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• • 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
  • C12N9/88—Lyases (4.)
• • 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
  • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
  • C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
  • C12H1/003—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages by a biochemical process

Definitions

• the present invention relates to the cloning of the structural gene of the malolactic enzyme of Lactococcus lactis, to its complete nucleotide sequence and to the deduced protein sequence, as well as to DNA fragments carrying the sequence coding for this gene. It also relates to the expression of this gene in different prokaryotic or eukaryotic hosts, and in particular in Saccharomyces and Schizosaccharomyces.
• Malolactic fermentation a secondary fermentation which is observed during the vinification process in addition to the alcoholic fermentation carried out by yeasts, consists of the degradation by certain lactic bacteria of malic acid.
• lactic acid and CO2 The bacterial species naturally present in the fermentation flora and responsible for malolactic fermentation belong to the genera Lactobacillus, Leucono ⁇ toc and Pediococcu ⁇ .
• WILLIAMS et al. [App. About. Microbiol. 47: 288-293 (1984)], as well as European Application 103300 describe the cloning of a 5 kb DNA fragment carrying the gene for the malolactic enzyme of L. delbrueckii in S. Cerevi ⁇ iae and in E. coli.
• a malolactic enzyme having an activity of a level comparable to that of the malolactic enzyme of bacteria of the genera
• Lactococcus lactis It is a protein of around 230 kDa, made up of subunits of around 56 kDa. Its pHi is approximately 4.3, and its Km for malic acid is approximately 10 to 12 mM. Its N-terminal sequence has also been determined [RENAULT, Malolactic fermentation:
• the inventors identified a DNA fragment of 2684 bp including the entire gene for the malolactic enzyme, and determined the sequence of this fragment, which is represented in the list of sequences in the appendix under the number SEQ.ID.N0. 1.
• expression of the malolactic gene was obtained in different hosts, prokaryotes and eukaryotes.
• the present invention relates to a nucleic acid fragment, characterized in that it comprises the sequence of the gene for the malolactic enzyme of Lactococcus lactis.
• sequence of the gene for the malolactic enzyme of Lactococcus lactis is meant in particular the coding sequence extending from nucleotides 466 to 2085 (inclusive) of the sequence SEQ. ID. NO 1 represented in the annexed list of sequences, as well as any sequence which, taking into account the degeneracy of the genetic code, codes for the same polypeptide; this definition also includes the sequences carrying minor modifications intended to allow better expression of the gene in a given host.
• the invention also encompasses nucleic acid fragments comprising a sequence homologous to or complementary to the gene sequence for the malolactic enzyme of Lactococcus lactis, as defined above, or of a fragment of at least 10 mothers. , preferably at least 20 mothers, of said sequence, and usable in particular as probes for the localization of said gene and / or as primers for its amplification.
• the invention also relates to expression cassettes, comprising the sequence of the gene for the malolactic enzyme of Lactococcus lactis, under the control of sequences capable of regulating the expression of said gene.
• sequences regulating the expression of a gene is meant promoter and terminator type sequences active in the host in which it is desired to obtain the expression of said gene. Promoters and terminators of different genes can be used, combined in different combinations. Among the sequences which can be used to obtain the expression in yeast of the gene for the malolactic enzyme of Lactococcus lactis, mention will be made, by way of nonlimiting example, of the promoters and terminators, known in themselves, of the genes of the alcohol dehydrogenase I (ADHI), phosphoglycerate kinase (PGK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
• ADHI alcohol dehydrogenase I
• PGK phosphoglycerate kinase
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• the expression cassettes according to the invention can be carried by plasmids, or integrated into the chromosomal DNA of the host yeast.
• the invention also relates to recombinant vectors characterized in that they comprise at least one DNA fragment comprising the sequence of the gene for the malolactic enzyme of Lactococcus lactis, as defined above.
• said vectors are expression vectors, comprising an expression cassette as defined above.
• said vectors comprise regulatory sequences active in yeast.
• the vectors in accordance with the invention are shuttle vectors, also having an origin of bacterial replication and a mark. selection in a bacterium (for example, antibiotic resistance gene).
• vectors can be selected on the basis of the nature and the strength of the regulatory elements which enter the expression cassette.
• the promoters and terminators described above can be chosen, or any other sequence making it possible to control and regulate the expression of a gene in yeast.
• Another criterion for the choice of vectors resides in the number of copies thereof, which is conditioned by the choice of the origin of replication.
