EP1346047A2

EP1346047A2 - Yeasts transformed by genes enhancing their cold stress tolerance

Info

Publication number: EP1346047A2
Application number: EP01995796A
Authority: EP
Inventors: Emmanuelle Bourgeois; Marie-Françoise Gautier; Philippe Joudrier
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de la Recherche Agronomique INRA
Priority date: 2000-12-27
Filing date: 2001-12-26
Publication date: 2003-09-24
Also published as: AU2002226511A1; FR2818660A1; FR2818660B1; WO2002051978A2; WO2002051978A3; US20040132191A1

Abstract

The invention concerns yeasts whereof the cold stress tolerance is enhanced by transformation with at least one gene coding for a protein selected among a group 2 LEA protein, a WALI protein, or a LTP.

Description

YEAS TRANSFORMED BY GENES INCREASING TOLERANCE TO COLD STRESS

The invention relates to obtaining yeasts that are resistant to stress, and in particular to cold stress. We use more and more frequently, in bakery or pastry, dough pieces which are previously sown with live yeasts and generally undergo a beginning of fermentation. They are then frozen and stored in this form until needed. However, cold stress, caused by freezing and storage at negative temperatures, causes a significant loss of the fermentative power of the yeast in these dough pieces. In the case of doughs containing live yeasts, it is important to have yeasts capable of adapting to cold stress, caused by freezing and storage at negative temperatures.

All living cells naturally have systems for adapting to different stresses, which aim to ensure the protection of the cell, its survival under stress, and the repair of damage caused by stress. These systems depend on a set of complex mechanisms, which sometimes act separately, sometimes jointly, to allow adaptation to one or more types of stress. Yeasts thus have a set of complex mechanisms (for review cf. ATTFIELD, Nature Biotech, 15, 1351-1357, 1997) which sometimes act separately, sometimes jointly, to allow adaptation to different types of stress. Cold stress involves several components: an osmotic component and an ionic component, linked to the dehydration which occurs during freezing, and a component linked to the lowering of the temperature, which slows down the whole metabolism and in particular the transmembrane exchanges , and by the formation of ice crystals, can damage cellular structures.

The factors involved in cold stress tolerance in yeast are still poorly understood. The most of the research carried out to increase the tolerance of yeasts to stress, and in particular to cold stress, has aimed at increasing their trehalose content. It has thus been proposed to increase the synthesis of trehalose by overexpressing in yeast strains genes coding for trehalose synthases (LONDESBOROUGH et al., Patent

US 5,422,254), or else limit its catabolism by inactivating the ATH1 gene which codes for a trehalase (KLIONSKY et al., Patent US 5,587,290; KIM et al., Appl. Environ. Microbiol., 62, 1563-1569 , 1996).

Many genes potentially involved in stress resistance have been identified in higher plants, and it has been proposed to use them to transform yeasts in order to increase their tolerance to stress.

Among these genes, mention will be made in particular of the genes coding for proteins of the LEA family (Late Embryogenesis Abundant) which are strongly expressed in plants during the maturation of the embryo. These genes are said to play a role in the tolerance of the embryo to desiccation.

Different groups of LEAs have been distinguished, according to their structure, and according to their potential function (BRAY, Plant Physiology, 103, 1035-1040, 1993): - the LEAs of group 1 could by their strong capacity for binding water, prevent it from leaking during drying; group 2 LEAs, also known as dehydrins, play a role as chaperone proteins, contributing to the maintenance of the structural integrity of other proteins;

- the LEA of group 3 and group 5 would ensure the sequestration of the ions; LEA group 4 would have an osmoprotective role, and would contribute to maintaining the integrity of the membranes.

IMAI et al. (Gene, 170, 243-248, 1996) report that the expression of the LE25 protein (group 4 LEA) of tomato improves tolerance of S. cerevisiae to ionic stress and cold stress (freezing). The same team ZHANG et al. (J. Biochem., 127, 611-616, 2000), also report that expression in S. cerevisiae protein LE4 from tomato (group 2) and HVAl from barley (group 3) also confers protection against cold stress, but to a lesser degree than LE25.

SWIRE-CLARK et al. (Plant Mol. Biol., 39, 117-128, 1999) observed that the expression of the wheat protein Em (LEA of group 1) improves the resistance of S. cerevisiae to osmotic stress, but has no effect on resistance to cold stress.

The inventors have isolated and cloned different cDNAs corresponding to genes induced in durum wheat (Tri ticum durum) under conditions of abiotic stress (ionic, osmotic, cold or thermal stress).

They studied the expression of these genes in S. cerevisiae and found that some of them could improve the tolerance of S. cerevisiae to different abiotic stresses. Among these, some brought in particular a very significant improvement in tolerance to cold stress.

Thus, among the cDNAs tested, they selected: - a cDNA, called pTd38, coding for a LEA protein of group 2, described previously as being induced in durum wheat by desiccation (LABHILLI et al., Plant Sci., 112 , 219-230, 1995); the nucleotide sequence of the pTd38 cDNA is represented in the annexed sequence list under the number SEQ ID NO: 1, the corresponding peptide sequence is represented under the number SEQ ID NO: 2.

- a cDNA called pTd64 coding for a WALI protein (for: “Wheat Aluminum Induced”), very close to the WALI7 protein of common wheat (Tri ticum aestivum), described by RICHARDS et al. , (Plant Physiol., 105, 1455-1456, 1994; GenBank); expression of the wali genes was initially observed in the roots of common wheat in response to stress from aluminum. Their potential role in responding to other types of stress are not known. The nucleotide sequence of the durum wheat pTd64 cDNA is represented in the annexed sequence list under the number SEQ ID NO: 3, the corresponding peptide sequence is represented under the number SEQ ID NO: 4.

