Cloning and Expression of the Gene of the Malolactic Enzyme of Lactococcus Lactis

The present invention relates to the cloning of the structural gene for 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 encoding this gene. It also relates to the expression of this gene in various prokaryotic or eukaryotic hosts, and in particular in Saccharomyces and Schizosaccharomyces.

Malolactic fermentation, a secondary fermentation which is observed during the process of winemaking in addition to the alcoholic fermentation performed by yeasts, consists in the degradation, by certain lactic acid bacteria, of the malic acid present in wines, to lactic acid and CO.sub.2. The bacterial species naturally present in the fermentative flora and which are responsible for the malolactic fermentation belong to the genera Lactobacillus, Leuconostoc and Pediococcus.

This reaction has two main advantages:

It leads to a deacidification of the wine by conversion of a diacid (L-malate) to a monoacid (L-lactate), the consequences of which are advantageous at the organoleptic level, for the majority of red wines and some white wines,

It permits the microbiological stabilization of the wine, and therefore a better preservation, by using a fermentable substrate, which prevents subsequent fermentations by other strains of undesirable microorganisms. Finally, malolactic fermentation can have an influence on the quality of the wine from the aromatic point of view.

This reaction poses, however, a certain number of problems because of its slowness and its uncontrollable character.

Malolactic fermentation occurs spontaneously only at the end of the natural wine-making process; indeed, the pH, the ethanol content and the presence of SO.sub.2 in the wine considerably slow down the growth of the lactic acid bacteria naturally present in the fermenting mass and which are responsible for the malolactic fermentation. In order to try to accelerate this fermentation, the technique most commonly used lies in the addition, to the fermenting medium, of an inoculum of lactic acid bacteria.

However, this technique does not always resolve the problems of implantation of the lactic acid bacteria because of the difficulty which they have in adapting to the wine medium. Moreover, the introduction of lactic acid bacteria amounts to adding, in addition to the malolactic enzyme whose activity is desired, numerous other enzymes whose action on the numerous substrates present in the wine can result in a development of the organoleptic qualities of the product which is very difficult to control.

It would therefore be desirable to have the possibility of carrying out the malolactic fermentation so as to arrive rapidly, and while removing uncontrollable variables, at an improvement of the product obtained.

With the aim of better controlling the malolactic fermentation, it has been proposed to clone the genes for the malolactic enzymes of lactic acid bacteria, and to introduce and express them in the microorganism which is normally involved in the manufacture of wine, such as S. Cerevisiae.

WILLIAMS et al. [App. Environ. 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 L. delbrueckii into S. Cerevisiae and into E. coli.

The gene for the malolactic enzyme of L. oenos has also been cloned into E. Coli [LAUTENACH et al., Microbiol., 39:29-39 (1984)].

However, the strains of microorganisms transformed express the gene only at a very low level, insufficient to permit its use in the manufacture of wine.

In addition, in both cases, an instability of the DNA cloned into E. Coli was observed, even going as far as bringing about the complete loss of the gene.

Moreover, a malolactic enzyme, having an activity of a level comparable to that of the malolactic enzyme of bacteria of the genera Leuconostoc, Pediococcus and Lactobacillus mentioned above, have recently been purified from Lactococcus lactis. It is a protein of about 230 kDa, consisting of subunits of about 56 kDa. Its pHi. is about 4.3, and its Km for malic acid is about 10 to 12 mM. Its N-terminal sequence has also been determined [RENAULT, Malolactic fermentation: Genetics and Genetic Engineering, GIM90, 6th International Symposium on Genetics of Industrial Microorganisms, Strasbourg (1990)].

From a purified preparation of this malolactic enzyme, the Inventors obtained preparations of polyclonal antibodies specifically directed against this enzyme. A Lactococcus lactis DNA library was then prepared in the vector .lambda.gt11, in E. coli. The screening of this library with the antibodies obtained made it possible to clone DNA fragments, which represent between the whole lot 4.5 kb of the same region of genomic DNA of Lactococcus lactis, containing the malolactic gene, and a fragment of the gene for malate permease, these genes being organized into operon. The Inventors identified a 2684 bp DNA fragment including the entire gene for the malolactic enzyme, and determined the sequence of this fragment, which is represented in the sequence listing in the annex under the number SEQ.ID.NO.1. In addition, the expression of the malolactic gene has been obtained in various prokaryotic and eukaryotic hosts. Thus, the introduction of the Lactococcus lactis malolactic gene into E. coli, under the control of its own regulatory elements as well as the expression of the said gene in yeast under the control of yeast regulatory elements have been achieved.

The subject of the present invention is a nucleic acid fragment, characterized in that it comprises the sequence of the gene for the malolactic enzyme of Lactococcus lactis.

