention comprises at least one peptide sequence polymorphism in the sequence of each of the proteins encoded by the four genes YLL041C, YGL008C, YDL054C and YDL237W located on the chromosomes 4, 7 and 12.
• YLL041C YGL008C
• YDL054C YDL237W located on the chromosomes 4, 7 and 12.
• said strain comprises four peptide sequence polymorphisms at the level of four amino acids selected from the following positions in the reference genome: the amino acid (Lys 158) of the protein encoded by the YLL041C gene, the amino acid ( Pro74) of the protein encoded by the YGL008C gene, the amino acid (Ser63) of the protein encoded by the YDL054C gene and the amino acid (Glu 165) of the protein encoded by the YDL237W gene.
• the yeast strain of the species Saccharomyces cerevisiae comprises at least one peptide sequence polymorphism in each of the sequences of five to six proteins encoded by the seven genes YLL041C, YGL008C, YDL054C, YKL029C, YOR090C , YBR218C and YDL237W located on chromosomes 2, 4, 7, 11, 12 and 15.
• YLL041C YGL008C, YDL054C, YKL029C, YOR090C , YBR218C and YDL237W located on chromosomes 2, 4, 7, 11, 12 and 15.
• said strain comprises five peptide sequence polymorphisms at the level of five amino acids selected from the following positions in the reference genome: the amino acid (Lys158) of the protein encoded by the YLL041C gene, the acid amino (Pro74) of the protein encoded by the YGL008C gene, the amino acid (Ser63) of the protein encoded by the YDL054C gene, the amino acid (Ile605) at the level of the protein encoded by the YKL029C gene, the acid amino (Met419) at the level of the protein encoded by the YOR090C gene and the amino acid (Glu 165) of the protein encoded by the YDL237W gene.
• the amino acid (Lys158) of the protein encoded by the YLL041C gene the acid amino (Pro74) of the protein encoded by the YGL008C gene
• the amino acid (Ser63) of the protein encoded by the YDL054C gene the amino acid (Ile605) at the level
• said strain comprises six peptide sequence polymorphisms at the level of six amino acids selected from the following positions in the reference genome: the amino acid (Lys158) of the protein encoded by the YLL041C gene, the acid amino (Pro74) of the protein encoded by the YGL008C gene, the amino acid (Ser63) of the protein encoded by the YDL054C gene, the amino acid (Ile605) at the level of the protein encoded by the YKL029C gene, the acid amino (Met419) at the protein encoded by the YOR090C gene, the amino acid (Glu722) of the protein encoded by the YBR218C gene and the amino acid (Glu165) of the protein encoded by the YDL237W gene.
• the amino acid (Lys158) of the protein encoded by the YLL041C gene the acid amino (Pro74) of the protein encoded by the YGL008C gene
• said strain comprises two to six substitutions of amino acids in the reference genome among the following: (Glu 158) of the protein encoded by the YLL041C gene, (Leu74) of the protein encoded by the gene YGL008C, (Leu63) of the protein encoded by the YDL054C gene, (Leu419) of the protein encoded by the YOR090C gene, (Lys722) of the protein encoded by the YBR218C gene and (Ala165) of the protein encoded by the YDL237W gene.
• Such a strain selection is advantageously used to obtain a strong reduction in the level of malic acid, and to increase the pH of a wine.
• Such a selection of individuals makes it possible to obtain a content of malic acid consumed in excess of 80% and which produce a wine whose pH is modified by a value of up to 0.4 units depending on the strain from the same starting grape juice.
• said strain comprises from one to two substitutions of amino acids in the reference genome from among the following: (Glu200) of the protein encoded by the gene and YGL008C and (Val605) at the level of the protein encoded by the YKL029C gene.
• Such strain selection makes it possible to obtain a strong increase in malic acid, and to decrease the pH of a wine.
• Such a strain selection is advantageously used to obtain a strong increase in the level of malic acid, and to reduce the pH of a wine.
• Such selection of individuals makes it possible to obtain a production of malic acid greater than 35% of the value of malic acid initially present. This production makes it possible to reduce the pH of the wine by a value of up to 0.4 units depending on the strains used from the same starting grape juice.