• Replicative vector with a high number of copies, having as origin of replication an ARS chromosomal sequence.
• Replicative vector with low copy number, having a chromosomal ARS sequence and a centromeric sequence.
• the vectors in accordance with the invention also include markers for selection in yeast, such as auxotrophy markers: URA3, LEU2, HIS3, TRP ⁇ , ADE, etc ... and / or markers for resistance to antibiotics (G418, hygromycin B, chloramphenicol, phleomycin), to herbicides (sulfo eturon methyl), to copper, etc ...
• markers for selection in yeast such as auxotrophy markers: URA3, LEU2, HIS3, TRP ⁇ , ADE, etc ... and / or markers for resistance to antibiotics (G418, hygromycin B, chloramphenicol, phleomycin), to herbicides (sulfo eturon methyl), to copper, etc ...
• Vectors in accordance with the invention carrying the gene coding for the malolactic enzyme of Lactococcus lactis can be introduced into any strain of yeast, by different transformation techniques.
• transformation techniques that can be used are the protoplast technique, the permeabilization technique with lithium salts and electroporation.
• the method of this construction comprises the following stages: construction of an expression cassette comprising the gene for the malolactic enzyme and regulating elements of variable force; - introduction of this cassette either in mono ⁇ copy, or in multicopy in yeast.
• YIp integrative vector
• the present invention also relates to strains of transformed eukaryotic or prokaryotic cells, characterized in that they contain at least one heterologous DNA fragment carrying at least one copy of a gene coding for the malolactic enzyme of Lactococcus lactis, under the control of sequences regulating the expression of said gene in said cell.
• said cells are yeast cells.
• the yeast strains in accordance with the invention find numerous applications in the food industry. be silent and in particular in the field of oenology to accomplish malolactic fermentation.
• said yeasts belong to the genus Saccharomyces.
• said yeasts belong to the genus Schizosaccharomyces.
• Schizosaccharomyces is sometimes used in oenology for its ability to degrade malate.
• its use is limited, because this degradation which is carried out by the pathway of the malic enzyme does not produce lactate, but ethanol and CO2, which leads to a significant deacidification, which can have negative influences on the characteristics of the wine.
• Schizosaccharomyces strains can have many uses in oenology; they can be used for example:
• the malolactic enzyme was purified as described by RENAULT (1990, cited above); cf. also C. ROULLAND, (DEA in oenology-ampelology, September 1988, University of Bordeaux II)
• Lactococcus lactis (IL 1441) is cultivated in the following medium:
• the pH is adjusted to 5%.
• the sterile medium is inoculated with 10% (v / v) of a preculture.
• the cells were cultured in 3 liters of the above medium to 28 "C for 24 hours, harvested by centrifugation at 1000 g for 10 min at 4 ° C and washed 2 times with 0.9% NaCl.
• the pellet is taken up in 30 ml of phosphate buffer (0.1 M, pH 6.0).
• the cells are ground ultrasonicated for 6 min at 0 ° C. After centrifugation
• the malolactic activity is measured by the decarbo ylation of malic acid followed by a CO 2 electrode (EISCHWEILER).
• the supernatant from the 70% precipitation has no activity.
• the pellet taken up in 2 ml of 0.1 M phosphate buffer pH 6.0, and which contains all the malolactic activity is deposited on a filtration column on ACA 34 gel (ULTROGEL; fractionation range from 20,000 to 350,000 ) 40 cm long and 2.6 cm in diameter, previously balanced by the elution buffer: 0.01 M phosphate buffer, pH 6.0, 0.1 M KC1.
• the elution is carried out with a flow rate of 18 ml / h.
• the volume of the fractions collected is 2.4 ml.
• the most active fractions correspond to fractions 50 to 54. They are combined for further purification by electrofocusing. 1 electrofocusing is carried out on a 110 ml LKB column. The pH gradient is formed by the ampholines used at 2% and stabilized by a glycerol density gradient established during the filling of the column. The most active fraction which focuses at pH 4.3, pHi of the protein, is recovered, then undergoes an additional purification step by chromatography ion exchange (anion exchange column SEPHAROSE Q, PHARMACIA).
• the elution is carried out by a gradient from 0 to 0.5 M NaCl, in a 50 mM Tris-Hcl buffer, pH 7.6, 0.1 mM EDTA, 1 mM ⁇ -mercaptoethanol.