By "sequence of the gene for the malolactic enzyme of Lactococcus lactis", there is understood in particular the coding sequence stretching from nucleotides 466 to 2085 (inclusive) of the sequence SEQ. ID. NO 1 represented in the sequence listing in the annex, as well as any sequence which, taking into account the degeneracy of the genetic code, encodes the same polypeptide; this definition also comprises the sequences carrying minor modifications intended to permit a better expression of the gene in a determined host.

The invention also encompasses nucleic acid fragments comprising a sequence homologous or complementary to the sequence of the gene for the malolactic enzyme of Lactococcus lactis, as defined above, or to a fragment of at least 10-mer, preferably of at least 20-mer, of the said sequence, and which can be used in particular as probes for the location of the said gene and/or as primers for its amplification.

The subject of the invention is also 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 the said gene.

By "sequences regulating the expression of a gene", there is understood sequences of the promoter and terminator type which are active in the host in which it is desired to obtain the expression of the said gene. The promoters and terminators of various genes can be used, associated in various combinations.

Among the sequences which can be used to obtain the expression, in yeast, of the gene for the malolactic enzyme of Lactococcus lactis, there may be mentioned, as non-limitative example, the promoters and terminators, known per se, of the genes for alcohol dehydrogenase I (ADHI), for phosphoglycerate kinase (PGK), and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The expression cassettes in accordance with the invention can be carried by plasmids, or integrated into the chromosomal DNA of the host yeast.

The subject of the invention is also recombinant vectors characterized in that they comprise at least one DNA fragment containing the sequence of the gene for the malolactic enzyme of Lactococcus lactis, as defined above.

According to a preferred embodiment of the present invention, the said vectors are expression vectors, comprising an expression cassette as defined above.

According to a preferred embodiment of the present invention, the said vectors comprise regulatory sequences which are active in the yeast.

Advantageously, the vectors in accordance with the invention are shuttle vectors, also possessing a bacterial replication origin and a selectable marker in a bacterium (for example, gene for resistance to an antibiotic).

These vectors can be selected on the basis of the nature and the strength of the regulatory elements which enter into the expression cassette. The promoters and terminators described above, or any other sequence which makes it possible to control and regulate the expression of a gene in yeast, can be chosen.

Another criterion for the choice of vectors lies in the number of copies thereof, which is determined by the choice of the replication origin.

By way of example, a choice can be made between the following vectors:

Replicative vector (YEp) at high copy number, possessing as replication origin in yeast, a portion of the endogenous 2 .mu. plasmid.

Replicative vector (YRp) at high copy number, possessing as replication origin, a chromosomal ARS sequence.

Linear replicative vector (YLp) at high copy number, possessing telomer sequences as replication origin.

Replicative vector (YCp) at low copy number, possessing a chromosomal ARS sequence and a centromere sequence.

Preferably, the vectors in accordance with the invention also contain selectable markers in yeast, such as markers for auxotrophy: URA.sub.3, LEU.sub.2, HIS.sub.3, TRP.sub.1, ADE, and the like and/or markers for resistance to antibiotics (G418, hygromycin B, chloramphenicol, phleomycin), to herbicides (sulphometuron-methyl), to copper, and the like.

Vectors in accordance with the invention, carrying the gene encoding the malolactic enzyme of Lactococcus lactis can be introduced into any yeast strain, by various transformation techniques.

Among the most common transformation techniques which can be used, there may be mentioned the protoplasts technique, the technique of permeabilization to lithium salts and electroporation.

In all cases, the process for this construction comprises the following steps:

construction of an expression cassette comprising the gene for the malolactic enzyme and the regulatory elements of variable strength;

introduction of this cassette either as single copy, or as multiple copies into the yeast,

The choice can also be made to integrate the gene for the malolactic enzyme of Lactococcus lactis, equipped with its control sequences, into the genome of the yeast, in which case an integrative vector (YIp) possessing no replication origin in the yeast will be chosen for example; it is also possible to integrate this gene using other techniques, for example co-transformation.

It is possible to modulate the level of expression of the gene encoding the malolactic enzyme of Lactococcus lactis, by adjusting especially the number of copies of the gene which are introduced into the yeast, and/or the strength of the regulatory elements associated therewith.

The subject of the present invention is also 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 encoding the malolactic enzyme of Lactococcus lactis, under the control of sequences regulating the expression of the said gene in the said cell.

According to a preferred embodiment of the present invention, the said cells are yeast cells.

The yeast strains in accordance with the invention find numerous applications in the food industry and in particular in the field of oenology for carrying out malolactic fermentation.

According to a preferred arrangement of this embodiment, the said yeasts belong to the genus Sacharomyces.

According to another preferred arrangement of this embodiment, said yeasts belong to the genus Schizosaccharomyces.

Schizosaccharomyces is sometimes used in oenology for its capacity to degrade malate. However, its use is limited because this degradation which is carried out using the malic enzyme does not produce lactate, but ethanol and CO.sub.2, which results in a substantial deacidification, which may have negative influences on the characteristics of the wine.