- a cDNA called pTd6.48, coding for a lipid transfer protein of the LTPs family (for: “lipid transfer protein”) of 9 Da. 9 kDa LTPs have the property of transferring charged lipids between two membranes in vitro. Their role in vivo is still poorly understood. It was observed that their expression was induced under stress conditions (abiotic or biotic); they inhibit the growth of different pathogenic microorganisms, and could thus play a role in the response to biotic stress (microbial attack). They could also intervene in the formation of the waxy cuticle of plants. The nucleotide sequence of the durum wheat pTd6.48 cDNA is represented in the sequence list in the appendix under the number SEQ ID NO: 5; the peptide sequences of the protein precursor, encoded by this cDNA, and of mature LTP6.48 are represented under the numbers SEQ ID NO: 6, and SEQ ID NO: 7.

The subject of the present invention is the use of at least one nucleic acid molecule chosen from: a) a nucleic acid molecule coding for a group 2 LEA protein whose polypeptide sequence has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with the sequence SEQ ID NO: 2; b) a nucleic acid molecule coding for a WALI protein whose polypeptide sequence has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90 % identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with the sequence SEQ ID NO: 4; c) a nucleic acid molecule coding for an LTP precursor or for a mature LTP, the polypeptide sequence of which has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with, respectively, the sequence SEQ ID NO: 6 or the sequence SEQ ID NO: 7; to transform a yeast to improve its tolerance to cold stress.

The percentages of identity or similarity between two polypeptide sequences can be determined according to methods known in themselves to those skilled in the art; in particular, they can be determined using the BLASTP program (ALTSCHUL et al., Nucleic Acids Res., 25, 3389-3402, 1997).

These nucleic acid molecules can be used separately. They can also be combined in the same yeast cell, to further increase its tolerance to cold stress, by combining different mechanisms participating in this tolerance; they can also be associated with nucleic acid molecules coding for proteins involved in mechanisms of tolerance to stresses other than cold stress. For example, to improve the tolerance of a yeast cell to ionic stress, it is possible to express in this cell a nucleic acid molecule coding for a LEA of group 2 whose polypeptide sequence has at least 70% identity or 75 % similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with the sequence SEQ TD NO: 9. The Inventors have in fact observed that the LEA of group 2, called Td27e, which is represented by the sequence SEQ ID NO: 9, has no effect on the tolerance yeasts with cold stress, but increases their tolerance to ionic stress. Likewise, to improve the tolerance of a yeast cell to osmotic stress, it is possible to express in said cell a nucleic acid molecule chosen from:

a nucleic acid molecule coding for a WALI protein whose polypeptide sequence has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with the sequence SEQ ID NO: 11;

a nucleic acid molecule encoding an LTP precursor or a mature LTP whose polypeptide sequence has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and very preferably at least 95% identity or 98% similarity, with, respectively, the sequence SEQ ID NO: 13 or the sequence SEQ ID NO: 14.

The inventors have indeed found that the WALI1 protein of durum wheat, which is represented by the sequence SEQ ID NO: 11, as well as the LTP called TdD2 which is represented by the sequence SEQ ID NO: 14, or its precursor, represented by the sequence SEQ ID NO: 13 have no effect on the tolerance of yeasts to cold stress, but increase their tolerance to osmotic stress.

The present invention also relates to a yeast transformed with at least one nucleic acid molecule as defined above.

Transformed yeasts in accordance with the invention can be obtained from yeast strains of different species. By way of nonlimiting examples, mention will be made of species of the genera Saccharomyces, 'Schi zosaccharomyces, Kl uyveromyces, Torulaspora, Pichia, Hansenula, Yarrowia, Candida, Geotrichum, etc. Advantageously, said yeast belongs to a species which can be used in baking or in pastry making for the production of leavened dough. This is usually a species of the genus Saccharomyces, such as Saccharomyces cerevisiae.

Said yeast can thus be transformed with a nucleic acid molecule a) and / or a nucleic acid molecule b) and / or a nucleic acid molecule c) as defined above, optionally combined, as indicated below. above, to one or more other nucleic acid molecules allowing it to confer resistance to stresses other than cold stress. Transformed yeasts in accordance with the invention can be obtained by the usual methods, known in themselves to those skilled in the art. Conventionally, the DNA sequence coding for the protein which it is desired to express is inserted into an appropriate expression vector, under transcriptional control of an active promoter in a yeast cell. The vector thus loaded is then introduced into the yeast cell, by any suitable method, for example by electroporation or transformation with lithium acetate. Extrachromosomal replicating vectors can be used; by way of nonlimiting examples, mention will be made of the vectors of the Yep series (MYERS et al., Gene, 45, 299-310, 1986) which have the origin of replication of the endogenous 2 μ plasmid, the Yrp vectors which have as origin of replication a chromosomal ARS sequence, etc. One can also use integrative vectors such as the vectors Yip, (MYERS et al., 1986), or Yiplac (GIETZ and SUGINO, Gene 74, 527-534, 1988), which do not have an origin of functional replication in the yeast. By way of example, mention will be made of expression systems such as those described by BITTER et al. (Methods Enzymol., 153, 516-544, 1989) or TUITE (Expression of heterologous genes, 169-212, In MF TUITE, and SG OLIVER (ed.), Biotechnology Handbooks, Vol. 4, Saccharomyces., Plenum Press, New York, 1991).

The chosen gene can for example be placed under the control of elements for regulating transcription in yeast such as those of the alcohol dehydrogenase ADH1 genes. (RUOHONEN et al., J. Biotechnol., 39, 193-203, 1995), phosphoglycerate kinase PGK1 (HITZEMAN et al., Nucl. Acids Res., 10, 7791-7808, 1982), glyceraldehyde-3-phosphate dehydrogenase TDH1 (BITTER and EGAN, Gene, 32, 263-274, 1984), actin ACT1 (GALLWITZ et al., Nucl. Acids Res., 8, 1043-1059, 1980), etc.