• the present invention also relates to a method for selecting oenological yeast strains as described above in the context of the present invention, comprising the steps of:
• step 3- obtain meiotic descendants from the hybrids of step 1- by induction of sporulation and isolation of spores;
• 6- carry out genotyping of the genome of the descendants making it possible to know the genotype of at least two loci; 7- carry out a genetic mapping from the data obtained in step 4- and in step 6-; and
• 8- select at least one group of individuals among individuals carrying demalicizing alleles and individuals carrying missing alleles.
• selection means the identification and isolation of oenological yeasts, that is to say yeasts involved in the process of making wine, and in particular in the metabolism of acids and bases present in wines.
• malignant allele means the allele which confers lower consumption of malic acid content during alcoholic fermentation, or even production of malic acid during alcoholic fermentation.
• genotyping means determining the inheritance of a portion of DNA at a specific point in the genome. In the context of the present invention, genotyping corresponds to the identification of a nucleotide sequence which is associated with the demalicizing character or the malignant character.
• Step 1- preferably consists in obtaining a hybrid by crossing two strains A and B of the species Saccharomyces cerevisiae according to the method of pairing spores with the micromanipulator described in the article by Marullo et al. (Marullo et al. 2009 FEMS Yeast Research, 9, 8: 1148-1160).
• the resulting hybrid is called H1, it is the product of the conjugation of spores resulting from the cross between strain A and strain B.
• the resulting hybrid H1 is heterozygous at many points of its genome.
• the technique of hemizygous hybrids implemented in step 2- is the method described in the article by Steinmetz et al., 2002, Nature, 416, 6878: 326-330.
• the principle of said method consists of eliminate by homologous recombination one of the two parental copies of the studied gene, by inserting at the locus of said studied gene, a gene for resistance to an antibiotic.
• hemizygous hybrids express only one of the two parental copies of the gene studied. If there is a significant difference (at the 10% threshold) between the two hybrids, the allele has an effect on the studied phenotype.
• the genes tested, the effect of which is significant on the percentage of malic acid consumption at the end of alcoholic fermentation are presented in Table 1.
• L-malic acid is quantified by enzymatic assay after having observed the complete cessation of the alcohol. alcoholic fermentation according to the method described in Peltier, et al., 2018, PLOS ONE, 13 (1), 191353 (https://doi.org/10.1101/191353).
• the allele noted DEMAL is the one which consumes the most malic acid
• the allele noted MAL is the one which preserves the malic acid content the most.
• the results presented were measured in a Merlot must (Merlot 2 sample from Table 1) and in a Sauvignon must (Sauvignon Blanc 1 sample from Table 1) whose initial malic acid contents are respectively estimated at 2.28 and 4. , 32 g / L.
• step 3- is carried out by microdissection or mass purification of spores.
• Step 3- involves induction of sporulation of a hybrid, the aforementioned H1 hybrid, on a medium preferably comprising 1% potassium acetate for 3 days at 24 ° C.
• the use of microdissection of ascospores is preferably carried out using a micromanipulator, for example according to the method published in Marullo et al., 2009, FEMS Yeast Research, 9, 8: 1148-1160.
• ascospores are isolated in bulk after partial purification, preferably using the method published in the article by Curran B.P.G. et al., 2006, “Basic Investigations in Saccharomyces cerevisiae”, Methods in Molecular Biology, vol 313, 1-13.
• step 4- the level of malic acid consumed is measured at the end of alcoholic fermentation by an enzymatic test.
• the measurement of the percentage of malic acid consumed for a set of 94 descendants of the H1 hybrid is carried out by enzymatic assay of L-malic acid after having observed the complete cessation of alcoholic fermentation according to the method described in Peltier and al., 2018, PLOS ONE, 13 (1), 191353 (https://doi.Org/10.1101/191353).
• step 5- the DNA of the descendants is extracted.
• the DNA of each descendant is advantageously extracted using extraction kits such as the DNA-Wizard Promega kit according to the protocol described in Marti-Raga et al. 2017, G3 February, 399-412.
• amplification of the genomic DNA is carried out between steps 5- and 6-.
• Amplification is carried out with a kit, preferably the Nextera XT kit or any other kit suitable for sequencing small genomes.
• the procedure used makes it possible to index, using specific DNA primers, 96 individuals and to amplify their DNA in order to build a Library for Illumina sequencing.
• the protocol used is that provided by the manufacturer, it is described in Marti-Raga et al. 2017, G3 February, 399-412.
