WO1995014774A2 - Toxicity-resistant molasses gene, related products and use thereof for the analysis and enhancement of microorganisms and molasses - Google Patents

Abstract

Sequence of nucleic acids characterized by expressing, within a cell using molasses as a substrate in a culture medium, a protein or peptide capable of conferring on said cell increased resistance to molasses toxicity. The nucleic acid sequence is selected from the group consisting of a) the sequence illustrated in figure 1 or its complementary strand; b) the sequence between nucleotides 664 and 1591 of figure 1 or the complementary strand thereof, said sequence being the coding region; c) the sequence between nucleotides 664 and 2235 of the sequence of figure 1 or the complementary strand thereof; d) the sequence between nucleotides 1 and 1591 of figure 1 or its complementary strand; e) the sequence derived from one or the other of the sequences defined in (a), (b) or (c) by substitution of one or more of their nucleotides, and even addition or deletion of a number of codons, as long as ssaid sequence continues to code for a protein conferring the above-mentionned property on the host in which it is expressed.

Description

GENE OF RESISTANCE TO TOXICITY OF MELASS, RELATED PRODUCTS AND THEIR USE FOR ANALYZING AND IMPROVING MICROORGANISMS AND MELASSES

The present invention relates to nucleotide sequences and their use for analyzing and improving microorganisms and molasses.

It is known that yeasts, fungi and certain bacteria can multiply (in the case of biomass production) and / or ferment (in the case of distilleries or other production) on by-products from sugar factories. These constitute for the micro-organisms a substrate which is widely used in industry.

These secondary products of sweets can be either products derived from sugar beet or from sugar cane.

Two different substrates from sugar beet are used: beet juice; it is obtained by diffusion of beetroot in hot water (70 ° C) and contains 12 to 16% sucrose; and molasses (or sewers); it corresponds to sugar residue. The first step in making sugar begins with obtaining beet juice, which is then processed to extract the white sugar. The residue from this extraction is called molasses and still contains around 50% (W / W) of sucrose.

The main difference between beet juice and molasses, apart from their difference in sucrose concentration, is that molasses is much more concentrated, by a factor of 4 to 5, in various non-sugar substances (mineral and organic) than beet juice. Sugar extraction successful the concentration of non-sugar substances in molasses. These considerably interfere with the metabolism of microorganisms, in particular growth.

Another usable substrate is sugar cane molasses. It differs from beet molasses on several points: the metabolism of the original plant is very different from that of beet so the various non-sugar nutrients are of another nature; the sugars (around 50%) are divided into sucrose (30 to 40%) and reducing carbohydrates (15 to 20%) consisting of glucose and fructose; many mineral and organic acids are present which, unlike beet molasses, gives a pH below 7; many gums and various polyglucosides are listed: pentosan, dextran. These products constitute about 2 to 4% of molasses and come from the plant or are synthesized by various microorganisms. These molecules cannot be used by yeast; mineral matter (around 5-10%) is often more important than in beet molasses; proteinaceous materials (2 to 3%) are on the contrary in lower quantity.

A decrease in growth was observed on molasses, from which the concept of toxic molasses appeared.

In order to understand what are the elements involved in this inhibition phenomenon, various works have been carried out to carry out an inventory of the constituents of molasses.

While the amino acids, sugars and mineral compounds of molasses had already been studied in detail, various volatile compounds not yet listed have been analyzed. Thus it has been demonstrated the presence in molasses of acetic acid, propionic acid, and benzyl alcohol as well as various other acids. Many combinations containing pyrazines, pyrrols, furans and phenols have been identified, probably resulting from chemical reactions relating to heating (Maillard reactions). On the other hand, aliphatic or aromatic organic acids have also been identified (Von Tressl et al. 1976).

Additives, used in sugar refinery to improve extraction yields, can be found in molasses as inhibiting elements. Assays of these additives (emulsifiers, disinfectants, flocculating agents, descaling agents and wetting agents) have been undertaken (Bronn and Fattohi 1988 and Fiedler 1981). It turns out that each additive can contribute to the inhibition of yeasts but their concentrations in molasses are below the inhibition threshold. Thus the causes of the toxicity of molasses remain still obscure and little known.