The transformed yeasts according to the invention have better tolerance to cold stress, which results in a survival rate after freezing and storage at -20 ° C., higher than that of the wild strain from which they come. Advantageously, their survival rate after freezing and storage at -20 ° C. is at least double that of the wild strain from which they come.

The transformed yeasts expressing a group 2 LEA as defined in a) above also exhibit better tolerance to ionic stress as well as to thermal stress, than the wild strain from which they originate; transformed yeasts expressing a WALI7 protein as defined in b) above also exhibit better tolerance to thermal stress than the wild strain from which they originate; transformed yeasts expressing an LTP of 9 kDa as defined in c) above also exhibit better tolerance to ionic stress, as well as to thermal stress, than the wild strain from which they originate.

Processed yeasts in accordance with the invention can be used in particular for the manufacture of bread leavening agents. The present invention also relates to baking leavens comprising said yeasts. The present invention also includes bakery or pastry products, and in particular baking or pastry doughs comprising said yeasts.

The present invention further relates to a lipid transfer protein chosen from: - an LTP precursor or a mature LTP whose polypeptide sequence has at least 90% identity or 95% similarity, and preferably at least 95% identity or 98% similarity, with, respectively, the sequence SEQ ID NO: 6 or the sequence SEQ ID NO: 7

- an LTP precursor or a mature LTP whose polypeptide sequence has at least 70% identity or 75% similarity, preferably at least 80% identity or 85% similarity, advantageously at least 90% identity or 95% similarity, and most preferably at least 95% identity or 98% similarity, with, respectively, the sequence SEQ ID NO: 13 or the sequence SEQ ID NO: 14.

The present invention also encompasses any nucleic acid sequence encoding one of said lipid transfer proteins.

A lipid transfer protein according to the invention can, as indicated above, be expressed in yeasts in order to increase their tolerance to cold shock. The present invention will be better understood with the aid of the additional description which follows, which refers to examples describing the production of transformed yeast strains in accordance with the invention.

EXAMPLE 1: CONSTRUCTION OF EXPRESSION VECTORS AND SELECTION OF RECOMBINANT YEASTS.

The cDNAs coding for group 2 LEA Td38, the durum wheat protein WALI7, and the LTP Td6.48 were isolated from a cDNA library of roots of durum wheat seedlings, obtained after induction by desiccation. The sequences of these cDNAs are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. Vectors allowing the expression of these genes in S. cerevisiae were constructed from the E. coli / S shuttle expression vector. cerevisiae pYES2 (InVitrogen) having the ura3 gene, and the origin of replication 2μ, by insertion under transcriptional control of the gal l promoter inducible by galactose and repressed by glucose, and the cyclic transcription termination sequence. Construction of a Group 2 LEA expression vector

The complete sequence of the cDNA pTd38 (SEQ ID NO: 1) encoding the protein Td38 (SEQ ID NO: 2) was inserted into the vector pYES2, previously digested with the enzymes No tl and KpnI and dephosphorylated.

The complete sequence of the cDNA called pTd27e (SEQ ID NO: 8) coding for another LEA protein of group 2, called Td27e (SEQ ID NO: 9) whose expression in the roots of seedlings of durum wheat is also induced by desiccation, and whose peptide sequence has 58.1% and 66.7% similarity to the sequence of the Td38 protein, was also cloned between the NotI and KpnI sites of pYES2. Construction of a WALI protein expression vector

The complete coding sequence of the pTdt64 cDNA (SEQ ID NO: 3) coding for a protein similar to the WALI7 protein of common wheat was inserted into the vector pYES2, previously digested with the enzymes Sac1 and SphI and dephosphorylated.

The complete coding sequence of the cDNA called pTd79b (SEQ ID NO: 10), coding for a WALI1 protein, identical to the WALI1 protein of common wheat (SNOWDEN and GARDNER, Plant Physiol., 103, 855-861, 1993), and whose expression in the roots of durum wheat seedlings is also induced by desiccation, has been cloned in pYES2 between the NotI and KpnI sites.

Construction of an expression vector for a 9kDa LTP

The complete coding sequence of the cDNA pTd6.48 (SEQ ID NO: 5) coding for the precursor of LTP6.48 (SEQ ID NO: 6) was inserted into the vector pYES2, previously digested with the enzymes £ coRI and SphI and dephosphorylated.

For comparison, a cDNA called pTdD2 (SEQ ID NO: 12), coding for the precursor of LTPD2 (SEQ ID NO: 13), which has 50% identity and 69% similarity with the precursor of LTP6 .48, and whose expression in the roots of seedlings of durum wheat is also induced by desiccation, was cloned into pYES2 between the Λfotl and Kpnï sites.

The vectors thus obtained were used to transform the strain of Saccharomyces cerevisiae INVScl (haploid strain of genotype MATα, his3-Δl, leu2, trpl-289, ura3-52). This strain is cultivated on YNB-Gal medium composed of 1.7 g of YNB-AA / AS (DIFCO LABORATORIES), 5 g of (NH ₄ ) ₂ SO ₄ , 20 g of galactose, 20 mg of uracil, 20 mg L-histidine, 60 mg L-leucine, 40 mg L-tryptophan per 1 liter. For the selection of recombinant yeasts, the same medium is used without uracil. After selection of the yeasts, the presence of the transcripts corresponding to the cDNAs is verified by hybridization with specific probes after induction by galactose. EXAMPLE 2: STRESS TOLERANCE OF RECOMBINANT YEASTS.

Tolerance to cold stress has been studied in the wild strain INVScl, in the strain INVScl transformed with the empty plasmid pYES, and in the recombinant strains expressing a type 2 LEA, a WALI protein, or a 9kDa LTP obtained as described in Example 1 above.

Cold stress:

Stress conditions

Tolerance to cold stress is evaluated by measuring the survival of yeasts after application of cold stress (freezing at -20 ° C, storage at this temperature for 24 hours, and thawing). The conditions described by IMAI et al. (1996, publication cited above) were used.