Strains of Schizosaccharomyces obtained in accordance with the invention and expressing the gene for the malolactic enzyme, carry out the malolactic fermentation with degradation of L-malate to L-lactate, and thus have the advantage of permitting a much less substantial deacidification than that which results from the malate/ethanol conversion.

These Schizosaccharomyces strains can have numerous uses in oenology; they can for example be used:

in co-culture with an oenological strain of Saccharomyces cerevisiae;

as immobilized cells.

A more complete appreciation of the invention and many of he attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.

FIG. 1 shows the restriction maps of four of five DNA fragments of digested Lactococcus lactis DNA having a common region ligated into vector .lambda.gt11, with the inserts subcloned into plasmid pUC18;

FIG. 2 shows the combination of a region of DNA coding for malolactic gene with oligonucleotide primer 1 or 12, respectively containing XhoI and BamHI sites, and with oligonucleotide primers 3 or 13, on the 3' side, respectively having XbaI and SacI sites;

FIG. 3 is a restriction map of vector pM1 containing malolactic gene; and

FIG. 4 is a restriction map of vector pD4 of an amplified DNA fragment containing malolactic gene with primer 12 (5' end) having a BamHI site and primer 13 (3' end) having a SacI site incorporated into vector pART1.

The present invention will be understood more clearly with the aid of the additional description below, which refers to examples describing the cloning of the gene for the malolactic enzyme of Lactococcusa lactis and its expression in bacteria and yeasts.

Malolactic enzyme was purified as described by RENAULT (1990, communication cited earlier); cf. also C. ROULLAND, (DEA in oenology-ampelography, September 1988, University of Bordeaux II).

The general techniques for manipulation of nucleic acids and molecular cloning to which reference is made in the examples below are those described by SAMBROOK et al. [Molecular Cloning: A Laboratory Manual (second edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)].

The extraction procedure is briefly recalled below:

Strain--culture conditions

Lactococcus lactis (IL 1441) is cultured in the following medium:

The pH is adjusted to 5%. The sterile medium is inoculated with 10% (v/v) of a preculture.

The cells are cultured in 3 liters of the above medium at 28.degree. C., for 24 hours, harvested by centrifugation at 1000 g for 10 min at 4.degree. C., and washed twice with 0.9% NaCl.

The pellet is taken up in 30 ml of phosphate buffer (0.1M, pH 6.0). The cells are ground by ultrasound for 6 min at 0.degree. C. After centrifugation (30,000 g, 15 min, at 4.degree. C.) the supernatant constitutes the crude extract.

The proteins are assayed by BCA Protein Assay Reagent (PIERCE).

The malolactic activity is measured by the decarboxylation of malic acid monitored using a CO.sub.2 electrode (EISCHWEILER).

After treatment with DNAse I (1 mg/ml and 15 min of incubation at room temperature), the crude extract is subjected to a fractional precipitation with ammonium sulphate (35% saturation, then 70%).

The supernatant from the precipitation at 70% has no activity. The pellet, taken up in 2 ml of 0.1M phosphate buffer, pH 6.0, and which contains all the malolactic activity, is deposited onto a filtration column on ACA 34 gel (ULTROGEL; fractionation range from 20,000 to 350,000) 40 cm in length and 2.6 cm in diameter, previously equilibrated with elution buffer: 0.01M phosphate buffer, pH 6.0, 0.1M KCl. The elution is performed with a flow rate of 18 ml/h. The volume of fractions collected is 2.4 ml.

Under these conditions, the most active fractions correspond to fractions 50 to 54. They are combined for an additional purification by isoelectric focusing. The isoelectric focusing is performed on a 110 ml LKB column. The pH gradient is formed by means of the ampholines used at 2% and is stabilized by a glycerol density gradient which is established during the filling of the column. The most active fraction which focuses at pH 4.3, the phi of the protein, is recovered and then undergoes an additional step of purification by ion-exchange chromatography (SEPHAROSE Q anion-exchange column, PHARMACIA).

The elution is performed by a gradient from 0 to 0.5M NaCl, in a 50 mM Tris-Hcl buffer, pH 7.6, 0.1 mM EDTA, 1 mM .beta.-mercaptoethanol.

The fraction containing the malolactic activity is eluted at an NaCl concentration of 0.33M. It is this fraction which is used for the production of antibodies.

The antibodies were obtained by injecting into two rabbits a portion of the purified preparation of malolactic protein according to the following procedure:

Subcutaneous injection of 2 ml of solution of malolactic protein (100 .mu.g in 0.3M NaCl buffer) supplemented with 2 ml of complete Freund's adjuvant.

Second injection 40 days later, with the same quantity of malolactic protein and 2 ml of incomplete Freund's adjuvant.