Concerning this toxicity, the inventors have demonstrated that there is above all a primary specific element, limiting the growth of yeasts, which corresponds to the major inhibiting effect. This effect is the most important and can be dissociated from the inhibition effect by non-sugar linked to osmotic pressure. In addition, this toxicity is variable according to the lots of molasses.

By the specific term, or specific toxicity, is meant any element of molasses whose toxicity on microorganisms can be canceled or reduced by the presence in said microorganism of the nucleotide sequences of the invention, preferably in at least guatre copies. The means currently used to circumvent this phenomenon of toxicity are: the use of naturally resistant microorganism strains (which limits the number of usable microorganisms). Indeed, the inventors observed that different industrial strains exhibited different natural sensitivities with regard to toxic molasses,

- classical genetic improvement of strains, so as to widen the range of usable strains. This improvement consists in creating either by mutation or by crossing a large quantity of strains and systematically testing their performance. Because of the multiplicity of characters and genes involved, this selection is tedious and unproductive, the judicious use of molasses. This use can consist either in a mixture of molasses having very different degrees of toxicity to reduce the level of toxicity of the whole, or in the introduction of different batches of molasses at different stages of the process (for example: start fermentation yeast on a little toxic molasses and when the metabolism is very productive, finish the fermentation with a much more toxic molasses) or finally simply to dilute highly toxic molasses. But these are piecemeal solutions that must be adapted to each fermentation.

All of these techniques remain dependent on an eminently variable raw material, thereby leading to unpredictable hazards in production yields, which is detrimental to the industry in question. •

Various approaches have made it possible to improve yeast strains with respect to toxic elements limiting the growth and understanding of these phenomena. Thus it has been shown that the resistance of Saccharomyces strain at high copper concentration was directly correlated to the number of gene copies at the CUP1 locus. (elch et al 1983). On the other hand, the overexpression of the HAL1 gene makes it possible to improve the growth of yeast on a medium with a high concentration of NaCl. Indeed, the growth of the strains is very sensitive to high concentrations of NaCl which then constitutes an inhibitory element in relation to the osmotic pressure (Gaxiola et al 1992). Furthermore, concerning the inhibition by osmotic pressure, the HOG genes 1 to 4 have been identified and effectively intervene in response to the increase in osmotic pressure (Brewster et al 1993).

Beyond the inhibition due to the osmotic pressure, the element or elements, contained in molasses, inhibiting the growth of microorganisms remain an imperfectly solved problem.

The authors highlighted that the degree of inhibition of the growth of a microorganism on a molasses was in most cases correlable to the number of copies of a particular gene in said microorganism, and by way of consequence to the quantity of a protein expressed by said gene. They have implemented methods for measuring the sensitivity of microorganisms to molasses and / or the toxicity of these molasses.

The solution is provided by the isolation of a nucleotide sequence and its use.

The present invention relates to a nucleic acid sequence, characterized in that it allows, in a cell using molasses as substrate in a culture medium, the expression of a protein or a peptide capable of conferring to said cell increased resistance to the toxicity of molasses, said nucleic acid sequence being chosen from the group consisting of: a) the sequence represented in FIG. 1 or its complementary strand, b) the sequence between nucleotides 664 and 1591 of FIG. 1 or its complementary strand, said sequence being the coding region, called RTM1 gene, c) the sequence included between nucleotides 664 and 2235 of the sequence of Figure 1 or its complementary strand, d) the sequence between nucleotides 1 and 1591 of Figure 1 or its complementary strand, e) ^' the sequences derived from one or the other of the sequences defined in (a), (b), (c) or (d) by substitution of one or more of their nucleotides, or even addition or deletion of certain codons, since this sequence continues to code for a protein conferring the above property on the host in which it is expressed.