Each of the strains is cultured in YNB-Galactose medium (50 ml). In the middle of the exponential growth phase (Absorbance at 600 nm = 1), 1 ml of culture is taken. After centrifugation. (3500 g, 3 min, 4 ° C.) the cell pellet is resuspended in 1 ml of complete YPD medium. 100 μl aliquots are separated in microtubes. Part of the aliquots is stored without treatment, and the other part is frozen in an absolute ethanol bath previously cooled and stored at -20 ° C. for 24h. The cells are thawed quickly by placing the tubes at 30 ° C a few seconds before spreading.

The untreated cells or the thawed cells are then diluted in complete YPD medium (dilution 1/10000 and 1/20000 for the controls, and 1/400 and 1/2000 for the cells having undergone cold stress). The dilutions thus obtained are spread on complete agar medium (YPD agar) and the dishes incubated at 30 ° C for 48 h. The colonies formed on each dish are counted, and the number of CFUs (colony forming units) is calculated taking into account the dilution.

The survival of each strain is evaluated by the percentage of surviving CFUs, that is to say the number of CFUs after stress divided by the number of control CFUs. For each experiment, the number of PDUs corresponds to the average of 4 spreads.

The results are summarized in Table I below:

Table I

In the case of the wild strain INVScl, the survival rate is 5.5%; the INVScl-pYES2 recombinant strain has a similar survival rate of 6.8%.

Recombinant yeasts expressing a group 2 LEA protein

The recombinant yeast INVScl-pYES2-r 27e has a cold stress survival rate of 6%, which is therefore similar to those observed with the wild strain INVScl or the strain INVScl-pYES2.

In contrast, the recombinant yeast INVScl-pYES2-Td38 has a cold stress survival rate of 20%. Recombinant yeasts expressing a WALI protein

The recombinant yeast INVScl-pYES2-Td79 _J has a cold stress survival rate of 3.5%, comparable to that of the wild strain INVScl or of the strain INVScl-pYES2.

In contrast, the recombinant yeast INVScl-pYES2- Tdt 64 has a cold stress survival rate of 27%.

Recombinant yeasts expressing LTP

The recombinant yeast INVScl-pYES2-1 tpD2 has a cold stress survival rate of 1.8%. The expression of this LTP therefore does not induce any improvement in survival from cold stress.

On the other hand, the recombinant yeast INVScl-pYES2-ltp6.48 has a cold stress survival rate of 28%.

Thermal stress :

Stress conditions

Tolerance to thermal stress is evaluated according to the same protocol as for cold stress: instead of freezing stress, the cells are incubated for 10 minutes at 50 ° C. The cells having undergone thermal stress are diluted to 1/4000 and to 1/10000 before spreading. The survival rate is calculated as described above for cold stress. The results are illustrated in Table II below.

Table II

In the case of the wild strain INVScl, the survival rate is 27.5%; it is 24% for the recombinant strain INVScl-pYES2. All the proteins expressed induce an increase in the survival rate. This increase is particularly significant in the case of Td38 and LTP6.48. It is also important in the case of Td27e. Ionic stress and osmotic stress:

Stress conditions

Tolerance to ionic stress or to osmotic stress is evaluated by comparing the growth kinetics at 30 ° C. under normal conditions (YNB-galactose medium), and under ionic stress conditions (YNB-galactose medium + 1.5 M Nacl), or osmotic stress (YNB-galactose medium + 2.5 M sorbitol).

In the case of the wild strain INVScl, or of the recombinant strain INVScl-pYES2, the duration of the latency phase (defined here as the time between the cultivation and the time when the absorbance at 600 nm reaches a value of 0.2), is 3 h under normal culture conditions. In the presence of 1.5 M NaCl, the duration of the lag phase is 87 h. In the presence of 2.5 M of sorbitol, the lag phase is 182 h.

Recombinant yeasts expressing a group 2 LEA protein

• INVScl-pYES2-Td27e

In the presence of 1.5 M NaCl, the latency phase of the growth kinetics of the yeast INVScl-pYES2-Td27e is 70 h less than that of the control yeasts (wild strain INVScl, or recombinant strain INVScl-pYES2) in same conditions.

On the other hand, in the presence of 2.5 M sorbitol, the latency phase of the transformed yeast is 48 h longer than that of the control yeasts.

• INVScl-pYES2-Td38

In the presence of 1.5 M NaCl, the latency phase of the growth kinetics of the yeast INVScl-pYES2-TdJS is 44 h less than that of the control yeasts. In the presence of 2.5 M sorbitol, no reduction in the lag phase is observed compared with the control yeasts.

It therefore appears that the yeast INVScl-pYES2-Td27e, and to a lesser extent the yeast INVScl-pYES2-Td38, have an increased tolerance to ionic stress.

On the other hand, none of these transformed yeasts shows improvement in tolerance to osmotic stress. The yeast INVScl-pYES2-Td27e appears even more sensitive than the control strains.

Recombinant yeasts expressing a WALI protein

• INVScl-pYES2-Tdt64

Under osmotic stress conditions, the growth kinetics of the yeast INVScl-pYES2-Tdt 4 are not modified. Only a slight decrease (10 h) in the latency phase is observed in the presence of 1.5 M NaCl.

• INVScl-pYES2-rd79b

Compared to the control yeasts, the latency phase of the transformed yeast INVScl-pYES2-Td79 _J is reduced by 10 h in YNB-Galactose medium supplemented with

1.5 M NaCl and 107 h in YNB-Galactose medium supplemented with 2.5 M sorbitol

It therefore appears that the expression of the protein

Td79b (WALI1), confers increased tolerance to osmotic stress, and a small increase in tolerance to ionic stress. The expression of the protein (WALI7) only confers a slight increase in tolerance to ionic stress.