The blood of the two rabbits is recovered 12 days after the 2nd injection, left for 24 h at 4.degree. C. to allow coagulation, centrifuged at 10,000 g for 10 min at 4.degree., and the serum recovered is aliquoted and stored at -20.degree. C.

The antibodies to malolactic enzyme were then tested by ELISA test carried out on both sera (called 120 and 119) obtained from rabbit serum according to the procedure described by SAMBROOK et al. [(1989)]. This analysis made it possible to determine that the optimal dilution for the use of the antibodies was 1/1000th.

The specificity of the antibodies was then determined by immunoelectrophoretic transfer (Western blotting).

The proteins from a crude extract of Lactococcus lactis (prepared as described in Example 1), on the one hand, and from the purified preparation of malolactic enzyme, on the other hand, were separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose membrane.

The polyclonal antibodies obtained are then bound to the membrane and revealed by anti-rabbit antibodies coupled to alkaline phosphatase.

The serum 120 recognizes, on the crude extract and on the purified fraction, a predominant band of molecular weight of about 60 kD corresponding to the malolactic enzyme, and several contaminant bands of lower molecular weight.

On the other hand, the serum 119 recognizes on the purified fraction, as well as on the crude extract, a single band, corresponding to the malolactic enzyme; this highly specific serum was therefore used for the subsequent experiments.

a) Extraction of Lactococcus lactis DNA

The Lactococcus lactis genomic DNA library was prepared in the vector .lambda.gt11.

The genomic DNA of Lactococcus lactis IL 1441 was, in a first stage, extracted and purified. For that, 500 ml of a culture in exponential phase are centrifuged at 5000 g for 10 min. The pellet is taken up in 5 ml of TE (10 mM Tris, 1 mM EDTA, pH 8) containing 150 mM NaCl and 1 mg/ml of lysozyme, and incubated for 10 min at 37.degree. C. 1 ml of SDS, 25% in EDTA, 0.1M, pH 8, are added and the mixture is incubated for 15 min at 60.degree. C. The DNA is purified by two extractions with phenol-chloroform, followed by two others with chloroform-isoamyl alcohol (24:1). A final purification is carried out on caesium chloride gradient in the presence of ethidium bromide. The 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 using a rod and dissolved in 1 ml of TE. The DNA concentration in solution was determined (1 .mu.g/.mu.l) by measurement of the OD at 260 nm.

b) Digestion of the Lactococcus lactis DNA

125 .mu.g of Lactococcus lactis DNA are partially digested with two units of enzyme AluI and two units of HaeIII for 35 min at 37.degree. C. so as to generate blunt ends. The digestion is stopped by addition of EDTA to 0.1M final.

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 .mu.l of TE and the fragments separated on sucrose gradient for 15 hours at 25,000 rpm.

Fractions of 0.5 ml are harvested and analysed on 0.8% agarose gel. The fraction containing fragments of 1.5 and 4 Kb is dialysed against TE for 4 hours.

c) Ligation of the digested DNA into the vector

.lambda.gt11

The vector .mu.gt11 (derived from bacteriophage .lambda.) was chosen to prepare the expression library. The principle of the preparation of the library consists in inserting DNA fragments into the 3' region of the phase encoding the .beta.-galactosidase gene, so as to express hybrid proteins under the control of the .beta.-galactosidase promoter.

A kit for cloning into .lambda.gttII (AMERSHAM) was used according to the procedure recommended by the supplier.

1 .mu.g of (blunt-ended) DNA digested and dialysed as described above was inserted into the vector .lambda.gt11 according to the following principle:

The DNA is ligated to dephosphorylated adaptors having a blunt end and another EcoRI cohesive end. After removal of the free adaptors by purification on exclusion column, the "adapted" DNA fragments are phosphorylated and ligated to the arms of the vector which were also digested with EcoR1 and dephosphorylated.

The in vitro 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.sup.- km.sup.+ k) lac U169, ProA.sup.+, lon.sup.-, araD139 StrA, SupF, trpC22: Tn10 (pMC9)] with an aliquot fraction of the phage suspension, and the transformants selected on LB medium (bactotryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l)+ampicillin (50 .mu.g/ml) at 43.degree. C. (to induce bacterial lysis). The lysis plaques are obtained after 4 h of incubation.

The library thus obtained represents about 3 times the Lactococcus lactis genome.

a) Plating of the library and preparation of the filters

75,000 lysis plaques were obtained by plating the library on LB+ epicillin+MgCl.sub.2 medium and incubation for 3 h 30 min at 43.degree. C. (conditions for non-expression). The plates are covered with nitrocellulose filters (C extra, AMERSHAM), impregnated with 10 mM IPTG and incubated for 4 h at 37.degree. C., so as to induce the expression.

The filters are rinsed in TNT (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20), and then incubated with gentle stirring for 30 min at room temperature in the same buffer.

