Classifications

• • 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
  • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
  • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C12/00—Processes specially adapted for making special kinds of beer
  • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
  • C12C12/004—Genetically modified microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C12/00—Processes specially adapted for making special kinds of beer
  • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
  • C12C12/006—Yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
  • C12H6/00—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages
  • C12H6/02—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages by distillation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to transformed yeast strains belonging to the genus Saccharomyces which comprise a heterologous nucleic acid encoding a water-producing NADH oxidase, and to their use in fermentation processes for transforming sugars with a production yield of reduced ethanol compared to wild-type, unprocessed Saccharomyces strains.
• scientific knowledge and know-how in viticulture and oenology have led to a very significant improvement in the organoleptic qualities of wines.
• Current viticultural practices favor the production of wines with high qualitative potential by delaying the moment of the harvest. A major consequence is the increase in the sugar content of musts, and therefore the alcohol content of wines (frequently above 14 °).
• S. cerevisiae yeasts especially oenological S. cerevisiae yeasts, convert sugars into alcohol with a yield of 0.47 g / g, which varies little according to the strain used. Therefore, obtaining a low-yielding S. cerevisiae yeast requires the implementation of genetic strategies to divert a portion of the sugars to the formation of other by-products.
• yeasts do not have NADH oxidase.
• the inventors have demonstrated that the introduction into a Saccharomyces yeast of a heterologous gene coding for a NADH oxidase producing water induces a modification of the metabolism of ethanol.
• the invention thus relates to a yeast Saccharomyces transformed with a heterologous gene encoding a NADH oxidase producing water, and its uses, including oenology.
• yeast means a yeast of the genus Saccharomyces.
• Said yeast may for example be chosen from one of the following species: Saccharomyces bayanus, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces pastorianus, or Saccharomyces uvarum.
• Saccharomyces bayanus Saccharomyces carlsbergensis
• Saccharomyces cerevisiae Saccharomyces paradoxus
• Saccharomyces pastorianus Saccharomyces uvarum.
• yeast Saccharomyces according to the invention is Saccharomyces cerevisiae (S. cerevisiae).
• oenological strain is meant a S. cerevisiae strain. A large number of oenological S. cerevisiae strains are commercially available or have been described in the prior art.
• a "coding sequence” or sequence "encoding" an expression product such as an RNA, a polypeptide, a protein, or an enzyme, is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, leads to the production of that expression product.
• RNA, polypeptide, protein, or enzyme is a nu
• a heterologous nucleic acid sequence refers to a nucleic acid sequence (gene, cDNA or RNA) that is not naturally contained by the cell, i.e., a sequence foreign or exogenous to the cell.
• water-producing NADH oxidase or “Nox, H 2 O” is meant an enzyme that catalyzes the reaction: 2NADH + 2H + + O 2 ⁇ 2NAD + + 2H 2 O. It may be particular of a bacterial enzyme. Indeed, a certain number of NADH oxidases producing water have been identified in bacteria, and have notably been listed in Table 1 of the article Riebel et al., 2002.
• a heterologous nucleic acid encoding an NADH oxidase producing water may for example consist of, or comprise, a coding sequence selected from the group consisting of the Nox, H 2 O enzyme genes identified in Lactococcus lactis (Hoefnagel et al., 2002; Genbank accession number AY046926 SEQ ID No.1), Enterococcus faecalis (Ross and Clairbone, 1992; Genbank X68847 Access Point; SEQ ID No. 2), Mycoplasma genitalis (Peterson et al., 1993, Genbank accession number U39707, SEQ ID No.
• Streptococcus mutans (Matsumoto et al., 1996, accession number Embl 815515, SEQ ID No. 4), Mycoplasma pneumoniae (Himmelreich et al., 1996, Embl accession number MPAE44, SEQ ID No. 5), Methanococcus japanicus (BuIt et al., 1996; Embl accession number MJU67512, SEQ ID No. 6), and Leuconostoc mesenteroides (Koike et al., 1985).
• the heterologous nucleic acid encoding a water-producing NADH oxidase may comprise regulatory or control sequences, such as an initiation codon, a codon stop, a promoter, signal, secretion sequence or other sequences used by the yeast genetic machinery.
• alcoholic fermentation refers to the sequence of reactions of conversion of pyruvate to ethanol, and by extension all reactions of transformation of sugars into ethanol.
• This reduction is accompanied by other metabolic changes, namely a reduction in the production of glycerol, ⁇ -ketoglutarate and hydroxyglutarate, and an accumulation of acetaldehyde, as well as acetate and acetoin.
• the invention thus relates to a transformed yeast strain belonging to the genus Saccharomyces which comprises a heterologous nucleic acid encoding a NADH oxidase producing water. ⁇
• the invention also relates to a process for the preparation of a transformed yeast strain belonging to the genus Saccharomyces which has, in alcoholic fermentation, a reduced yield of ethanol production compared to the wild-type Saccharomyces strain, which has not been transformed.