Recombinant yeasts expressing LTP • INVScl-pYES2-ltpD2

Compared to that of control yeasts, the latency phase of the yeast INVScl-pYES2-2tpD2 is shortened for 50 h in YNB-Galactose medium supplemented with 1.5 M NaCl, and 25 h in the presence of 2.5 sorbitol Mr. • INVScl-pYES2-ltp6.48

The latency phase of the yeast INVScl-pYES2- l tp6. 48 is shortened by 50 h, compared to that of the control yeasts, in the presence of 1.5 M NaCl. It does not differ from those of the control yeasts in the presence of 2.5 M sorbitol.

It therefore appears that tolerance to ionic stress is improved by the expression of the LTPD2 protein or the LTP6.48 protein. On the other hand, only the expression of LTPD2 confers increased tolerance to osmotic stress. EXAMPLE 3 COLD STRESS TOLERANCE OF RECOMBINANT YEASTS OBTAINED FROM AN INDUSTRIAL STRAIN

Host yeast strain:

Industrial strain used in bread-making = Saccharomyces cerevisiae CLIB 320 (cellobiose-, D-galactose +, D-glucose +, lactose-, maltose +, melezitose +, melibiose-, raffinose +, sucrose +, trehalose-).

CLIB = collection of strains of biotechnological interest from Grignon.

Expression vector: PVT100-U-ZEO PVT100-U-ZEO is derived from the vector pVTU (VERNET et al., Gene, 52, 2325-2333, 1987). The characteristics of this vector are as follows:

- shuttle vector with origin of replication 2 μ for yeast and ori for E. coli; - ampicillin resistance (AmpR) for selection in E. coli and phleomycin resistance (ZEO) for selection in yeast;

- Adh promoter and Adh terminator.

Construction of expression vectors and selection of recombinant yeasts:

The vector PVT100-U-ZEO is digested by the enzymes Xbal and Xhol and dephosphorylated. The same Xbal and Xhol sites are created, by PCR, at the ends of the sequences encoding the LEA of group 2 Td38 (SEQ ID NO: 2), the protein WALI7 of durum wheat Tdt64 (SEQ ID NO: 4), the LTP Td6. 48 (SEQ ID NO: 6), and LTP Td6.48 without the signal peptide (SEQ ID NO: 7), in order to allow the oriented cloning of these cDNAs. The constructions obtained were verified by sequencing.

Each of the recombinant vectors as well as the empty vector are used for the transformation of the CLIB320 strain. The lithium acetate transformation method described by GRISHIN and KORSHUNOVA (Yeast Genetics and

Molecular Biology, The Hague, 1990) was used.

The yeast culture is done on YEPD medium (10 g of yeast extract, 20 g of peptone (DIFCO laboratories), 20 g of D (+) glucose for 1 1). An agar medium is obtained by adding 20 g of bactero-agar to the preceding composition. The selection of transformants is based on resistance to phleomycin. For this, 100 μg / ml of phleomycin (CAYLA) are added to the YEPD medium. Stress tolerance of recombinant yeasts:

Stress conditions:

The tolerance to cold stress of each strain of yeast is evaluated by the survival rate after application of the stress. Two types of stress were applied: rapid freezing at -20 ° C and storage at -20 ° C for variable periods of time, then thawing at room temperature or thawing by passing at 4 ° C for 24 h.

Experiments ^' :

A preculture of each recombinant strain as well as the wild strain is carried out by seeding, with an isolated colony, of 50 ml of YEPD medium + 100 μg / ml of phleomycin and culture at 28 ° C., 220 rpm, 48 h. 100 ml of YEPD medium + 100 μg / ml of phleomycin are seeded with

1/20 of the volume of the preculture and cultivated under the same conditions until reaching an absorbance at 660 nm of 4, plateau phase of the growth kinetics. Spreads after dilution are carried out at time 0 on medium

YEPD supplemented with 100 μg / ml of phleomycin, then incubated in an oven at 30 ° C for 48 h. Aliquots of 500 μl of culture are frozen at -20 ° C. by placing them in a tank of cold absolute ethanol. Every 24 h, an aliquot of each sample is thawed at temperature room temperature and another is placed at 4 ° C for 24 h for gentle thawing. Spreads at serial dilutions are made and then incubated in an oven at 30 ° C for 48 h. For each duration of freezing, the number of colony forming units (cfu) relative to the number of cfu developing at time 0 makes it possible to determine the survival rate (expressed in%).

For each freezing time and each stress condition, the number of cfu corresponds to the average of the results obtained for two clones and for two spreads per clone. The results expressed by the survival rate (%) are summarized in Tables III and IV below.

Claims

1) Use of at least one nucleic acid molecule chosen from: a) a nucleic acid molecule coding for an LTP precursor or a mature LTP whose polypeptide sequence has at least 70% identity or 75% of similarity with, respectively, the sequence SEQ ID NO: 6 or the sequence SEQ ID NO: 7; b) a nucleic acid molecule coding for a WALI protein whose polypeptide sequence has at least 70% identity or 75% similarity to the sequence SEQ ID NO: 4; c) a nucleic acid molecule coding for a group 2 LEA protein whose polypeptide sequence has at least 70% identity or 75% similarity to the sequence SEQ ID NO: 2; to transform a yeast to improve its tolerance to cold stress.

2) Yeast transformed with at least one nucleic acid molecule as defined in claim 1.

3) transformed yeast according to claim 2, characterized in that it is further transformed by at least one molecule of nucleic acid chosen from:

- a nucleic acid molecule coding for a group 2 LEA whose polypeptide sequence has at least 70% identity or 75% similarity to the sequence SEQ ID NO: 9;

- A nucleic acid molecule coding for a WALI protein whose polypeptide sequence has at least 70% identity or 75% similarity to the sequence. SEQ ID NO: 11;

- a nucleic acid molecule coding for an LTP precursor or a mature LTP whose polypeptide sequence has at least 70% identity or 75% similarity with, respectively, the sequence SEQ ID NO: 13 or the sequence SEQ ID NO: 14. 4) transformed yeast according to any one of claims 2 or 3, characterized in that said yeast belongs to the genus Sa ccha romyces.