In order to limit the detection of "false positive" clones which can hybridise in a non-specific manner during the screening of the library, a second series of filters, constituting duplicates of the first series, were deposited on the plates, incubated and treated in the same manner.

b) Depletion of the serum 119

The immunological screening of the library is carried out with the aid of the serum 119 previously depleted against the E. coli proteins. The depletion of the serum is performed as follows:

A culture of E. coli Y1090 is carried out in 100 ml of LB+ampicillin for 24 h at 37.degree. C. The culture is centrifuged at 5000 g for 10 min at 4.degree. C. The pellet is taken up in 3 ml of buffer (50 mM Tris, 10 mM EDTA, pH 8), and the suspension frozen at -20.degree. C. and thawed (several times). The cells are completely lysed by sonication at 0.degree. C. (six times 20 seconds), then centrifuged at 12,000 g for 10 min at 4.degree. C. The lysate is recovered and incubated for 4 h at room temperature in the presence of 1 ml of antibody 119 diluted 1/10th in TNT buffer+1% gelatin+3% BSA+5% powdered skim milk.

The depleted serum is stored at 4.degree. C. in the presence of sodium azide (0.05%).

c) Immunological screening of the library

The nitrocellulose filters are then incubated in the presence of the depleted serum 119 as described above, and the revealing of the bound antibodies is carried out with the aid of anti-rabbit antibodies coupled to alkaline phosphatase, as previously described.

Only the clones showing a positive signal on the two filters are taken into consideration. 24 positive clones were thus selected.

In order to verify the specificity of the signal obtained, the positive clones were subcloned and a second detection with the aid of antibodies was carried out. Around each positive lysis plaque, an agarose carrot is removed and incubated in 500 .mu.l of SM buffer (NaCl 6 g/l, MgSO.sub.4 7H.sub.2 O 2 gl, Tris base 6 g/l, gelatin 0.01%, pH 7.5), for 2 hours at 4.degree. C. in order to allow diffusion of the phage particles. This suspension is again used to infect E. coli 1090 and the mixture plated on LB+ampicillin+MgCl.sub.2 plate as above, so as to obtain well isolated lyses plaques. A second screening by the serum 119 carried out as above made it possible to eliminate 14 clones out of the 24 tested, which no longer exhibited significant signals. The other 10 clones, very clearly positive were selected.

d) Characterization of the recombinant clones

The phage DNA from the 10 positive clones was extracted and analysed. A well isolated positive lysis plaque (subclone) was systematically used as starting material.

The DNA is extracted from confluent lysis plaques on LB+ampicillin plates. The plates are covered with 5 ml of SM buffer and stirred at 4.degree. C. for 2 hours. The suspension is recovered, supplemented with few drops of chloroform and centrifuged at 4000 g for 10 min at 4.degree. C. in order to remove the 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 in order to determine their size.

Two oligonucleotides [leda gt11 Primer (forward) 24-MER and lambda gt11 Primer (reverse) 24-MER, company BIOLABS] corresponding to .beta.-galactosidase sequences surrounding the EcoRI cloning site were used as primers for amplification of the inserts.

Analysis of the amplification products on agarose gel (1.6%) made it possible to determine that the 10 .lambda.gtt11 clones selected had inserts of between 2 and 3.5 kb in size.

In order to establish the restriction map of the cloned DNA fragments, the inserts of the .lambda.gt11 vectors were subcloned into the plasmid pUC18 [YANISHPERRON et al. Gene No. 33, p. 103-119, (1985)]. It was thus possible to obtain the subcloning of 6 inserts.

The restriction map of the 6 inserts was prepared; 5 of the 6 inserts have a common region, suggesting that these 5 inserts come from the same genomic fragment. This was verified by Southern hybridization of restriction fragments of Lactococcus lactis genomic DNA separated on agarose gel and transferred onto Nylon membrane, with the inserts radioactively labelled with .sup.32 p.

The restriction maps of 4 of these 5 inserts are represented in FIG. 1.

The sizes of the inserts represented are 3 kb, 2.7 kb, 2 kb and 3 kb. As these inserts overlap, the total size of the cloned region is 4.5 kb.

The 6th clone, which has an insert having a restriction map without any similarity with that of the other 5 clones, comes from a different genomic DNA fragment.

In order to determine whether one of the two types of cloned DNA fragments contained the 5' region of the gene encoding the malolactic enzyme of Lactococcus lactis, a Southern hybridization of the inserts separated on agarose gel and transferred onto nylon membrane, with a probe of oligonucleotides derived from the protein sequence of the NH.sub.2 -terminal end of the malolactic enzyme (P. RENAULT, communication cited earlier) was carried out. The probe chosen is a mixture of oligonucleotides deduced from the sequence including the first 6 amino acids:

The 5 inserts coming from the same DNA fragment hybridize with the probe; which proves that the 5' end of the gene encoding the malolactic enzyme of Lactococcus lactis is contained in the 5 cloned inserts. On the other hand, no hybridization was detected on the 6th clone.

e) Sequence of the clone P153A

The nucleotide sequence of one of the 5 clones P153A having a 2.7 kb insert was determined by dideoxy-termination method in an Applied Biosystem sequencer. The total sequence of the insert is represented in the annex, 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 frame is capable of encoding a protein of 53 kD, this is completely compatible with the size of the malolactic enzyme of Lactococcus lactis estimated by gel filtration and SDS-PAGE electrophoresis (about 60 kD).