• the antibodies were obtained by injection into two rabbits of part of the purified preparation of malolactic protein according to the following protocol: - Subcutaneous injection of 2 ml of malolactic protein solution (100 ⁇ g in 0.3 M NaCl buffer) added with 2 ml of complete Freund's adjuvant.
• the blood of two rabbits was collected 12 days after the second injection, left for 24 h at 4 ° C to allow clotting, centrifuged at 10,000 g for 10 min at 4 °, and the recovered serum is aliquoted and stored at -20 " C.
• the anti-malolactic enzyme antibodies were then tested by ELISA test carried out on the two sera (called 120 and 119) obtained from the rabbit serum according to the protocol described by SAMBROOK et al. ((1989)]. determined that the optimal dilution for the use of antibodies was 1 / 1000th.
• the specificity of the antibodies was then determined by immunoelectrophoretic transfer (Western blot).
• polyclonal antibodies obtained are then fixed on the membrane and revealed by anti-rabbit antibodies coupled to alkaline phosphatase.
• Serum 120 recognizes on the crude extract and on the purified fraction, a majority band of molecular weight approximately 60kD corresponding to the malolactic enzyme, and several contaminating bands of lower molecular weight.
• serum 119 recognizes on the purified fraction, as on the crude extract, a single band, corresponding to the malolactic enzyme; this very specific serum was therefore used for subsequent experiments.
• the genomic DNA of Lactococcus lactis IL 1441 was first extracted and purified. For this, 500 ml of a culture in the exponential phase are centrifuged at 5000 g for 10 min. The pellet is taken up in 5 ml of TE (10 mM Tris 1 M EDTA pH 8) containing
• Ethidium bromide is extracted with isoamyl alcohol. After dilution with 2 volumes of water, the DNA is precipitated with 6 volumes of ethanol. The precipitate is recovered at the baguette and dissolved in 1 ml of TE. The concentration of DNA in solution was determined (1 ⁇ g / ⁇ l) by measuring the OD at 260 nm.
• the digested DNA is precipitated with 1/20 vol of 3M sodium acetate and 2 volumes of ethanol. After centrifugation, the DNA is taken up in 300 ⁇ l of TE and the fragments separated on a sucrose gradient for 15 hours at 25,000 rpm. 0.5 ml fractions are collected and analyzed on 0.8% agarose gel. The fraction containing 1.5 and 4 Kb fragments is dialyzed against TE for 4 hours. c) Ligation of the digested DNA to the vector ⁇ gtll The vector ⁇ gtll (derived from the bacteriophage ⁇ ) was chosen to produce the expression library.
• the principle of production of the library consists in inserting DNA fragments into the 3 ′ region of the coding phase of the ⁇ -galactosidase gene, so as to express hybrid proteins under the control of the ⁇ -galactosidase promoter.
• a cloning kit in ⁇ gtll was used according to the protocol recommended by the supplier.
• DNA is ligated to dephosphorylated adapters having a blunt end and another EcoRI cohesive end. After removal of the free adapters by purification on an exclusion column, the "adapted" DNA fragments are phosphorylated and ligated to the arms of the vector also digested with EcoRl and dephosphorylated.
• the in vi tro packaging of the phage particles is then carried out.
• the library thus obtained was titrated by infection of the bacterial strain E. coli 1090 [hSd (r-km + k) lac U169, ProA + , Ion-, araDl39, StrA, SupF, trpC22: TnlO (pMC9)] aliquot fraction of the phage suspension, and the transformants selected on LB medium (bacterotryptone 10 g / 1, yeast extract 5 g / 1, NaCl 10 g / 1) + ampicillin (50 ⁇ g / ml) at 43 ° C (to induce bacterial lysis). The lysis ranges are obtained after 4 h of incubation.
• the bank thus obtained represents approximately 3 times the genome of Lactococcus lactis.
• plaques were obtained by plating the library on LB medium + ampicillin + MgCl2 and incubating 3:30 to 43 ° C (under non-expression).
• the dishes are covered with nitrocellulose filters (C extra, AMERSHAM), impregnated with 10 mM IPTG and incubated for 4 h at 37 "C, so as to induce expression.
• the filters are rinsed in TNT (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.05% Tween 20), then incubated with light agitation for 30 min at room temperature in the same buffer.