5) transformed yeast according to claim 4, characterized in that said yeast belongs to the species

Saccharomyces cerevisiae.

6) Baking leaven, comprising a yeast transformed according to any one of claims 2 to 5.

7) Bakery or pastry product comprising transformed yeasts according to any one of claims 2 to 5.

LIST OF SEQUENCES

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BOURGEOIS, Emmanuelle GAUTIER, Marie-Françoise JOUDRIER, Philippe

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<212> DNA

<213> Triticum durum

<400> 1 acacaaccaa gacaagtaaa cagcagcact agtagatttc ccgagtgaca agttcagcgc 60 aacatggagc accagggaca cggcaccggc gagaagaagg gcatcatgga gaacatcaag 120 gagaagctcc ccggtggcca aggtgaccac cagcagaccg ctggcaccca cgggcagcat 180 ggacacactg gaatgacagg cacggagatg catgacacca cggccaccgg cggcacccat 240 gggcagcagg ggcttaccgg aacgactggc actgggacac acggcaccgg tgagaagaag 300 agcctcatgg acaaggtgaa ggagaagctg cctggacagc actaagctcg gtctgcccac 360 ggccgccacc tttgcagaat aatactccac cgtatatgaa ttgatctgag tctagttcac 420 ctagctcact tggtcgttgg aggagcaaat gtatctctgg tttaagtttt cacggacaac 480 agtgtgttca cagttttcgt ctatttacac tccgtcatgc aaatttcctt tttgttccaa 540aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaing

<210> 2

<211> 93

<212> PRT

<213> Triticum durum

<400> 2

Met Glu His Gin Gly His Gly Thr Gly Glu Lys Lys Gly Ile Met Glu 1 5 10 15

Asn Ile Lys Glu Lys Leu Pro Gly Gly Gin Gly Asp His Gin Gin Thr 20 25 30

Ala Gly Thr His Gly Gin His Gly His Thr Gly Met Thr Gly Thr Glu 35 40 45

Met His Asp Thr Thr Ala Thr Gly Gly Thr His Gly Gin Gin Gly Leu 50 55 60

Thr Gly Thr Thr Gly Thr Gly Thr His Gly Thr Gly Glu Lys Lys Ser 65 70 75 80 Leu Met Asp Lys Val Lys Glu Lys Leu Pro Gly Gin His 85 90

<210> 3

<211> 891

<212> DNA

<213> Trit icum durum

<400> 3 agtgaggaag. gccacaatca gcaactgacc tgtaatacct tacctagcta ggtgtactat 60 gtttgagcca agatgttggg ggtgttcagc ggcgaggtgg tggaggtgcc ggcggagctg 120 gtggcggccg ggagccggac gccatcgccc aagacacggg cgtcggagct ggtgaagcgc 180 ttcctcgccg gcaacgacct ggccgtgtcc gtggagctgg gatcactggg caacctcgcc 240 tactcccacg ccaaccagtc cctcctcctc ccaaggtctt tcgctgcaaa ggatgagatc 300 ttctgcctgt tcgagggagt cctggacaac ttggggcggt tgagccagca gtacggcctc 360 tccaagggcg gcaacgaggt gctcctcgtg atcgaggcct acaagacgct gagggacaga 420 gccccctatc ccgccagctt catgctctcc cagctcaccg gcagctacgc cttcgtgctc 480 ttcgacaagt ccacctcctc cctcctcgtc gcatccgacc cggagggcaa ggtgccgctc 540 ttctggggaa tcaccgccga cggctgcgtc gccttctccg acgacatcga cctgctgaaa 600 ggatcttgcg gcaagtcact ggcgcctttc ccgcaaggtt gcttctactg gaacgctctt 660 ggaggcctca agtcgtacga gaatcccaag aacaaggtca ccgctgtgcc tgcagatgag 720 gaggaaatct gtggtgcaac tttcatggtg gaaggatcta ccgttgtcgc ggcacttcag 780 taggagattc tcttgtctcc gtcgctgggc aaagcaggca aggccgttcg tgtgtagatg 840 gtggtggtgt aatataatgc aacaaggcgc gtgtgctact ctcttgtgat c 891

<210> 4

<211> 236

<212> PRT

<213> Triticum durum

<400> 4

Met Leu Gly Val Phe Ser Gly Glu Val Val Glu Val Pro Ala Glu Leu 1 5 10 15

Val Ala Ala Gly Ser Arg Thr Pro Ser Pro Lys Thr Arg Ala Ser Glu 20 25 30

Leu Val Lys Arg Phe Leu Ala Gly Asn Asp Leu Ala Val Ser Val Glu 35 40 45

Leu Gly Ser Leu Gly Asn Leu Ala Tyr Ser His Ala Asn Gin Ser Leu 50 55 60

Leu Leu Pro Arg Ser Phe Ala Ala Lys Asp Glu Ile Phe Cys Leu Phe 65 70 75 80

Glu Gly Val Leu Asp Asn Leu Gly Arg Leu Ser Gin Gin Tyr Gly Leu 85 90 95

Ser Lys Gly Gly Asn Glu Val Leu Leu Val Ile Glu Ala Tyr Lys Thr 100 105 110

Leu Arg Asp Arg Ala Pro Tyr Pro Ala Ser Phe Met Leu Ser Gin Leu 115 120 125

Thr Gly Ser Tyr Ala Phe Val Leu Phe Asp Lys Ser Thr Ser Ser Leu 130 135 140 Leu Val Ala Ser Asp Pro Glu Gly Lys Val Pro Leu Phe Trp Gly Ile 145 150 155 160