Within the meaning of the present invention, the term “derived sequence” means both a DNA sequence or a single-stranded or double-stranded RNA, as well as their complementary sequences.

The sequence of FIG. 1 notably comprises the following restriction sites: BglII, HindIII, EcoRV, ClaI and BamHI, as indicated in FIG. 2, and useful for the preparation of detection probes.

Also part of the invention are the homologous sequences comprising nucleotides variants with respect to those defined above and which include certain localized mutations insofar as these variant nucleic acids hybridize with the previously defined nucleic acids or with the fragments of these.

The subject of the invention is also the fragments of said sequence useful for the expression and detection of the gene, which are in particular probes or PCR amplification primers, and are capable of hybridizing with all or part of the sequence defined in FIG. 1.

According to an advantageous embodiment, the sequence of said probe is homologous or complementary to a segment of at least 10 bp of sequence I.

For the purposes of the present invention, "homologous sequence" includes not only sequences identical to sequence I, or to a fragment (part) thereof, but also those differing only by substitution, deletion or l adding a small number of nucleotides, provided that the sequences thus modified have specificity for hybridization to the sequence I or of the segment considered.

Likewise, the term “complementary sequence” is understood to mean not only the strictly complementary sequences of the sequence I or of its fragments, but also modified sequences, as indicated above, having a specificity of hybridization to the strictly complementary sequences.

The hybridization conditions are those which are commonly known and applied both for short probes (from 10 to 100 nucleotides) and for long probes (greater than 100 nucleotides).

The probes can be chosen more particularly from the group consisting of: a) the sequences defined in (a) to (e) above, b) the restriction fragments of the sequence shown in FIGS. 1 and 2, in particular:

the fragment between nucleotides 492 and 1538 of the sequence of FIG. 1,

the fragment between nucleotides 1537 and 2167 of the sequence of FIG. 1, c) any sequence homologous or derived from a sequence defined in (a) or (b) by substitution, deletion or addition of one or more nucleotides since this sequence specifically hybridizes with all or part of the nucleotide sequence represented in figure 1.

The fragments, probes or primers can, if necessary, be covalently associated with a labeling substance detectable directly or indirectly in particular by an optical or luminescent radioactive signal.

Detection can be carried out by conventional amplification methods today, the most widespread method being PCR (Polymerase Chain Reaction).

The pairs of DNA polymerization primers constituted by probes as defined above chosen so that they allow the amplification of a fragment included in the sequence of FIG. 1 are part of the invention.

An example of a pair of primers that can be used consists of the following two sequences: 5'ATGTCAAATGACTCTAGTGG3 'and 5'GGTTTCAAGGACTGACACTC3'.

In all that follows, the following definitions are adopted.

The term “elements allowing the expression of a sequence” means the promoter and terminator regulatory sequences, whether they are specific to the gene or whether any sequence is suitable for the expression of the minimum coding sequence in the chosen host cell.

The term “host cell” means any appropriate microorganism, in particular Sac cha -omy ces, other yeasts, fungi and bacteria in which at least one nucleotide sequence in accordance with the specified invention above, will be introduced for replication and / or expression.

By transformation is meant any process allowing the introduction into the host cell of one or more copies of the nucleotide sequence according to the invention specified above.

Such a method can be used for overexpression of the RTM1 gene.

This overexpression can be achieved either by a multicopy vector, or by simple or multiple integration at the same locus or at different loci and / or under the control of a strong and / or inducible promoter.

The present invention also relates to pressure vectors containing one or more copies of the nucleic acid sequences, in particular those described from (a) to (e), under the control of promoters suitable for expression in the micro- organism in leguel it is intended.

Preferably, and for the resistance to the toxicity of molasses to be notable, the number of copies of the sequence is equal to or greater than 4, and the vector used is a multicopy vector.