• the genotyping carried out in step 6 - implements sequencing, which sequencing is preferably carried out using a sequencing kit which consists firstly in amplifying the DNA in order to construct a Library for Illumina MySeq sequencing using the MySeq Reagent Kit V2 sequencing kit (Pair End 2x250 bases) according to the instructions provided by the manufacturer as described in Marti-Raga et al. 2017, G3 February, 399-412.
• the sequence reads obtained are preferably filtered by bioinformatics methods using the FASTX-Toolkit suite (http: // hannonlab.cshl.edu/fastx_toolkit/).
• the readings retained (quality Q> 20) are then aligned with the BWA software (Li, H., and R. Durbin, 2010.
• SAM tools suite described in the article by Li, H. et al., 2009, Bioinformatics, 25, 2078-2079, the SNPs of each of the descendants are identified and positioned on the genome by Pileup software. These SNPs allow the construction of a very dense genetic map made up of 1000 bi-allelic markers. The method of construction of this type of map and the bioinformatic tools used are described in the article Marti-Raga et al. 2017, G3 February, 399-412.
• the genetic mapping used in step 7- of the method according to the invention is a method which establishes a statistical link between the inheritance of genetic markers and the variation of a quantitative character.
• the genetic markers which are statistically linked are “quantitative character loci” (the English translation of which is “Quantitative Trait Loci”, with the associated acronym “QTL”) such as those described in application EP 2 183 364.
• Quantitative character loci the English translation of which is “Quantitative Trait Loci”, with the associated acronym “QTL”
• QTL Quantitative Trait Loci
• step 7- the bi-allelic markers (they are in the order of a thousand) from step 6- are used to perform genetic mapping.
• a statistical link is found between the variation in quantitative character and four genetic markers with a very high significance level (ll_150, XV_1052, IV_31 and IV_360).
• the statistical test used is based on mapping by the so-called "interval mapping” method using a Haley-Knott regression (Peltier et al. 2018., BMC Genomics 19, 772).
• the genomic position of the markers, the gene concerned and the value of the probability of the test (risk of the first species) are listed in table 2.
• the effect of the MAL and DEMAL allele is also indicated, expressed as a percentage of malic acid consumed.
• the DEMAL allele corresponds to the "demalicizing trait" which means the allele that confers the phenotype responsible for the elimination of the highest content of malic acid (unlike the MAL allele).
• step 2- of the method the genes and / or the QTLs exhibiting nucleotide variations which are linked to the percentage by mass of consumption of malic acid during the alcoholic fermentation of grape juice by the yeast Saccharomyces cerevisiae are directly identified.
• nucleotide variations affect the sequence of genes YDL237W, YDL054C, YKL029C, YDL021W, YOR380W, YBR218C, YOR090C, YLL041C i YGL037C, YGL008C and YBL036C.
• These nucleotide sequence variations can easily be genotyped within a large population of individuals by genetic typing methods, that is to say any biochemistry or molecular biology method capable of identifying the presence of a variation.
• nucleotide or protein sequence such as PCR, DNA sequencing, protein sequencing, and / or a method based on the detection of SNP using mass spectrometry, according to the technology qualified as sequenom® Gabriel, S., 2009, Current Protocol in Human Genetics, Suppl. (60. doi.org/10.1002/ 0471142905. hg0212s60).
• the MAL and DEMAL alleles used are determined from the reference sequence sacCer3 (sacCer3, version April 2011) derived from the strain S288C and available on the SGD database ("Saccharomyces Genome Data base") with a link accessible via https://www.yeastgenome.org/.
• the nomenclature used is that of the description of the genetic mutations described in the article by den Dunnen JT et al., 2000, Human Genome Variation Society, Hum.Mutat. 15: 7-12.
• pools of alleles are determined which will make it possible to reduce or preserve the malic acid content of the wines at the end of alcoholic fermentation.
• pools of alleles are defined and are designated by the following acronyms: Q2demal, Q2mal, Q4demal, Q4mal, Q6demal, Q6mal, Q7demal, Q7mal, Q6c and Q7c.
• the numbers (2, 4, 6 or 7) correspond to the number of locations taken into account in each pool.
• the Q2demal, Q4demal, Q6demal and Q7demal pools contain only alleles linked to the demalizing trait.
• the Q2mal, Q4mal, Q6mal and Q7mal pools only contain alleles linked to the malignant trait.