• the method comprises the step of transforming a so-called "wild-type" yeast strain of Saccharomyces by introducing at least one heterologous nucleic acid encoding a water-producing NADH oxidase.
• transform or “transformation” means the introduction of a foreign (i.e., exogenous) RNA or DNA gene or sequence into a host Saccharomyces yeast, so that the yeast host expresses the gene or sequence introduced to produce the desired substance, in this case a NADH oxidase producing water.
• the yeast strain that has received and expresses the nucleic acid encoding a water-producing NADH oxidase has been "transformed".
• the sequence coding for NADH oxidase producing water can be
• They may be promoter-type and terminator sequences active in yeast, for example the promoters and terminators of the alcohol-dehydrogenase 1 (ADH1), phosphoglycerate kinase (PGK) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes.
• ADH1 alcohol-dehydrogenase 1
• PGK phosphoglycerate kinase
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• the invention therefore also relates to an expression cassette comprising a nucleic acid encoding a NADH oxidase producing water, preferably of bacterial origin, associated with sequences for regulating the expression of said NADH oxidase producing sequence. water in Ia. yeast.
• Nucleic acid encoding NADH oxidase producing water, or a cassette containing it, may be carried by a vector or integrated into the genome (chromosomal DNA) of the transformed yeast.
• a "vector” can be used to convey the nucleic acid sequence encoding NADH oxidase producing water in the yeast host, or the cassette containing it, so as to transform the yeast and facilitate expression of the sequence introduced. It may be for example a plasmid-type DNA vector.
• Yeast transformation generally employs "shuttle" vectors in which the nucleic acid sequence encoding NADH oxidase producing water can be combined with a sequence allowing its expression in yeast, such as a yeast promoter.
• These shuttle vectors include other additional sequences to allow expression in bacteria, such as E. coli, or other microorganisms. These additional sequences that do not come from yeasts are used only for the construction of the vectors.
• the invention therefore also relates to a vector comprising an expression cassette comprising a nucleic acid encoding a NADH oxidase producing water, preferably of bacterial origin, associated with sequences for regulating the expression of said coding sequence NADH oxidase producing water in the yeast.
• the DNA vector can be introduced by any technique known to those skilled in the art, in particular by lithium acetate transformation, by electroporation or by means of protoplasts.
• lithium acetate transformation method described by Schiestl and Gietz, (1989) can be used.
• said yeast strain of the genus Saccharomyces is an industrial strain used in oenology, brewery, bakery or cider house.
• said yeast strain of the genus Saccharomyces is a Saccharomyces cerevisiae strain, preferably the oenological strain S. cerevisiae V5 (also called ScV5M, deposited on June 18, 1992 in the National Collection of Microorganism Cultures, held by the Institut Pasteur, under number 1-1222).
• NADH oxidase producing water is a bacterial NADH oxidase.
• Said nucleic acid encoding a NADH oxidase producing water may comprise a sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No .5 and
• nucleic acid encoding a NADH oxidase producing water comprises the gene of the enzyme Nox, H 2 O identified in Lactococcus lactis (SEQ ID No.1).
• the yield of ethanol production, in alcoholic fermentation, of the genus Saccharomyces strain transformed with respect to the wild-type Saccharomyces strain can be reduced by 10 to 20%, preferably by approximately 15%, which represents a reduction of at least 0.5 °, preferably at least 1 °, more preferably at least 2 °, depending on the initial sugar content in the fermentation medium.
• the degree of alcohol represents the number of ml of pure ethyl alcohol contained in 100 ml of a liquid, measured at 20 ° C. 1 ° of alcohol corresponds to 7.80 g / l of ethanol.
• Saccharomyces Yeasts are capable of transforming the sugar of their culture medium by producing a smaller amount of ethanol relative to the corresponding wild-type Saccharomyces yeasts.
• yeast strains are therefore particularly useful in processes for the preparation of fermented beverages in order to obtain beverages having a reduced ethanol content relative to beverages prepared by alcoholic fermentation using unconverted Saccharomyces yeasts. These yeasts can also be used in bakery baking processes.
• the invention therefore relates to the use of a yeast strain Saccharomyces according to the invention for carrying out an alcoholic fermentation.
• Said alcoholic fermentation can be implemented in a baking, winemaking, brewery, cider house or distillery process.
• a yeast strain according to the invention is therefore particularly useful for the preparation of a fermented beverage, such as wine, champagne, beer, cider, or brandy, or in breadmaking, especially for bread preparation.
• the invention also relates to an alcoholic fermentation process that allows the transformation of sugars with a reduced ethanol production yield.
• the method comprises the steps of: a) inoculating a medium containing a high sugar content with at least one Saccharomyces yeast strain according to the invention; O b) cultivating said yeast strain Saccharomyces and allowing the fermentation to proceed to transform the sugars into alcohol.