The sequence SEQ ID NO 1 comprises, in addition, 465 nucleotides upstream of the codon for initiation of translation. This sequence comprises the entire promoter of the gene for the malolactic enzyme. The Lactococcus lactis promoters have a size of the order of 150 nucleotides upstream of the initiation codon. Furthermore, the signals characteristic of initiation of transcription of the Lactococcus lactis genes are present.

Downstream of the open reading frame, 596 nucleotides were sequenced, and another open reading frame of 581 nucleotides was detected in 3' of the malolactic gene in another reading frame.

The determination of homologies with the genes or known proteins was carried out by comparison of the nucleotide sequence SEQ ID NO 1, and the protein sequences deduced with databases.

As regards the open reading frame of 1620 nucleotides, very substantial amino acid and nucleotide conservations were detected with various malic enzymes, which are responsible for the decarboxylation of malate to pyruvate.

Moreover, a consensus sequence for binding of NAD was found.

These homologies are perfectly consistent with the function of the malolactic enzyme, which uses, as malic enzyme, malate as substrate, and requires NAD for the conversion of the substrate to lactate.

As regards the beginning of the reading frame identified downstream of the malolactic gene, amino acid homologies, which were also substantial (of the order of 40%), were identified with citrate permeases. It appears extremely probable that this open reading frame may correspond to the 5' region of a malate permease.

It therefore appears that the structural gene encoding the malolactic enzyme, and a portion of the malate permease gene are present on the DNA fragment sequenced, and that these genes are organized into operon.

The expression of the malolactic gene was studied in E. coli, in various transformants of the bacterial strain DH5.alpha.: p153A (clone sequenced), p5A, p191A (see restriction maps, FIG. 1).

The transformants as well as the control strain DH5.alpha. transformed with pUC18, were inoculated into a range of culture media at increasing malate concentration: 3 ml of M9 medium (Na.sub.2 HPO4, 7H.sub.2 O 6 g/l, KH2PO4 3 g/l, NaCl 0.5 g.l, NH4Cl 1 g/l, glucose 4 g/l), supplemented with thiamine 1 mg/l, aspartic acid 20 mg/l and ampicillin 20 mg/l, and containing 0; 0.3; 0.5 or 1% malate (w/v). The medium is buffered to pH 6.5 by the addition of citric acid.

The cultures were incubated at 37.degree. C., with stirring, for 48 h.

As control, Lactococcus lactis was cultured in 8 ml of M9 medium containing 5 g/l of "YEAST EXTRACT" and the same malate range. The cultures were incubated at 28.degree. C., for 48 h, under anaerobic conditions.

The cultures were then centrifuged for 10 min at 4000 g. The supernatants were recovered and tested for the presence of L-lactate. The L-lactate assay was performed with the aid of a kit (BOEHRINGER), according to the procedure described by the supplier.

The results, presented in Table I below (expressed in g/l) show a very clear production of lactate in the culture supernatant of the transformants p153A and p191A, whereas no trace of lactate is detected in the supernatant of the control strain (E. coli DH5 transformed with pUC18). Another transformant, p5A, which was tested under the same conditions, does not, on the other hand, produce lactate. This is explained by the fact that the malolactic gene carried by the recombinant vector p5A has a 3' end from which 200 nucleotides have been truncated. This region therefore seems to be essential for the malolactic activity.

In the absence of malate, there is no significant production of lactate in the culture supernatant of the transformants p153A and p191A, nor of the control strain transformed with pUC18. This is explained by the fact that E. coli does not produce L-lactate under these culture conditions.

In the presence of malate, no production of lactate is observed for the control strain. On the other hand, as regards the transformants p153A and p191A, a significant production of lactate is observed in the presence of 0.1, 0.3 and 1% malate.

The level of lactage produced is quite low when it is compared with that produced by the control strain L. lactis tested under the same conditions and starting with the same malate concentrations. However, it should be noted that the production of L-lactate in L. lactis corresponds to the L-lactate produced by malolactic fermentation, and also to a high proportion of the L-lactate produced by degradation of sugars, via L-LDH which converts pyruvate to lactate.

Moreover, it is difficult to determine the portion of malate which is really available for the malolactic fermentation. Indeed, under the culture conditions used for E. coli, a significant portion of the malate can be used via the tricarboxylic cycle.