• the Q6c and Q7c pools contain the pooled Q4demal alleles with 2 and 3 missing alleles, respectively.
• the method according to the invention advantageously comprises genotyping implementing a detection of polymorphism at the level of a nucleotide (SNP) by mass spectrometry, preferably according to the technology qualified as sequenom® ( sequenom®IPLEX® Gold) with the MassARRAY® Analyzer Chip Prep Module 384 apparatus, marketed by the company Agena Biosciences® and according to the protocol described in the article by Gabriel S. et al., 2009, Current Protoco / s in Human Genetics, Suppl. 60. (doi.org/10.1002/ 0471142905. hg0212s60).
• SNP nucleotide
• the design of the primers is carried out by the MassARRAY® Assay Design tool (version 4.0.0.2) allowing to amplify fragments of less than 120 bases and by selecting mass differences between alleles between 16 and 70 Da. About 15 ng of DNA are needed to genotype individuals using the MassARRAY® iPLEX® platform (Agena Biosciences®).
• the method according to the invention advantageously comprises a step of isolating at least one strain.
• the method according to the invention advantageously comprises a step of isolating at least two strains, respectively designated by the letters X and Z, for example the X28 and Z8 clones, from the descendants of two populations from hybrids obtained: for example, by crossing strains A and B on the one hand, and strains C and D on the other hand.
• the hybrids and their descendants are obtained according to steps 1 and 3 as described above in the context of the invention.
• said two clones X28 and Z8, obtained according to the method according to the invention are crossed and the resulting hybrid is sporulated in order to obtain large offspring by implementing steps 1 and 3 as described above. .
• the DNA of this progeny is extracted by a method described by Albertin et al.
• the present invention also relates to a use of a Saccharomyces cerevisiae yeast comprising a strain as described above in the context of the present invention for modifying the pH of a wine, and preferably for increasing the pH of a wine.
• the method according to the invention advantageously comprises a step of isolating at least two strains, respectively designated by the letters X and Z, for example the clones X8 and Z1, derived from the progeny of two populations resulting from hybrids obtained: for example, by crossing strains A and B on the one hand, and strains C and D on the other hand.
• the hybrids and their descendants are obtained according to steps 1 and 3 as described above in the context of the invention.
• said two clones X8 and Z1, obtained according to the method according to the invention are crossed and the resulting hybrid is sporulated in order to obtain a large progeny by implementing steps 1 and 3 as described above. .
• the DNA of this progeny is extracted by a method described by Albertin et al. which allows rapid extraction compatible with the sequenom® genotyping technique (Albertin et al., 2018, Yeast, 35, 141-156).
• sequenom® described in detail in the experimental part.
• the selection of clones having an appropriate genotype makes it possible to choose individuals with an extreme phenotype without resorting to fermentation tests.
• the present invention also relates to a use of a Saccharomyces cerevisiae yeast comprising a strain as described above in the context of the present invention for reducing the pH of a wine.
• YGL037C, and YBL036C are those described in the SGD database (Saccharomyce Genome Database - www.yeastgenome.org-).
• H2 hybrid by crossing two strains C and D of the species Saccharomyces cerevisiae according to a method described in Marullo, P. et al., 2009, FEMS Yeast Research, 9, 8: 1148-1160 : the H2 hybrid obtained is the product of the conjugation of spores resulting from the crossing between strain C and strain D.
• Isolation of an X28 clone From the H1 hybrid resulting from the crossing of strains A and B, the X28 descendant from a population of 94 descendants isolated by micromanipulation is isolated (Marullo et al., 2009, FEMS Yeast Research, 9, 8: 1148-1160). This technique consists of dissecting yeast asci formed from a culture on Potassium Acetate ACK medium for 3 days at 24 ° C. The asci are digested with cytohelicase (2mg / mL) and the spores are isolated using a micromanipulator (Singer TM MS200). The malic acid content of the 94 descendants is measured at the end of alcoholic fermentation, which fermentation is carried out as follows:
• the fermentations were carried out in 20 ml flasks marketed by the company Fisher Scientific® under the reference 20-HSV. They were used to ferment 10 mL of must from the Merlot 2 sample with a starting malic acid content of 2.280 g / L.
• the vials were closed with screw caps with a 3 mm HT silicone / PTFE septum marketed by Fisher Scientific®. Hypodermic needles were inserted into the septum for the release of CO 2 .