• the culture can be carried out with a supply of oxygen (microoxygenation), which can be continuous or discontinuous, and where oxygen is provided in limiting or non-limiting conditions.
• a supply of oxygen microoxygenation
• the yeast strain according to the invention is cultured under non-limiting oxygen conditions, that is to say under conditions where there remains oxygen. dissolved in the medium, not consumed by the yeasts.
• medium containing a high sugar content is meant a medium containing at least 30 g / l of sugars, preferably at least 50 g / l of sugars, more preferably at least 80 g / l of sugars.
• sucrose refers to carbohydrates in general, and more specifically to monosaccharides such as glucose or fructose, or polysaccharides such as sucrose or maltose, for example.
• Said alcoholic fermentation process may be a brewing process.
• the medium containing a high sugar content is then a malting must, prepared from a mixture of barley and hops.
• Said alcoholic fermentation process can also be a breadmaking process.
• the medium containing a high sugar content is then the dough raised, for example a bread dough, brioche, etc.
• said alcoholic fermentation process is a winemaking process.
• the medium containing a high sugar content is then a must of grapes.
• the sugar content of the grape must used in winemaking is generally between 140 and 260 g / l.
• the most used yeasts in winemaking are S. cerevisiae yeasts, which generally have an ethanol production yield of the order of 0.47 g per g of sugars.
• the use of these yeasts in winemaking (under traditional conditions, that is to say in anaerobiosis) thus produces wines having an alcohol content between 8 and 15 °.
• the inventors have shown that yeasts S.
• cerevisiae transformed according to the invention have a yield of conversion of sugars into alcohol of 0.39 g per g of sugars, which represents a reduction of about 15% relative to the alcohol yield of wild strains.
• the use of these yeasts can lead to wines having an alcohol content of between 7 and 13 °.
• the inventors have shown that the use of a S. cerevisiae transformed yeast strain according to the invention, for carrying out a fermentation under controlled oxygenation conditions, from a synthetic medium having a content in sugars of 200 g / 1 makes it possible to obtain a reduction of 1 ° of the ethanol content with respect to the content that would have been obtained by fermentation under anaerobic conditions with the wild S.
• yeast growth phase is decoupled from the NADH oxidase activity phase, that is, if the NADH oxidase activity only manifests itself at the end. yeast growth phase (stationary phase).
• the cultured yeasts multiply until exhaustion of the culture medium in one of their substrate.
• the nitrogen is in limiting amount in the medium compared to the sugars (approximately 300 to 500 mg / l of assimilable nitrogen against approximately 200 g / 1 in sugars). Fermentation takes place from the beginning of the yeast multiplication phase and continues once the yeasts have stopped growing. For such fermentation media, the stopping of growth caused by the depletion of the medium in nitrogen and micronutrients occurs when about 30% of the sugars have been consumed.
• the invention more particularly proposes a winemaking process comprising the steps of: a) inoculating a must of grapes with at least one Saccharomyces yeast strain according to the invention; b) cultivating said Saccharomyces yeast strain and allowing the fermentation to proceed to transform the sugars into alcohol; wherein said Saccharomyces yeast strain is initially grown under anaerobic conditions, and then grown under aerobic conditions when the stationary growth phase of the yeasts is reached, especially when substantially all of the assimilable nitrogen of the grape must is consumed.
• anobic conditions is meant culture conditions in the presence of oxygen, preferably under non-limiting oxygen conditions for the cultured Saccharomyces strain.
• non-limiting oxygen conditions for the cultured Saccharomyces strain.
• those skilled in the art are able to determine if they are in the presence of non-limiting culture conditions by simply detecting the presence of dissolved oxygen in the culture medium. For example, for a V5 strain transformed with the L.
• lactis noxE gene grown on a synthetic medium mimicking a grape must as described in the examples (MS medium comprising 180-200 g / l of glucose, 6 g / l of malic acid, 6 / I of citric acid, 460 mg / l of nitrogen, in the form of NH 4 Cl (120 mg / l) and amino acids (340 mg / l)), a transfer of oxygen of 10 mg / l / h corresponds to non-limiting microoxygenation conditions.
• said yeast strain Saccharomyces is a S. cerevisiae strain, more preferably a S. cerevisiae oenological strain such as strain V5.
• the invention also relates to the use of a yeast Saccharomyces according to the invention for regenerating NAD + from NADH, for example during biotransformations.
• Biotransformation is the use of living organisms to perform reactions that are difficult to implement by laboratory chemical methods.
• one or more precursor molecules are provided to the living organism, and after a period of time sufficient for the metabolism to occur, a product or products are isolated from the culture medium or the biomass, which differ from the precursor molecules by one or a small number of enzymatic modifications
• biotransformations used for the production of pure enantiomers (alcohols, hydroxy acids, amino acids, etc.) or other compounds for fine chemistry (green chemistry), involve reduction reactions requiring an electron acceptor, typically a cofactor of NADH or NADPH type.