Thr Ala Asp Gly Cys Val Ala Phe Ser Asp Asp Ile Asp Leu Leu Lys 165 170 175

Gly Ser Cys Gly Lys Ser Leu Ala Pro Phe Pro Gin Gly Cys Phe Tyr 180 185 190

Trp Asn Ala Leu Gly Gly Leu Lys Ser Tyr Glu Asn Pro Lys Asn Lys 195 200 205

Val Thr Ala Val Pro Ala Asp Glu Glu Glu Ile Cys Gly Ala Thr Phe 210 215 220

Met Val Glu Gly Ser Thr Val Val Ala Ala Leu Gin 225 230 235

<210> 5

<211> 782

<212> DNA

<213> Triticum durum

<400> 5 aatacgactc actataggga aagctggtac gcctgcaggt accggtccgg aattcccggg 60 tcgacccacg cgtccggaaa atctagctat ctcatcatct ctgcctgagc tcactaccac 120 tactattgct agcttgatcg agatggcccg ttctgctgtt gctcaggtcg tgctcgtcgc 180 cgtggtggct gctatgctcc tcgcagtcac ggaggcggct gtatcgtgcg gtcaggtgag 240 ctctgccttg agcccctgca tctcctatgc acgcggcaac ggcgccagcc catctgcggc 300 ctgctgcagc ggcgttagga gtctagccag ctcagcccgg agcaccgctg acaagcaagc 360 ggcgtgcaag tgcatcaaga gcgctgctgc tgggctcaac gctggcaagg ccgccggcat 420 ccccacaaag tgcggcgtta gcgtccctta cgccatcagc tcttcggtcg actgctctaa 480 gattcgctga tcgagcactt gctgccatcg ctgttgccat cgtcccctac gccatcgttg 540 ctggatctac gcttagtacg ttgaggtcac acacacgcac acccacatat atatatgaat 600 aaatgctctc atattatctc actgcgtgag agagagagga gtacgtacgt ccaagcagct 660 ctgcatggcc ggccacactg ttgtatcgat gtttggttgt tcttccactc cccgagtttg 720 ctgtactttg taccatgtgt acttttgata tatggattgt gtactcagct gatcagctct 780 aa 782

<210> 6

<211> 116

<212> PRT

<213> Triticum durum

<400> 6

Met Ala Arg Ser Ala Val Ala Gin Val Val Leu Val Ala Val Val Ala 1 5 10 15

Ala Met Leu Leu Ala Val Thr Glu Ala Ala Ala Val Ser Cys Gly Gin 20 25 30

Val Ser Ser Ala Leu Ser Pro Cys Ile Ser Tyr Ala Arg Gly Asn Gly 35 40 45

Ala Ser Pro Ser Ala Ala Cys Cys Ser Gly Val Arg Ser Leu Ala Ser 50 55 60 Ser Ala Arg Ser Thr Ala Asp Lys Gin Ala Ala Cys Lys Cys Ile Lys

65 70 75 80

Ser Ala Ala Ala Gly Leu Asn Ala Gly Lys Ala Ala Gly Ile Pro Thr

85 90 95

Lys Cys Gly Val Ser Val Pro Tyr Ala Ile Ser Ser Ser Val Asp Cys

100 105 110

Ser Lys Ile Arg

115

<210> 7

<211> 90

<212> PRT

<213> Triticum durum

<400> 7

Ala Val Ser Cys Gly Gin Val Ser Ser Ala Leu Ser Pro Cys Ile Ser 1 5 10 15

Tyr Ala Arg Gly Asn Gly Ala Ser Pro Ser Ala Ala Cys Cys Ser Gly 20 25 30

Val Arg Ser Leu Ala Ser Ser Ala Arg Ser Thr Ala Asp Lys Gin Ala 35 40 45

Ala Cys Lys Cys Ile Lys Ser Ala Ala Ala Gly Leu Asn Ala Gly Lys 50 55 60

Ala Ala Gly Ile Pro Thr Lys Cys Gly Val Ser Val Pro Tyr Ala Ile 65 70 75 80

Ser Ser Ser Val Asp Cys Ser Lys Ile Arg 85 90

<210> 8

<211> 751

<212> DNA

<213> Triticum durum

<400> 8 caaagagcaa aagctaaagc cacaaccaag tccagtttag gaagaggcag agatggagtt 60 ccaagggcag cacgacaacc ccgccaaccg cgtcgacgag tacggcaacc cgttcccgct 120 ggccggcggc gtggggggag gacacgccgc tcctggcacc ggcgggcagt tacaggcccg 180 caggggagag cacaagaccg gtgggatcct gcatcgctcc ggcagctcca gctccagctc 240 gtcttccgag gacgacggca tgggcgggag gaggaagaag ggcatgaaag agaagatcaa 300 ggagaagctc cccggcggcc acaaggacaa ccagcagcac atggcgactg gtacagggac 360 tggaggagcc tacgggccgg ggactggaac tggtggagcc tacgggcagc aaggccacgc 420 aggaatggcc ggcgccggca ctggcaccgg cgagaagaag gggatcatgg acaagattaa 480 ggagaagctg ccgggacagc actgagccga cggctccggc tggccgcttc ctttgcatag 540 ctacacgcgt caatgccttc tagttccacg tgatcttttt gttcaataat aataagatga 600 agcagaacga aaacttgtct ctgatctcgt ctgtgtcagg gacacttttc tgtatacagc 660 gtgcgtcgtg tttgttatgt tttgtgtgtt gtgtcttcat gttgaaacaa atttagtgta 720 caactgaaaa aaaaaaaaaa aaaaaaaaaa a 751