• the fermentations were initiated with the inoculation of 2.10E6 viable cells.mL -1 of culture in liquid phase (YPD) carried out in 1 mL deepwell microplates marketed by the company Fisher Scientific®.
• the concentration of viable cells was estimated by flow cytometry using an apparatus of the Cell Lab Quanta TM brand sold by the company Beckman Coulter®.
• the fermentation temperature was maintained at 24 ° C. using an incubator sold by the company Binder TM GmbH.
• the flasks are placed under agitation at 175 rpm. / min. during fermentation using an orbital stirrer such as that marketed by the company Stuart® under the reference SSL1. Details of the method are given in the article by Peltier et al. , 2018, PLOS ONE, 13 (1), 191353. (https://doi.org/10.1101/191353).
• the percentages of consumption indicated in the present description are calculated relative to the total content of malic acid present at the start in the sample considered.
• the distribution of the content of malic acid consumed - on the abscissa, expressed as a percentage by mass calculated relative to the total mass of malic acid present at the start - is shown in FIG. 1 for the progeny of H1 and in particular for the X28 clone in the Merlot must of the Merlot 2 sample.
• Isolation of a Z8 clone In the same way as for the H1 hybrid, the H2 hybrid obtained by crossing the strain C and D. From its progeny (obtained as described above in the context of obtaining X28) the Z8 clone is isolated, which also exhibits an extreme phenotype for the consumption of malic acid.
• the distribution of the content (said content being expressed as a percentage by mass calculated relative to the total mass of malic acid present at the start) of malic acid consumed is shown in FIG. 1 for the progeny of H2 and in particular for the Z8 clone. in the Merlot must of the Merlot 2 sample.
• Strain B is a strain which degrades the most malic acid with 45% degraded malic acid in comparison with strains A, C and D.
• the level of malic acid consumption of strains X28 and Z8 is shown in Figure 2 (in which the values read on the ordinate correspond to the average percentage of malic acid consumed in a must of Merlot (Merlot 2) and Sauvignon Blanc (Sauvignon Blanc 1), the initial malic acid contents of which are respectively estimated at 2.28 and 4.32 g / L); malic acid consumption values are also shown for the four parental strains A, B, C and D and for the H1 and H2 hybrids.
• the selected X28 clone shows a consumption percentage of 66%, which illustrates the effectiveness of the crossing / selection methods implemented within the framework of the invention to improve the quantitative characteristics.
• the level of malic acid consumption of strains X8 and Z1 is shown in Figure 12 (in which the values read on the ordinate correspond to the average percentage of malic acid consumed in a must of Merlot (Merlot 2) and Sauvignon White (Sauvignon Blanc 1), the initial malic acid contents of which are respectively estimated at 2.28 and 4.32 g / L); malic acid consumption values are also shown for the four parental strains A, B, C and D and for the H1 and H2 hybrids.
• FIG. 12 shows that the consumption phenotype is extreme for the strains X8 and Z1, in comparison with the consumption phenotype of the other strains.
• the selected X8 clone shows a slight production of malic acid at the end of fermentation, which results in a negative content of the consumption percentage of -10%.
• cassettes are amplified by PCR (acronym for “Polymerase Chain Reaction”) using as template the genomic DNA of the strains of the Euroscarf deletion collection (http://euroscarf.de) previously deleted for the gene in question. .
• the cassettes were amplified with the 500 bases upstream and downstream of the target gene, 5 'and 3' of the coding sequence.
• the H1 hybrid was transformed with the cassettes using the protocol described by Gietz, et al., 2007, Nature Protocols, 2 (1), 31-34. (https://doi.org/10.1038/nprot.2007.13).
• the DNA of each descendant is advantageously extracted using extraction kits such as the DNA-Wizard Promega kit according to the protocol described in Marti-Raga et al. 2017, G3 February, 399-412.
• Amplification is carried out with a kit, preferably the Nextera XT kit or any other kit suitable for the sequencing of small genomes.
• the procedure used makes it possible to index 96 individuals using specific DNA primers and to amplify the DNA in order to construct a Library for Illumina sequencing.
• the protocol used is that provided by the manufacturer, it is described in Marti-Raga et al. 2017, G3 February, 399-412.
• Genotyping is then carried out which implements sequencing, which sequencing is carried out using a sequencing kit which consists firstly in amplifying the DNA in order to construct a Library for Illumina sequencing such as described above.