• a culture of E. coli Y1090 is carried out in
• the cells are lysed completely by sonication at 0 ° C (six times 20 seconds), then centrifuged at 12,000 g for 10 min at 4 ° C. The lysate was recovered and incubated for 4 h at room temperature in the presence of 1 ml of antibody 119 diluted 1/10 'in TNT buffer
• the exhausted serum is stored at 4 ° C. in the presence of sodium azide (0.05%).
• c) Immunological Screening of the Bank The nitrocellulose filters are then incubated in the presence of the exhausted serum 119 as described above, and the revelation of the fixed antibodies is carried out using anti-rabbit antibodies coupled to alkaline phosphatase, as previously described.
• an agarose core is taken and incubated in 500 ⁇ l of SM buffer (NaCl 6 g / 1, MgS0 7H 2 0 2 g / 1, Tris base 6 g / 1, gelatin 0.01 % pH 7.5) for 2 hours at 4 ° C to allow diffusion of phage particles. This suspension is again used to infect E. coli
• the phage DNA of the 10 positive clones was extracted and analyzed.
• a well isolated positive lysis range (subclone) was systematically used as starting material.
• the DNA is extracted from the confluent lysis plaques on LB + ampicillin dishes. The dishes are covered with 5 ml of SM buffer and shaken at 4 ° C. for 2 hours. The suspension is recovered, treated with a few drops of chloroform, and centrifuged at 4000 g for 10 min at 4 ° C to remove bacterial debris. The DNA is extracted from the phage suspension thus purified.
• the first analysis consisted in amplifying by PCR the inserts carried by the recombinant phages to determine their size.
• oligonucleotides [lambda gtll Primer (forward) 24-MER and lambda gtll Primer (reverse) 24-MER, Company BIOLABS] corresponding to ⁇ -galactosidase sequences surrounding the EcoRI cloning site were used as primers for amplification of the inserts.
• the sizes of the inserts shown are 3 kb, 2.7 kb, 2 kb, and 3 kb. These overlapping inserts, the total size of the cloned region is 4.5 kb.
• the 6th clone which has an insert with a restriction map without any similarity to that of the other 5 clones, comes from a different genomic DNA fragment.
• the nucleotide sequence of one of the 5 clones P153A exhibiting a 2.7 kb insert was determined by the dideoxitermination method on a sequencer Applied Biosystem. The total sequence of the insert is shown in the appendix, under the identification number SEQ ID NO 1, as well as the amino acid sequence which is deduced therefrom.
• the coding region of the malolactic gene is 1620 nucleotides. The first 20 amino acids deduced from the nucleotide sequence obtained are identical to the protein sequence determined from the purified enzyme, with the exception of the 14th amino acid (lysine instead of cysteine determined from the purified enzyme).
• This open reading phase is capable of coding for a protein of 53 kD, which is entirely compatible with the size of the malolactic enzyme of Lactococcus lactis estimated by gel filtration and by SDS-PAGE electrophoresis (approximately 60 kD).
• the sequence SEQ ID NO 1 also comprises 465 nucleotides upstream from the translation initiation codon. This sequence includes the entire promoter of the malolactic enzyme gene. Lactococcus lactis promoters are on the order of 150 nucleotides upstream of the initiation codon. In addition, the characteristic signals of initiation of transcription of the genes of Lactococcus lactis are present.
• malolactic enzyme which uses, like the malic enzyme, malate as a substrate, and requires NAD for the transformation of the substrate into lactate.
• the medium is buffered to pH 6.5 by addition of citric acid.
• L-lactate was carried out using a kit (BOEHRINGER), according to the protocol described by the supplier.
• the level of lactate produced is quite low if it is compared to that produced by the control strain of L. lactis tested under the same conditions and from the same malate concentrations.
• the production of L-lactate in L. lactis corresponds to the L-lactate produced by fermentation malolactic, and also a significant proportion of L-lactate produced by degradation of sugars, via L-LDH which converts pyruvate into lactate.
• the coding region was placed under the control of regulatory elements.
• the expression plasmid pVTlOO-U was used. This plasmid contains the origin of yeast replication 2 ⁇ , the selection marker URA3 and the strong regulatory elements ADH (promoter and terminator of alcohol dehyrogenase I), as well as the bacterial elements (origin of replication and gene for resistance to 'ampicillin).
• This plasmid has been described by VERNET et al [Gene 52; 225-233, (1987)]
• the coding region of the malolactic gene was amplified by PCR from the plasmid pl53A, using primers consisting of oligonucleotides derived from the sequence of the malolactic gene.