• a major problem that limits the use of biotransformations is the regeneration of NAD (P) from NAD (P) H.
• the coupling of the biotransformations with another enzymatic reaction for example the reaction catalyzed by alcohol dehydrogenase or by lactate dehydrogenase, allows efficient regeneration of the cofactors.
• problems remain to be solved, in terms of implementation, enzyme stability and overall cost.
• Figure 1 shows a reaction scheme of the metabolic pathway of glucose degradation.
• Figure 2 shows the structure of the expression plasmids used to express NADH oxidase NoxE in S. cerevisiae yeast.
• FIG. 3 shows the integration of the TDH3p-noxE-PGKt expression cassette at the URA3 locus.
• Figure 4 illustrates the monitoring of residual glucose and dissolved oxygen for the control strain V5 grown on MS medium with a supply of 10 mg / l / h of oxygen.
• Figure 5 illustrates the impact of oxidase expression on growth, glucose consumption and metabolic profiles of V5 and V ⁇ noxE strains cultured on MS medium with a constant supply of oxygen at 10 mg / l / hr.
• Figure 6 shows the monitoring of residual glucose and dissolved oxygen for V5 and V ⁇ noxE strains in the stationary phase with a 10 mg / l / h oxygen supply.
• Figure 7 shows the measurement of growth, glucose consumption and metabolic profiles of V5 and MSnoxE strains cultured on MS medium at an oxygen supply of 10 mg / l / h in stationary phase.
• the genomic DNA of L lactis MG1363 is placed under the control of yeast regulatory elements, either in yeast / E. coli shuttle vectors or integrated into the yeast genome.
• the expression plasmid pVT100-U ZEO was used (FIG. 2).
• This plasmid derives from the plasmid pVT100-U described by Vernet et al. (1987) which contains the 2 ⁇ yeast replication origin, the selection marker URA3 and the strong regulatory elements ADH 1 (promoter and terminator of alcohol dehydrogenase I), as well as bacterial elements (origin of replication and ampicillin resistance gene), in which the Phleomycin resistance-conferring Tn5 ble gene was inserted, as described by Remize et al., (1999).
• the vector pVTZEO-ADH1poxE was constructed by inserting the noxE gene (Hoefnagel et al., 2002) into the vector pVT100U-ZEO.
• the noxE gene was amplified by PCR as described in 5, from the total DNA isolated from L. lactis MG 1363, using the primers 5'-CGGCGCTCGAGATGAAAATCGTAGTTATCGGT-3 '(SEQ ID No.7) and 5'-CGGCGTCTAGATTATTTGGCATTCAAAGCTGC-3 '(SEQ ID No.8) in which the Xho ⁇ and XbaI sites (underlined) were introduced.
• pVTZEO-ADHInoxE The map of the recombinant plasmid obtained, called pVTZEO-ADHInoxE, is shown in FIG.
• the pVTZEO-TDH3 / 7 ⁇ xE vector was also constructed from plasmid pVT100U-ZEO, by replacing the ADH1p-ADH1t expression cassette with a cassette consisting of the noxE gene under the control of the yeast gene promoter TDH3 encoding glyceraldehyde. 3-phosphate dehydrogenase and the terminator of the PGK gene encoding phosphoglycerate kinase.
• the TDH3 promoter has been described as a very strong and constitutive promoter (Mumberg et al., 1995).
• Plasmid p VTZEO-TDH 3noxE was obtained using the intermediate plasmid pFL-TDH3nox. The latter was obtained by cloning the TDH3 promoter region into the yeast / coli shuttle vector pFL60 described by Minet et al., (1992).
• the TDH3 promoter region was amplified from I 1 genomic DNA of the yeast strain S. cerevisiae S288C, using the 5'CGGAGCTCCAGTTCGAGTTTATCATTATC-3 'oligonucleotides (SEQ ID No.9) and 5'CGGGATCCTCGAAACTAAGTTCTTGGTG -3 '(SEQ ID No.10) in which the SacI and BamHI sites (underlined) were introduced as described in 5.
• the coding region of the noxE gene was amplified by PCR, as described in 5, from the Chromosomal DNA of L. lactis MG1363 using the oligonucleotides 5'-CGGGATCCATGAAAATCGTAGTTATCGGT-3 '(SEQ ID No.11) and 5'-CGCTCGAGTTATTTGGCATTCAAAGCTGC-3' (SEQ ID No.12) allowing the introduction of BamHI sites and Xhol (underlined).
• the two PCR fragments thus generated were digested and then ligated into the plasmid pFL60 digested with SacI and XhoI as described in 6.
• the plasmid pFL-TDH3 noxE was thus obtained.
• the TDH3p ⁇ noxE-PGKt expression cassette was amplified, as described in 5, from this plasmid using the 5'-oligonucleotides.