• the amplification described above is carried out, and, it is followed by the complete sequencing of the genome of each descendant using an Illumina MySeq apparatus using the MySeq Reagent Kit V2 sequencing kit (Pair End 2x250 bases) according to the prescriptions supplied by the manufacturer as described in Marti-Raga et al. 2017, G3 February, 399-412.
• the sequence reads obtained are preferably filtered by bioinformatics methods using the FASTX-Toolkit suite (http: // hannonlab.cshl.edu/fastx_toolkit/).
• the readings selected have the quality parameter Q> 20, which defines an error rate of less than 1%. These readings are then aligned with the BWA software (Li, H., and R. Durbin, 2010. Bioinformatics 26 (5): 589-595) on the reference genome S288C (sacCer3, version April 2011) available on the basis of SGD data (https://www.yeastgenome.org/).
• the SAM tools suite described in the article by Li, H. et al., 2009, Bioinformatics, 25, 2078-2079, the SNPs of each of the descendants are identified and positioned on the genome by the pileup software. These SNPs allow the construction of a very dense genetic map made up of 1000 bi-allelic markers. The method of construction of this type of map and the bioinformatic tools used are described in the article Marti-Raga et al. 2017, G3 February, 399-
• a genetic mapping is then carried out, which mapping establishes a statistical link between the inheritance of genetic markers and the variation of a quantitative trait.
• the genetic markers which are statistically related are "quantitative trait loci" which the English translation is “Quantitative Trait Loci”, with a commonly used acronym which is "QTL”; such QTLs are described in particular in application EP 2 183364.
• the methods for detecting QTL in yeasts are described in the literature, for example in the article published by Peltier et al., 2018, BMC Genomics 19, 772.
• Bi-allelic markers identified during genotyping are used to carry out genetic mapping.
• a statistical link is found between the variation in quantitative character and four genetic markers with a very high significance level (ll_150, XV_1052, IV_31 and IV_360, see table 2).
• H3 hybrid Genetic selection of the progeny of the H3 hybrid: a cross between the Z8 and X28 clones is then carried out to give the H3 hybrid according to the same protocol as that described above for obtaining the H1 and H2 hybrids . From this H3 hybrid, bulk spores are isolated by a process using the hydrophobicity of their wall, and which is detailed in the description which follows directly.
• a spore preparation is obtained after 3 days of culture on potassium acetate (ACK) medium at 1% by mass at a temperature of 24 ° C .; then these spores are digested in a cytohelicase preparation (2 mg / mL) and the spores are purified by carrying out successive washings with water and with an aqueous solution of Nonidet P40 (also known by its name given by the IUPAC classification: " octylphenoxypolyethoxyethanol ') at 0.01%.
• the degree of spore enrichment varies from 80 to 99% depending on the samples. From this preparation, colonies are isolated on a petri dish. In the present case, substantially 1000 descendants are isolated. The DNA of these descendants is extracted by a method described by Albertin et al. (Albertin et al., 2018, Yeast, 35, 141-156) which allows rapid extraction compatible with the sequenom® genotyping technique.
• the phenotypes of the 1000 descendants isolated from the H3 hybrid are evaluated by the sequenom® method according to the protocol described by Gabriel S. et al., 2009, Current Protocol in Human Genetics, Suppl. 60. (doi.org/10.1002/ 0471142905. hg0212s60): the design of the primers is carried out by the MassARRAY® Assay Design version 4.0.0.2 tool allowing to amplify fragments of less than 120 bases and by selecting mass differences between alleles between 16 and 70 Da. Approximately 15 ng of DNA is required to genotype individuals using the MassARRAY® IPLEX platform (Agena Bioscience®).
• the PCR products are analyzed by mass spectrometry and the size of the alleles of each amplifiate is estimated using software from the MassARRAY® System suite.
• the genotyping is carried out on the allelic pools Q2demal, Q4demal, Q6demal and Q7demal which contain only alleles linked to the demalinizing trait.
• the individuals who are sought individuals carrying the Q4demal allelic pool which comprises the following alleles: Q4demal: YLL041C (p.Lys158Glu) + YGL008C (p.Pro74Leu) + YDL054C (p .Ser63Leu) + YDL237W (p.Glu165Ala).