<210> 9 <211> 150 <212> PRT <213> Triticum durum

<400> 9

Met Glu Phe Gin Gly Gin His Asp Asn Pro Ala Asn Arg Val Asp Glu 1 5 10 15

Tyr Gly Asn Pro Phe Pro Leu Ala Gly Gly Val Gly Gly Gly His Ala 20 25 30

Ala Pro Gly Thr Gly Gly Gin Leu Gin Ala Arg Arg Gly Glu His Lys 35 40 45

Thr Gly Gly Ile Leu His Arg Ser Gly Ser Ser Ser Ser Ser Ser Ser 50 55 60

Ser Glu Asp Asp Gly Met Gly Gly Arg Arg Lys Lys Gly Met Lys Glu 65 70 75 80

Lys Ile Lys Glu Lys Leu Pro Gly Gly His Lys Asp Asn Gin Gin His 85 90 95

Met Ala Thr Gly Thr Gly Thr Gly Gly Ala Tyr Gly Pro Gly Thr Gly 100 105 110

Thr Gly Gly Ala Tyr Gly Gin Gin Gly His Ala Gly Met Ala Gly Ala 115 120 125

Gly Thr Gly Thr Gly Glu Lys Lys Gly Ile Met Asp Lys Ile Lys Glu 130 135 140

Lys Leu Pro Gly Gin His 145 150

<210> 10

<211> 551

<212> DNA

<213> Triticum durum

<400> 10 catcatcctc gacaccaaag ctcatcttct tctccttgaa atctttttgg gttcatcaga 60 tttggaggat gtcttgcaac tgtggatccg gttgcagctg cggctcagac tgcaagtgcg 120 ggaagatgta ccctgatctg acggagcagg gcagtgccgc ggcccaggtc gccgccgtgg 180 tcgtcctcgg cgtggcgcct gagaacaagg cggggcagtt cgaggtggcc gccggccagt 240 ccggcgaggg ctgcagctgc ggcgacaact gcaagtgcaa cccctgcaac tgttaagctg 300 catgcactcg tgtgatggtg tgagagtata cgtgaataac gagcgtccct ctgatctgat 360 ggagtcgagc aagggtgcgt gtgcgtgtgc gtgtggttta cttgctcgct ctccgcctat 420 gctctgccct tggtgtcctt gtgtgtatgt gtgtgcacgt gtccctgtaa ttgcttcatc 480 tatctccact atggatggag tgatgaatat gtaagaatga atgatttacc taaaaaaaa 55a

<210> 11

<211> 75

<212> PRT

<213> Triticum durum

<400> 11 Met Ser Cys Asn Cys Gly Ser Gly Cys Ser Cys Gly Ser Asp Cys Lys

1 5 10 15

Cys Gly Lys Met Tyr Pro Asp Leu Thr Glu Gin Gly Ser Ala Ala Ala 20 25 30

Gin Val Ala Ala Val Val Val Leu Gly Val Ala Pro Glu Asn Lys Ala 35 40 45

Gly Gin Phe Glu Val Ala Ala Gly Gin Ser Gly Glu Gly Cys Ser Cys 50 55 60

Gly Asp Asn Cys Lys Cys Asn Pro Cys Asn Cys 65 70 75

<210> 12

<211> 591

<212> DNA

<213> Triticum durum

<400> 12 acatttccag caagcaagcc gaagcactag atcctcgatg gctcgcgtgg cactgctcgc 60 cgtgttcacc gtgctcgccg cactggcagt ggcggagatg gcgtctgggg cggtgacctg 120 cagcgacgtg acgtccgcca tcgcgccgtg catgtcctac gcaacggggc aagcgtcgtc 180 accctcggcg gggtgctgca gcggggtgag gaccctgaac ggcaaggcgt ccacctcggc 240 cgaccggcag gcggcgtgcc gctgcctcaa gaacctggcg gggtcgttca atggcatcag 300 catgggtaac gccgccaaca tccccggcaa gtgcggcgtc tccgtctctt tccccatcaa 360 caacagcgtc aactgcaaca accttcatta agttatctac gagcatcatc atcacaccag 420 gctagctagc ccactcggtg tctactgttg ctgctctctg cgtgtgttcg ttgttgtttt 480 ctgcatgtgt tccacctcca tctgttgtcc ttgttacaga tcgagcaatgggggggggggggg

<210> 13

<211> 117

<212> PRT

<213> Triticum durum

<400> 13

Met Ala Arg Val Ala Leu Leu Ala Val Phe Thr Val Leu Ala Ala Leu 1 5 10 15

Ala Val Ala Glu Met Ala Ser Gly Ala Val Thr Cys Ser Asp Val Thr 20 25 30

Ser Ala Ile Ala Pro Cys Met Ser Tyr Ala Thr Gly Gin Ala Ser Ser 35 40 45

Pro Ser Ala Gly Cys Cys Ser Gly Val Arg Thr Leu Asn Gly Lys Ala 50 55 60

Ser Thr Ser Ala Asp Arg Gin Ala Ala Cys Arg Cys Leu Lys Asn Leu 65 70 75 80

Ala Gly Ser Phe Asn Gly Ile Ser Met Gly Asn Ala Ala Asn Ile Pro 85 90 95

Gly Lys Cys Gly Val Ser Val Ser Phe Pro Ile Asn Asn Ser Val Asn 100 105 110 Cys Asn Asn Leu His 115

<210> 14

<211> 93

<212> PRT

<213> Triticum durum

<400> 14

Ala Val Thr Cys Ser Asp Val Thr Ser Ala Ile Ala Pro Cys Met Ser 1 5 10 15

Tyr Ala Thr Gly Gin Ala Ser Ser Pro Ser Ala Gly Cys Cys Ser Gly 20 25 30

Val Arg Thr Leu Asn Gly Lys Ala Ser Thr Ser Ala Asp Arg Gin Ala 35 40 45

Ala Cys Arg Cys Leu Lys Asn Leu Ala Gly Ser Phe Asn Gly Ile Ser 50 55 60

Met Gly Asn Ala Ala Asn Ile Pro Gly Lys Cys Gly Val Ser Val Ser 65 70 75 80

Phe Pro Ile Asn Asn Ser Val Asn Cys Asn Asn Leu His 85 90