As will be shown in detail in the examples which follow, an advantageous vector for transforming yeast, and in particular Saccharomyceε cerevisi ^' ae is the multicopy vector pFL44L, such as described in Bonneaud N. et al. (1991), in which one or more copies of a sequence chosen from the group consisting of: a) the sequence represented in FIG. 1 or its complementary strand, b. The sequence between nucleotides 664 and 1591 of the FIG. 1 or its complementary strand, said sequence being the coding region, called the RTM1 gene, c) ^'the sequence between nucleotides 664 and 2235 of the Figure 1 sequence, or its complementary strand, d) the sequence between nucleotides 1 and 1591 of Figure 1 or its complementary strand, e) the sequences derived from either of the sequences defined in (a), (b), (c) or (d) by substitution of one or more of their nucleotides, or even addition or deletion of certain codons, as soon as this sequence continues to code for a protein conferring the above property on the host in which it is expressed.

The vector having integrated the sequence of 2.8 b between the SphI and Kpnl sites is represented in FIG. 3 and is called pFL44L-2.8 is present in the strain LG16-2.8 deposited at the C.N.C.M. November 23, 1993 under No. 1-1376.

The invention also relates to the recombinant cellular hosts comprising one or more sequences such as described in sections (a) to (e) above, or a vector or several vectors according to the invention, these hosts being able to exhibit increased resistance. to the toxicity of molasses used in the composition of their culture medium.

These host cells of the invention are microorganisms of industrial interest; these may include:

- yeasts, in particular baker's yeasts, or wine or distillery yeasts,

- bacteria, in particular E. coli, streptomyces spp, Bacillus spp, Corynebacterium spp or Zymomonas mobilis,

- fungi, especially Aspergillus, Penicillium or Trichoderma reseii.

According to an advantageous embodiment, the method of overexpression of the RTM1 or equivalent gene, conferring resistance to the toxicity of molasses can be adapted for example to a multicopy vector containing an origin of replication of an appropriate host micro¬ organism (in particular a eukaryotic cell or a bacterium), at least one gene whose expression allows the selection either of bacteria or of eukaryotic cells having received said vector, an appropriate regulatory sequence, in particular a promoter allowing the expression of genes in said bacteria or eukaryotic cells and in which is inserted a total or minimum nucleotide sequence as defined above, which vector is a vector of expression conferring on the host cell an improvement in growth on molasses.

According to this advantageous embodiment, a transformed host cell is obtained, named DM 4126-2.8, deposited under No. 1-1377 on November 23, 1993 at the C.N.C.M.

The invention also relates to a protein or a polypeptide characterized in that it contains the amino acid sequence shown in FIG. 6, said sequence being capable of conferring on the cell which contains it an increased resistance to the toxicity of molasses.

The term “protein or polypeptide of the invention” is understood to mean, in addition to the sequence represented in FIG. 6, any sequence modified with respect to the sequence of FIG. 6 by deletion, insertion, replacement or addition of one or more amino acids therefore the polypeptide thus modified is involved in resistance to the toxicity of molasses entering the culture medium of a microorganism of industrial interest.

This protein or polypeptide has a membrane protein structure, similar in terms of structure to adrenergic receptors and has a hydrophilic part and a hydrophobic part. The hydrophilic part is a good candidate to represent an active part of the protein in its function of increasing the resistance to the toxicity of molasses.

It is also a good candidate as an immunogen with the aim of obtaining polyclonal or monoclonal antibodies specific for the protein of the invention.

The sequence between amino acids 1 and 309, or any sequence derived or modified with the meanings given above also forms part of the invention, from the moment when this sequence is functionally associated with the resistance to the toxicity of the molasses.

Finally, monoclonal or polyclonal antibodies which specifically recognize the protein or one of its fragments are also part of the invention.