• primers consisting of oligonucleotides derived from the sequence of the malolactic gene.
• the introduction of Xhol restriction sites upstream and Xbal downstream of the coding region was carried out during the amplifications.
• oligonucleotides used as primers are: - an oligonucleotide making it possible to isolate the coding region at the level of the ATG translation initiation codon (primer 1) and comprising an Xhol site
• the chosen oligonucleotide (primer 3') is located a few nucleotides downstream from the translation termination codon.
• Taq buffer 10X 10 ⁇ l Taq polymerase (5 ⁇ / ⁇ l) 0.5 ⁇ l dNTP2 ⁇ M 10 ⁇ l MgCl 2 25mM 6 ⁇ l pl53A 4 ⁇ l (40ng)
• Amplification conditions 30 seconds at 94 ° C, 30 seconds at 40 ° C, 1 min at 72 ° C. for 30 cycles.
• the size of the amplified fragments was verified by analysis of an aliquot on agarose gel. 100 ng of the amplified fragments were digested with Xhol and Xbal and ligated to 50 ng of vector pVTlOO-U previously digested with Xhol and Xbal and dephosphorylated.
• the vector pVTlOO-U obtained, called pMl, is represented in FIG. 3.
• pMl Yeast transformation
• the yeast strain Saccharomyces cerevisiae SCV5M was transformed with the vector pM1.
• Microorganisms held by the Institut Pasteur, under the number 1-1222. It is a haploid laboratory strain, ura “ , MATa, derived from an oenological strain.
• the two media used do not contain uracil, which ensures selection pressure for the plasmids.
• a transformant of ScV5M by the plasmid pVTlOO-U containing no insert was used as negative control.
• CULTURE AND DOSAGE CONDITIONS 8 ml of medium are inoculated with the yeast strains (transformants and control). Growth is performed in sterile 10 ml tubes, at 28 ° C, for 60 h, without shaking.
• the recombinant strains therefore degrade the malate to lactate by malolactic fermentation, and with greater efficiency at acid pH, therefore under conditions close to those of oenology.
• the expression of the malolactic gene has also been studied in Schizosaccharomyces pombe.
• the coding region of the malolactic gene was placed under the control of a promoter of S. pombe, in a shuttle vector S. pombe / E. coli. a) Introduction of qèn ? TM lactic acid on the PARTI multicopy plasmid
• the expression plasmid pART1 was used. This plasmid contains the origin of replication of yeast ARS1, the selection marker LEU2 and the promoter of S. pombe alcohol dehydrogenase, as well as bacterial elements (origin of replication and gene for resistance to ampicillin). This plasmid has been described by McLeod et al., (EMBO J. 6; 729-736, 1987).
• the coding region of the malolactic gene was amplified by PCR from the plasmid pl53A, using primers consisting of oligonucleotides derived from the sequence of the malolactic gene. In order to facilitate cloning, the introduction of BamHI restriction sites upstream and Sac1 downstream of the coding region was carried out during the amplifications.
• the oligonucleotides used as primers are:
• an oligonucleotide allowing the coding region to be isolated at the level of the ATG translation initiation codon (primer 12) and comprising a BamHI site - on the 3 ′ side, the chosen oligonucleotide (primer 12)
• the size of the amplified fragments was verified by analysis of an aliquot on agarose gel.
• transformants obtained One of the transformants obtained, called Sp / D42, was tested for its ability to carry out malolactic fermentation (conversion of malate to lactate).
• the transformant was cultivated under the following conditions:
• a transformant of the same strain by the plasmid pART1 containing no insert was used as a negative control.
• the cultures are centrifuged for 5 min at 4000 g and the supernatant tested for the production of L-lactate using a BOEHRINGER kit, as indicated previously.
• the transformant Sp / D42 is capable of degrading practically all of the L-malate in the medium (at pH 3.5, only 150 mg / 1 of L-malate have not been degraded) and moreover, to convert more than 80% of the malate present into L-lactate (3.05 g / 1).
• TABLE III DETERMINATION OF LACTATE IN S. POMBE
• AAAAGTAGAG TGATTTTACT CTACTTTTTT AGAATACTTT TATATAATAG GAAATATGAA 120
• AAAAAACTTA AAGAAACGAA AATATCGGGA ATTAGTCTTC CCTTATATGC CTTTTTCGTA 2165

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