• the empty plasmid pVTZEO-TDH3 used as a control was generated from the plasmid pFL-TDH3, which corresponds to the plasmid pFL60 in which the PGK promoter was replaced by a multi-site cloning, using the double-stranded oligonucleotide (MWG) ⁇ '-ATCCCCCGGGCTGCAGGTCGACC-S '(SEQ ID No.15), then in which the TDH3 promoter was cloned at the site SacI and BamHI as previously explained.
• MWG double-stranded oligonucleotide
• the TDH3p-noxE-PGKt expression cassette was amplified, as described in 5, from plasmid pFL-TDH3noxE using oligonucleotides 5'-CGGCGGATATCGCTCCAGTTCGAGTTTATCA-3 '(SEQ ID No.16) and 5'-CGGCGACTAGTTTTCACACAGGAAACAGCTA- 3 '(SEQ ID No.17) in which the EcoRV and Spel sites were introduced.
• the amplification fragment obtained was ligated to the plasmid pUG6 (Guldener et al., 1996) digested with EcoRV and SpeI and dephosphorylated as described in 6.
• the plasmid pUG6 / oxE was obtained.
• a PCR fragment carrying the loxP-kanMX4-loxP and the ⁇ oxE-PGKt TDH3p modules was amplified, as described in 5, from the plasmid pUG ⁇ noxE, using the oligonucleotides 5 TGATTCGGTAATCTCCGAGCAGAAGGAAGAACGAAGGAAGGCAGGTCGACAAC CCTTAAT-3 '(SEQ ID No.18) which has 20 nucleotides complementary to pUG6 and an extension of 40 nucleotides (underlined) corresponding to the region -157 to -117 upstream of the ATG of URA3, and '-
• the yeast strain Saccharomyces cerevisiae ScV5M (called V5) was transformed with the vectors p VTZEO-ADH 1noxE, pVTZEO-TDH 3noxE and with the vectors pVT100UZEO and pVTZEO-TDH3 (controls).
• the strains obtained are listed in Table 1.
• Table 1 List of plasmids and strains used.
• V ⁇ pVTZEO- 2 ⁇ , URA3, Ap R , G418 R This ADHp-NOxE-ZEO R ADHInoxE work
• strain V5 was transformed with 1, 4 ⁇ g of integration fragment prepared as described in 3. Integration at the URA3 locus was verified by PCR from the genomic DNA of G418R transformants obtained, using oligonucleotides located upstream and downstream of the integration site. A strain called ⁇ / 5noxE with the fragment integrated into the URA3 locus was obtained ( Figure 3):
• the strain SCV5M was deposited on June 18, 1992 at the National Collection of Cultures of Microorganisms, held by the Institut Pasteur, under the number 1-1222. It is a S. cerevisiae haploid strain, MATa, ura3, derived from an oenological strain.
• the transformation method used is that of lithium acetate described by Schiestl and Gietz, (1989).
• the selective medium used to select the strains transformed by the plasmids is YNB (0.67% Yeast nitrogen base,
• the clones that integrated the integration fragment carrying the kanMX and NADH oxidase modules were selected on rich medium YEPD (1% bacto yeast extract, 2% bactopeptone, 2% glucose) supplemented with 200 ⁇ g / ml geneticin G418 (Gibco, England).
• 50 ng of plasmid or 100 ng of genomic DNA are mixed with 500 nM of oligonucleotides, 5 ⁇ l of 1OX Mg 2+ -free DyNazyme EXT buffer buffer (FINNZYMES, Finland), 1.5 mM MgCl 2 , 200 ⁇ M of dNTPs, 1 unit of DyNAzyme EXT (FINNZYMES 1 Finland) in a total volume of 50 ⁇ l.
• the amplification conditions are as follows: 2 minutes at 94 ° C., 30 cycles of 30 seconds at 94 ° C., 30 seconds at 50 ° C., 2 minutes at 72 ° C., then 7 minutes at 72 ° C. on Perkin amplification. -Elmer Cetus model 9600.
• the digestion of the DNA with restriction enzymes is carried out as described by the supplier (Promega Corporation, USA). After digestion, the plasmids are dephosphorylated with 10 units of Bacterial Alkaline Phosphatase (Qbiogene, USA) according to the protocol described by the supplier. The dephosphorylation reaction is stopped by phenol / chloroform extraction (Sambrook et al., 1989). 50 ng to 100 ng of amplified and digested DNA are ligated to 100 ng of digested and dephosphorylated plasmid in a final reaction mixture of 10 ⁇ l, in the presence of 5 units of T4 DNA ligase (Biolabs, USA) overnight at 16 ° C.
• ligation mixture is used to transform the competent E. coli DH5 ⁇ bacteria (Library Efficiency DH5 ⁇ competent cells, Invitrogen, USA) according to the protocol described by the supplier.