The subject of the present invention is also a method of detecting and guantifying the presence, in a culture of industrial microorganisms, of a nucleic acid sequence according to the invention, characterized by the following sequence of steps:

- the DNA of the micro-organism is made accessible for hybridization either by extraction or by cell permeabilization, the DNA is hybridized with a probe associated directly or indirectly with a boundary substance detectable directly or indirectly in particular by a radioactive signal optical or luminescent,

- where appropriate, the detection method uses the PCR technique, the DNA being amplified using a pair of primers as defined above, namely homologous or complementary nucleotide sequences to all or part of the sequence defined in FIG. 1 and in particular the following oligonucleotides: 5'ATGTCAAATGACTCTAGTGG3 'and 5'GGTTTCAAGGACTGACACTC3'

The invention also relates to a method for detecting and measuring the presence of a polypeptide, the presence of which confers increased resistance to the toxicity of molasses or industrial microorganisms, characterized in that it uses antibodies polyclonal or monoclonal against proteins or polypeptides corresponding to the sequence of Figure 6 or fragments thereof, in an immunometric test including a sandwich type method, or competition.

The implementation of detection and / or quantification methods has the advantage of allowing in particular:

- evaluation of the sensitivity of micro-organisms towards molasses,

- the search for the number of copies of the RTM1 gene in various microorganisms (Saccharomyces, fungi or other yeast like Saccharomyces and bacteria),

- the characterization of microorganisms. Indeed, there is such variability concerning this gene that it is possible to provide a specific hybridization profile for each strain of Saccharomyces cerevisiae yeast analyzed.

A kit for detecting the presence of a polypeptide, the presence of which confers increased resistance to the toxicity of molasses to industrial microorganisms is also part of the invention and is characterized in that it comprises:

- antibodies, if any, labeled,

- a reagent for the detection of an immunological reaction of the antibody-antigen type,

- where appropriate, reagents to perform lysis of the cells of the sample tested. A PCR detection kit for the presence of a gene, the presence of which confers increased resistance to the toxicity of molasses to industrial micro¬ organisms whose culture medium contains molasses is part of the invention and is characterized in that it comprises in particular: at least nucleotide primers as defined above, where appropriate labeled,

- nucleotides triphosphate dATP, dCTP, dTTP and dGTP,

- a DNA polymerization agent.

The present invention also relates to a measurement method characterized in that the transformed host cells make it possible, specifically, to measure the toxicity due to or to the inhibitory elements contained in molasses.

According to an advantageous embodiment, the toxicity of molasses is measured by the differential growth between a cell (a microorganism) of sensitive origin and the same cell transformed by several copies (preferably by a number greater than 3) of the sequence defined above, and this under the same culture conditions.

This embodiment makes it possible to measure the toxicity of molasses, according to the batches and the storage time. It measures the presence of specific toxic element (s) in any fraction of the molasses. Finally, it makes it possible to measure (quantify) the sensitivity of any strain of microorganism, by manufacturing reference batches of molasses.

The present invention also relates to a process for improving microorganisms and in particular industrial microorganisms, characterized in that the transformation of any of these microorganisms by a nucleotide sequence or a vector containing said sequence provided elements appropriate regulators, improves its growth on all solid and / or liquid media containing molasses.

This method preferably consists in transforming the microorganism of industrial interest by a multicopy vector comprising at least one copy of a sequence and its derivatives as defined in FIG. 1.

An example of a microorganism improved by the method of the invention is given in Example 5 below in which a strain of Saccharomyces cerevisiae has been transformed by the multicopy vector pFL44L-2,8-14 DM and has been identified under reference 4126 to 2.8 DM and deposited at the CNCM under ^No. 1-1377 dated 23 November 1993.

This process makes it possible to respond specifically to the growth limitation of certain microorganisms on molasses.

The advantage of the present invention compared to conventional genetics, is that this method (the overexpression of said sequence in a host cell, in particular a yeast) brings a specific and significant improvement without obligation to put all of the others into play. strain characters.

The strains obtained will have better performance on most batches of molasses; increased growth rate, improved biomass yield and reproducibility of fermentations.

Thus the field of application covers, the industries producing yeasts (in particular baker's, distillery, wine and brewer's yeasts), industries using yeasts (especially for the production of alcohol such as distilleries), industries producing all kinds of metabolites (including proteins, enzymes, amino acids, organic acids, xantha e etc.) and other fermentation industries, in fact any process using molasses as a growth substrate for microorganisms of industrial interest.