• the individuals exhibiting the Q4demal allelic combination are analyzed for their fermentation properties. These 53 individuals have an average value of 56.15% degraded malic acid with 25% of individuals showing malic acid consumption.
• Met419Leu degrade more malic acid at the end of fermentation than those carrying the Q6c control pool: YLL041C (p.Lys158Glu) + YGL008C (p.Pro74Leu) + YDL054C (p.Ser63Leu) + YDL237W (p.Glu165Ala) + YKL029C ( p.lle605Val) + YOR090C (p. Met419).
• the selection on the basis of the genotype of the descendants of the H3 and H4 hybrids using the Q2demal, Q4demal, Q6demal and Q7demal pools makes it possible to identify individuals who have a very high degraded malic acid value.
• the selection of individuals from the same populations exhibiting the Q2mal, Q4mal, Q6c and Q7c pools allows the selection of individuals degrading a smaller mass of malic acid calculated in relation to the total mass present at the start (see Table 4). The numbers and average values of the pools are presented in Table 4.
• L-malic acid is quantified by enzymatic assay after observing the complete cessation of alcoholic fermentation according to the method described in Peltier, et al., 2018, PLOS ONE, 13 (1), 191353 (https: // doi .org / 10.1101 / 191353) using an enzymatic kit in accordance with the OIV-OENO-599-2018 method (Compendium of international methods of analysis - International Organization of Vine and Wine).
• This colorimetric assay is based on the measurement of the absorbance at 340 nm by UV spectrophotometry.
• H5 hybrid Genetic selection of a strain producing malic acid in the progeny of the H5 hybrid: a cross between the Z1 and X8 clones is then carried out to give the H5 hybrid according to the same protocol as that described above for obtaining the hybrids H1 and H2, H3 and H4. From this H5 hybrid, a preparation of spores is obtained according to the protocol linked to the hydrophobicity of their wall according to the method detailed for progeny of hybrids H3 and H4. From this preparation, 1000 progeny are isolated from which DNA is extracted according to details given for the progeny of the H3 and H4 hybrids.
• the genotype of the descendants isolated from the H3 H4 and H5 hybrids is evaluated by the sequenom® method and relates to the allelic pools Q6mal and Q7mal which contain only alleles linked to the missing trait.
• the numbers and average values of the pools are presented in Table 5.
• the average value reached for the Q6mal pool is -8%, that is to say that the yeast produces 8% more malic acid than that initially contained in the must. This production is greater than that of all the commercial yeasts, the distribution of which is shown in figure 3.
• the results synthesized in figure 13 show the difference in malic acid at the end of fermentation which exists between the strains carrying the allelic pools Q6mal and Q6demal overall descendants. These results are confirmed by the Wilcoxon statistical test, under the conditions described in Frank Wilcoxon, “Individuel compensons by ranking methods”, Biométries Bulletin (en), vol. 1, n ° 6, 1945, p. 80-83, which indicates a significant difference between the groups.
• the individuals which present the allelic combination Q7mal YLL041C (p.Lys158) + YGL008C (p.Asp200Glu) + YDL054C (p.Ser63) + YDL237W (p .Glu165) + YKL029C (p.lle605Val) + YOR090C (p.Met419) + YBR218C (p.Glu722) are analyzed for their fermentation properties. Individuals carrying the Q7mal pool show an additional production of malic acid compared to the initial quantity of malic acid contained in the must. This parameter is expressed as a final mass percentage calculated relative to the total mass of malic acid present at the start.
• the average value reached for the Q7mal pool is -22%, that is to say that the yeast produces 22% more malic acid than that initially contained in the must. Quite remarkably, individuals FMGS3_191 and FMGS3_13 respectively produce 37 and 42% more malic acid than the content initially contained in the must.
• Figure 13 it is shown that there is a difference between the strains carrying the Q7mal and Q7demal allelic pools in all the offspring.
• a cabemet sauvignon must is vinified with the strains FMGS3_191 and FMGS3_13 which carry the Q7mal pool as well as the control strains A, B and X8.
• the malic acid content is measured at the end of alcoholic fermentation.
• the values of the y-axis are expressed in mass concentration of acid malic per liter.
• the horizontal line indicates the initial malic acid content of the must, which is 3.34 g / L.
• the strains FMGS3_191 and FMGS3_13 produce a remarkable quantity of malic acid much greater than that of the strain X8 which is the best performing descendant of the H1 and H2 hybrids.

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