In order to carry out the expression of the malolactic gens cloned into yeast, the coding region was placed under the control of yeast regulatory elements (promoters and terminators), in yeast/E. coli shuttle vectors.

a) Introduction of the malolactic gens into the multicopy plasmid pVT10-U

The expression plasmid pVT100-U was used. This plasmid contains the replication origin of yeast 2 .mu., the selectable marker URA3 and the strong ADH regulatory elements (alcohol dehydrogenase I promoter and terminator), as well as the bacterial elements (replication origin and the gens for resistance to ampicillin).

This plasmid has been described by VERNET et al [Gens 52; 225-233, (1987)].

The coding region of the malolactic gens was amplified by PCR starting with the plasmid p153A, with the aid of primers consisting of oligonucleotides derived from the sequence of the malolactic gene. In order to facilitate the cloning, the introduction of restriction sites XhoI upstream and XbaI downstream of the coding region was carried out during the amplifications.

The oligonucleotides used as primers are:

an oligonucleotide which makes it possible to isolate the coding region at the level of the codon for initiation of translation ATG (primer 1) and containing a XhoI site.

On the 3' side, the oligonucleotide chosen (primer 3') is located a few nucleotides downstream of the codon for termination of translation.

The sequences as well as the exact positions of the primers used are indicated in FIG. 2.

The amplification was carried out in the following manner:

Amplification conditions: 30 seconds at 94.degree. C., 30 seconds at 40.degree. C., 1 min at 72.degree. C. for 30 cycles.

The size of the amplified fragments was checked by analysing an aliquot an agarose gel.

100 ng of the amplified fragments were digested with XhoI and XbaI and ligated to 50 ng of pVT100-U vector previously digested with XhoI and XbaI and dephosphorylated.

The vector pVT100-U obtained, called pM1, is represented in FIG. 3.

b) Transformation of the yeast

The yeast strain Saccharomyces cerevisiae SCV5M was transformed with the vector pM1.

The strain SCV5M was deposited on 18 June 1992 at the Collection Nationale de Cultures de Microorganismes, held by Institut Pasteur, under the number I-1222. It is a laboratory strain which is haploid, ura.sup.-, MATa, derived from an oenological strain.

The transformation method used is that of lithium acetate described by ITO et al. [J. Bacteriol. No. 153, pp. 163-168, (1983)].

c) Production of malolactic fermentation in yeast

Two of the transformants obtained, called ScV5M/pMIa and ScV5M/pMIb were tested for their capacity to carry out the malolactic fermentation (conversion of malate to lactate); these transformants were cultured under the following conditions:

on the one hand, on minimum synthetic medium YNB (6.7 g/l "yeast nitrogen base without amino acid" (DIFCO), 20 g/l glucose) containing 0, 0.6 or 1% malate, buffered either to pH 3, or to pH 6 with citric acid (6.3 g/l).

On the other hand, on synthetic medium mimicking the composition of a grape must, [SABLAYROLLES et BARRE, Sciences des Aliments, No. 6, p. 373-383 (1986)], buffered to pH 3.3.

The two media used do not contain uracil, which ensures a selection pressure for the plasmids.

A transformant of ScV5M by the plasmid pVT100-U containing no insert was used as negative control.

8 ml of medium are inoculated with the yeast strains (transformants and control). The growth is carried out in sterile tubes of 10 ml, at 28.degree. C., for 60 h, without stirring.

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 in Example 5.

The results obtained are presented in Table II.

In the minimum synthetic medium YNB (Table II), in the absence as in the presence of malate (0.3 and 1%), no trace of L-lactate is detected with the control yeast (ScV5M/pVTU). This is completely consistent with the fact that S. cerevisiae is incapable of carrying out the conversion of malate to lactate, and that, on the other hand, under anaerobic conditions, it produces only traces of lactate from glucose, mainly in the form of D-lactate.

The transformed strains pM1a and pM1b, in the absence of malate, produce traces of L-lactate of the order of about fifty to about one hundred milligrams. It is probable that this slight production results from the conversion, by the malolactic enzyme, of a portion of the endogeneous yeast malate, metabolized from glucose. In the presence of malate in the culture medium (0.6 and 1% of malate), and in YNB media buffered to pH 3, or in synthetic broth (pH 3.3, 3 g/l of malate), a very significant production of L-lactate (from 0.5 to 0.7 g/l for the clone pMla under these experimental conditions) is observed.

The highest production is observed in synthetic broth whose composition is very similar to that of the grape musts (0.7 g/l of L-lactate, equivalent to 7.7 .mu.mol/ml).

Under the same culture conditions (YNB) but at pH 6, the production of L-lactate is very low, of the same order of magnitude as in the absence of malate. The difference in production of L-lactate according to the pH of the culture medium could be explained by a problem of permeation of the malate in S. cerevisiae at pH 6. At pH 3 or 3.3, a large portion of the malate (pK 3.5) present in non-dissociated form can enter inside the cell by mere diffusion. On the other hand, at pH 6, the malate, mainly in dissociated form, would enter less easily inside the cell.