• the colonies obtained are selected on LB plates (1% bactotryptone, 0.5% bacto yeast extract, 1% NaCl) plus ampicillin (100 ⁇ g / ml).
• L 1 plasmid DNA of the clones obtained is then extracted by QIAprep Miniprep (Qiagen, USA) and analyzed by enzymatic digestion.
• MS medium was used for preculture and culture. This is a synthetic medium that simulates a standard grape must (Beyy et al., 1990).
• MS medium contains 18-20% glucose, 6 g / l malic acid, 6 / I citric acid, 460 mg / l nitrogen, as NH 4 Cl (120 mg / l) and amino acids (340 mg / l).
• the medium is supplemented with methionine (115 mg / l) and, if necessary, uracil (50 mg / l).
• the pH of the MS medium is 3.3.
• Anaerobiosis factors ergosterol (7.5 mg / l), oleic acid (2.5 mg / l) and Tween 80 (0.21 g / l) are added.
• the precultures are carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium at 28 ° C. with stirring (150 rpm) for 30 h.
• the reactors are inoculated from these precultures, at a cell density of 1.10 6 cells / ml, and maintained at a constant temperature of 28 ° C. with permanent stirring (500 rpm).
• the microxygenation conditions are obtained by aerating the reactor with air at a rate kept constant. Dissolved oxygen is measured using INGOLD Clark electrodes.
• the transfer coefficient (Kia) is measured according to the dynamic method (Dursun et al, 1999).
• the solubility of oxygen (C *) in the must is determined according to Sablayrolles and Barr (1986).
• the oxygen consumption is obtained by integrating the curve obtained during the calculation of the OUR.
• the exit gas passes through a refrigerated condenser to prevent evaporation of volatile compounds.
• the growth is followed by measuring the optical density at 600 nm and by counting the number of cells on a Coulter Counter (ZBI) type apparatus on a sample of an aliquot of culture medium.
• ZBI Coulter Counter
• the metabolites are assayed in the supernatant, after centrifugation at
• the concentration of glucose, glycerol, ethanol, pyruvate, succinate, acetate, ⁇ -ketogluatarate and 2-hydroxygluatarate is determined by high-pressure liquid chromatography (HPLC) using an HPX-87H type column (Bio-Rad).
• HPLC high-pressure liquid chromatography
• the concentration of acetaldehyde is determined by the enzymatic method described by Lundquist,
• the enzymatic activities are determined extemporaneously.
• the specific NADH oxidase activity in the cell extracts is measured spectrophotometrically at 25 ° C in a total volume of 1 ml containing 50 mM potassium phosphate buffer (pH 7), 0.3 mM NADH and 0.3 mM EDTA.
• the reaction is initiated by adding 5 to 50 ⁇ l of cell extract, followed by the decrease in absorbance at 340 nm.
• the protein concentration is determined using the BC Assay kit (Uptima, Interchim). d - Extraction and measurement of intracellular levels in NADH / NAD
• the metabolites are extracted as described by Gonzalez et al., (1997).
• Cofactor concentrations are determined from enzyme reactions coupled to NAD (H) - as described below.
• the amount of NADH produced during the reaction is determined by fluorescence spectrophotometry (excitation wavelength, 340 nm, emission wavelength, 460 nm) using a Perkin Elmer LS 5OB fluorescence spectrophotometer.
• the enzymatic reactions are carried out at 30 ° C. in a total volume of 2 ml of reaction buffer containing 4.25 mM Tris-NH 4 Cl (pH 7.0), 25 ⁇ M dihydroxyacetone phosphate, and 125 ⁇ M ⁇ -ketoglutarate, as described by Klingenberg (1974). Aliquots of 5 to 100 ⁇ l of samples are added to the reaction buffer. A baseline is obtained.
• glycerol-3-phosphate dehydrogenase (170 U.ml -1 , Roche) and then 1 ⁇ l of NADPH-dependent glutamate dehydrogenase (240 ⁇ l ml -1 Roche) are added successively. Each addition is carried out after obtaining a stable signal.
• the concentration of NAD is determined as previously described
• reaction buffer contains 1.8 ml of a mixture of 0.2 M glycine and 0.4 M hydrazine hydrate (pH 9), 85 mM ethanol and 5 to 200 ⁇ l of extract in a total volume of 2.01 g. ml.
• 1 ⁇ l of alcohol dehydrogenase (882 U. ml -1 , Roche) is added.
• the cofactor concentrations in the samples are calculated by an external calibration method, making it possible to determine the response coefficient of each cofactor.
• the measurements are made in triplicate. 2. Results 20
• Fermentations in batch mode under microoxygenation conditions were carried out in order to analyze the impact of the expression of the oxidase on the growth, the degradation of the sugar, the production of metabolites and the intracellular concentration.
• NAD N-oxidase
• NADH NADH cofactors
• the fermentations were carried out with the strains expressing NADH oxidase V ⁇ noxE, V5pVTZEO-TDH3nox £, V5pVTZEO-ADH1 noxE and control strains V5, V5pVTZEO-TDH3 and V5pVTZEO-ADH1.
• the air flow rate used in this experiment is kept constant throughout the fermentation at 17 ml / min, which corresponds to an oxygen transfer rate of 10 mg / l / h. Under these conditions, all the oxygen is consumed by the control strain V5 (FIG. 4).
• NADH oxidase The specific activity of NADH oxidase was measured in the different strains at 2 stages of fermentation (Table 2) in mid-exponential phase (17 h culture) and stationary phase (40 h).
• the growth phase is short.
• the depletion of assimilable nitrogen from the medium causes a rapid entry into the stationary phase (after about 30 h), whereas approximately 30% of the initial sugars are consumed.
• the stationary phase therefore represents an important phase during which the majority of the sugars (approximately 70%) are degraded.
• NADH oxidase activity was detected in cell extracts of the control strain and strains transformed by empty plasmids, whereas significant activity was measured in the strains expressing the noxE gene, indicating that the enzyme encoded by the bacterial gene noxE expresses well in S. cerevisiae.
• the maximum activity obtained (1.48 U / mg protein) is approximately 7 times greater than that measured in a cell extract of L. Lactis (Lopez de Felipe and Hugenholtz, 2001).
• NADH oxidase is expressed throughout the fermentation period, with a specific activity level approximately 3-fold higher in the growth phase compared to the stationary phase when the noxE gene is under the control of the TDH3 promoter.
• the level of specific activity is 2 times higher in the stationary phase than in the growth phase.
• the level of activity varies considerably depending on the promoter used and the number of copies of the gene.
• the TDH3 promoter makes it possible to obtain an activity 5 times greater than that obtained with the promoter ADH1 in the growth phase, whereas the levels of specific activity are similar for the 2 constructions in stationary phase.
• noxE is under the control of TDH3
• the activity obtained in multicopy is about 3 times greater than that obtained in the strain having integrated this cassette into a copy.
• Table 3 shows the yields of biomass and products obtained after stopping the fermentation.
• Table 3 Production yield of the main fermentative metabolites, biomass, carbon balance and degree of reduction of strains V5, V5 pVT100-UZEO, V5nox £, V5 pVTZEO-TDH3noxE, V5 pVT ZEO-ADH ⁇ noxE on MS medium with constant supply of 10 mg / l / h of oxygen
• V5 (0.009) (0.005) 0.001) (0.001) (0.001) (0.0001) (0.002) (0.0001) (0.010) (0.002) 0.064
• V5pVTZEO- (0.003) (0.002) (0.002) (0.000) (0.000) (0.0001) (0.000) (0.0007) TDH3
• V ⁇ noxE 0.5 0.025 101 (0.002) (0.001) (0.001) (0.003) (0.002) (0.0005) (0.000) (0.0000) (0.005) (0.000)
• ADHInoxE (0.002) (D 1 OOI) (0.006) (0.001) (0.004) (0.0005) (0.001) (0.0001)
• the 3 strains expressing the oxidase consume only half (about 100 g / l) of the sugars present, unlike the control strains that complete the fermentation.
• the effects of oxidase on the central metabolism were therefore analyzed by comparing biomass yields and main fermentation by-products at half-fermentation (reaction progress 0.5). For information, the yields obtained after degradation of all the sugars (200 g / l) are indicated for the control strains (reaction progress 1).
• glycerol is also decreased from the beginning of fermentation.
• Oxidase by reoxidizing part of the intracellular NADH, therefore competes with other yeast enzymes using this cofactor.
• ADH alcohol dehydrogenase
• GPDH glycerol 3-P dehydrogenase
• the first consequence of limiting the flow of carbon to ethanol synthesis is an increase in the production of acetaldehyde and acetate in the transformants.
• Acetate synthesis mainly generates NADPH via Alddep and Alddp acetaldehyde dehydrogenases located in the cytoplasm and in the mitochondria, respectively ( Saint-Prix et al., 2004).
• the V ⁇ noxE strain also shows a strong decrease in ⁇ -ketoglutarate production. This effect could be due to the surplus of NADPH linked to the increase of acetate synthesis.
• the decline in its production in the ⁇ / 5noxE strain may therefore stem from a lower availability of substrate ( ⁇ -ketoglutarate) and / or a lower availability of NADH due to competition with NADH oxidase.
• substrate ⁇ -ketoglutarate
• NADH NADH dependent
• the increase in acetate may be related to the accumulation of its precursor, acetaldehyde, whose production is increased drastically and very early. It is interesting to note an early arrest (around 20 h) of the growth of the V5noxE strain, whereas at this stage the concentration of acetaldehyde in the medium reaches 1.1 g / l instead of 0.2 g / l. for the control strain. The number of cells reached by V5nox® is three times lower than that of the wild-type strain.