The recombinant strains therefore degrade malate to lactate by malolactic fermentation, and with a higher efficiency at acidic pH, therefore under conditions similar to those in oenology.

The same experiment was carried out on synthetic broth, and the production of lactate measured after culturing for 10 days; 1.5 g/l (equivalent to 17 .mu.mol/ml) of lactate were produced under these conditions.

The expression of the malolactic gene was also studied in Schizosaccharomyces pombe. For this purpose, the coding region of the malolactic gene was placed under the control of an S. pombe promoter, in an S. pombe/E. coli shuttle vector.

a) Introduction of the malolactic gene into the multicopy plasmid pART1

The expression plasmid pART1 was used. This plasmid contains the yeast replication origin ARS1, the selectable marker LEU2 and the S. pombe alcohol dehydrogenase promoter, as well as bacterial elements (replication origin 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 starting with the plasmid p153A, with the aid of primers consisting of oligonucleotides derived from the sequence of the malolactic gene. In order to facilitate the cloning, the introduction of BamHI restriction sites upstream and SaCI downstream of the coding region was carried out during the amplifications.

The oligonucleotides used as primers are:

an oligonucleotide which makes it possible to isolate the coding region at the level of the codon for initiation of translation ATG (primer 12) (SEQ ID NO: 4) and containing a BamHI site

on the 3' side, the oligonucleotide chosen (primer 13) (SEQ ID NO: 6) is located a few nucleotides downstream of the codon for termination of translation and has a SacI site.

The sequences as well as the exact positions of the primers used are indicated in FIG. 2 (SEQ ID NO: 3-8).

The amplification was carried out in the following manner:

Amplification conditions: 30 seconds at 94.degree. C., seconds at 37.degree. C., 1 min at 72.degree. C. for 30 cycles.

The size of the amplified fragments was checked by analysing an aliquot on agarose gel.

100 ng of the amplified fragments were digested with BamHI and SacI and ligated to 50 ng of vector pART1 previously digested with BamHI and SacI and dephosphorylated.

The vector obtained, called pD4, is represented in FIG. 4.

b) Transformation of S. pombe

An S. pombe strain auxotrophic for leucine was transformed with the vector pD4 and with the vector pART1. It is the laboratory strain leul-32h+, which was supplied by J.KOHLI, Institute of General Microbiology, Raltzerstrasse 4, CH-3012, Bern, Switzerland.

The method of transformation used is that of transformation with lithium acetate adapted from Ito et al. (Journal of Bacteriology 153, 163-168, 1983).

c) Production of malolactic fermentation in S. pombe

One of the transformants obtained, called Sp/D42 was tested for its capacity to carry out the malolactic fermentation (conversion of malate to lactate). The transformant was cultured under the following conditions:

in minimum synthetic medium YNB containing 50 g/l of glucose and 5.5 g/l of L-malate, buffered either to pH 3.5 or to pH 6. This medium does not contain leucine, which ensures a selection pressure for the plasmids introduced.

A transformant of the same strain by the plasmid pART1 not containing insert was used as negative control.

8 ml of medium are inoculated with the yeast strains (transformant and control). The growth is carried out in sterile tubes of 10 ml, at 40.degree. C., for one week, without stirring.

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 above. The degradation of L-malate was studied by assaying the L-malate residual in the culture medium with the aid of a BOEHRINGER kit.

The results obtained are presented in Table III below.

Under the various culture conditions tested, the control yeast Sp/pART1 produces only traces L-lactate (of the order of 20 to 40 mg/l). Indeed, like S. cerevisiae, S. pombe is incapable of converting malate to lactate, and, on the other hand, produces only traces of lactate under anaerobic conditions from glucose.

The strain transformed with the malolactic gene (Sp/D42), in the absence of malate, produces very low quantities of L-lactate, of the order of 170 to 300 mg/l. This slight production very probably results from the conversion, by the malolactic enzyme, of a portion of the yeast endogenous malate, metabolised from glucose. This was also observed with the S. cerevisiae transformants (Table II). In the presence of 5.5 g/l of L-malate in the culture medium, a very high production of L-lactate is observed for the transformant Sp/D42, from 2.4 to 3 g/l according to the pH of the culture medium. The highest production is observed at acidic pH, therefore under pH condition [sic] similar to those in oenology- The production obtained at pH 6 is also very high, compared to that obtained for the S. cerevisiae transformants at the same pH.

On the other hand, in the presence of 5.5 g/l of L-malate and at acidic pH, the transformant Sp/D42 is capable of degrading practically all the L-malate of the medium (at pH 3.5, only 150 mg/l of L-malate were not degraded) and furthermore, of converting more than 80% of the malate present to L-lactate (3.05 g/l).