• Acetaldehyde is a toxic compound for yeast. It negatively affects the formation of biomass (Aranda and del Olmo, 2004, Liu and Pilone, 2000) and at a high concentration the fermentation rate (Roustan and Sablayrolles, 2002).
• acetaldehyde can be metabolized to acetoin and 2,3-butanediol ( Figure 1), non-toxic compounds for yeast.
• Acetoin is produced by the condensation of 2 molecules of acetaldehyde by pyruvate decarboxylase (PDC), then reduced to 2,3-butanediol by butanediol dehydrogenase (BDH). This reduction is NADH dependent (Gonzalez et al., 2000).
• PDC pyruvate decarboxylase
• BDH butanediol dehydrogenase
• the five oxygenation conditions tested on the 5noxE strain correspond to maximum transfer rates of 2, 4, 6, 7 and 10 mg / l / h of oxygen.
• Table 5 shows the effects obtained on ethanol production yield, acetaldehyde accumulation, glucose degradation, growth and oxygen consumption.
• Table 5 Dissolved O 2 , ethanol yield, acetaldehyde concentration, glucose consumption and final biomass for the V5 strain at 10m g / l / h of 0 2 transferred and for the V ⁇ noxE strain at different transfer rates (OTR).
• Residual strain 8 ethanol B final (mg / l / h) (% glucose
• the probe As a percentage of air saturation in the medium, the probe indicates 100% when the oxygen concentration in the medium (MS 20% glucose) is 6.4 mg / l. b g ethanol produced by glucose consumed
• the reduction in production yield of ethanol is accompanied by an acetaldehyde accumulation, correlated with a biomass reduction of about 60% and an incomplete consumption (about half) of the substrate.
• the lack of growth is also observed in the two cases where I 1 O 2 is not limiting, although less markedly for 2 mg / l / h of O 2 transferred.
• the glycerol production remains lower than that obtained for the wild-type strain cultured at 10 mg / l / h of 02, which indicates that the supply of O 2 is sufficient to allow operation oxidase.
• the activity phase of the NADH oxidase was decoupled from the growth phase.
• fermentations in batch mode were performed under anaerobic conditions up to 28 hours of fermentation (end of the growth phase), then under controlled microoxygenation conditions from the entry into the stationary phase and throughout this phase.
• the fermentations were carried out with the strain expressing NADH oxidase V ⁇ noxE and the control strain V5.
• all of the oxygen is consumed by the control strain V5, while the oxygen remains largely non-limiting for the VbnoxE strain (FIG. 6).
• the effects obtained on growth, glucose degradation, formation of ethanol, acetate, acetaldehyde, acetoin and butanediol are shown in Figure 7.
• the V5nox strain under these conditions, has a growth identical to that of the control strain, an identical final biomass (30 ⁇ 10 7 cells) and is capable of fermenting almost all the glucose present (close to 200 g / l). Dissociating the growth phase from the activity phase of NADH oxidase thus makes it possible to overcome the side effects observed previously, on growth and fermentability.
• the oxygen supply is expected to cause an increase in the formation of acetaldehyde, which however remains limited to 400 mg / l, a concentration which does not drastically affect the fermentability.
• the production of acetate is slightly increased compared to the control strain cultivated under the same conditions.
• the carbon flux is also reoriented towards the formation of acetoin, while the production of 2,3 butanediol remains similar between the two strains and is not affected by the O 2 input.
• the results obtained were compared with those obtained during a standard oenological fermentation, carried out under the same conditions, but in the absence of oxygen supply (conditions of strong anaerobiosis) (Table 6).
• Table 6 Final concentration of the main fermentative metabolites and biomass production of strains V5 and V5 ⁇ oxE on MS under 10 mg / l / h stationary phase oxygen and anaerobiosis conditions.
• Biomass 6 6 4 4 a CO 2 estimated from ethanol production
• the V ⁇ noxE strain behaves like the control strain, the oxidase not being active.
• the production of ethanol by the V ⁇ noxE strain under microoxygenation conditions limited to the stationary phase is decreased by 8 g / 1 compared with that of the wild-type strain under anaerobic conditions.
• the use of a strain expressing NADH oxidase under microoxygenation conditions controlled and decoupled from the growth phase allows a reduction in the degree of ethanol which in this example reaches 1 ° alcohol.
• the oxygen is limiting during the first hours of the supply, and then not limiting during most of the oxygenation phase (FIG. 6).
• Nicotinamide adenine dinucleotides (NAD, NADP, NADH, NADPH): spectrophotometric and fluorimetric methods in H. Bergmeyer, H. (Ed.), Methods ofenzymatic analysis, pp. 2045-2059.
• Acetaldehyd Betician Mit Aldehyd dehydrogenase, Methods of Enzymatic Analysis, Academy Press, Inc., pp. 1509-1513.
• Vernet T., Dignard, D. and Thomas, D. Y. (1987).

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