The invention also covers any kit for measuring the specific toxicity of a molasses on the growth of a microorganism, in particular a yeast of industrial interest, containing:

- an ampoule of a control microorganism,

an ampoule of the same transformed microorganism and containing at least 4 copies of the gene conferring resistance to the toxicity of molasses to said microorganism, these two ampoules being in a form allowing their conservation, in particular lyophilization,

- if necessary, molasses making it possible to prepare a reference medium.

The invention makes it possible to screen molasses which must enter into the composition of culture media for industrial microorganisms, by a process characterized in that the minimum inhibitory concentration of said molasses is measured on a cell line comprising at least four nucleotide sequences according to the invention compared to the MIC obtained with a cell line not comprising them.

The invention also makes it possible to identify the constituent (s) of molasses responsible for toxicity on the growth of industrial microorganisms, characterized by the implementation of the following steps:

- fractionation of molasses by gentle fractionation techniques,

- separation of the constituents of the fraction (s) for which the toxic effect is preserved, measurement of the toxic effect of each of these constituents, the toxicity measurements at each of the stages being carried out by measuring the MIC of a molasses on a reference line and on a line having incorporated at least 4 copies of one of the sequences of the invention, the index of a specific toxicity being brought by the differential effect of the constituent on the reference strain and the transformed strain.

An appropriate reference strain for this can be, for example, the strain LG16 and the transformed strain LG16-pFL44L-2.8.

The present invention finally relates to the use as a selection marker of the nucleotide sequence in accordance with FIG. 1, or its derivatives, fragments or complementary sequences in particular for yeasts, during transformation experiment.

It constitutes a positive selection system as a dominant marker, allowing the selection of microorganisms (in particular yeasts) on molasses.

Its advantageous use thus allows on the one hand the selection on molasses of the strain transformed by this sequence and on the other hand the improvement of the growth on molasses of said strain.

In addition to the foregoing arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refer to examples of implementation of the process which is the subject of the present invention, with reference to the appended figures, in which:

FIG. 1 represents the nucleotide sequence of 2.8 kb containing the RTM1 gene,

FIG. 2 represents the restriction map of a 2.8 kb fragment containing the RTM1 gene, the coding part of which is indicated by the arrow,

FIG. 3 is a representation of the plasmid pFL44L-2,8, FIG. 4 is a representation of the plasmid pFL44L-2,8-14DM, FIG. 5 represents the detection by autoradiography of the chromosomes of yeast Saccharomyces cerevisiae, hydrated by a probe specific for the RTM1 gene,

- Figure 6 shows the peptide sequence of the protein encoded by the RTM1 gene.

It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

Molecular techniques

General DNA manipulation techniques use the laboratory manual as a reference (Sambrook et al 1989).

The enzymes are used as recommended by the manufacturer and come from the BRL (Bethesda Research Laboratories) and Boehringer (Boehringer Mannheim) laboratories.

The techniques used essentially relate to the following stages:

- digestion with restriction enzymes,

- obtaining blunt ends of DNA from cohesive ends by the activity of the enzyme Klenow, ligation of DNA molecule by the ligase of bacteriophage T4, labeling of probe with α ³ P-dCTP by the method d '' random priming '', DNA transfer on membrane from agarose gel,

- DNA hybridization with the argues probes,

- preparation of chromosomes from the yeast Saccharomyces cerevisiae, the protocol of which is adapted from that described by Carie and Oison (1985), - separation of chromosomes from the yeast Saccharomyces cerevisiae: the separation conditions on a CHEF type device correspond to a migration at 200 V for 27 h in a buffer 0.5 X TBE (50 mM Tris, 50 mM borigue acid, 1 M EDTA ), with pulses of 70 s during a period of 15 h and then of 120 s during a period of 12 h.

Transformation techniques

The yeast transformation is carried out either by the LiCl method (Ito et al (1983) J. Bacteriol. 153, 163-168, or by electroporation (Delorme 1989 and Becker and Guarente 1990).

Preparation of culture media containing molasses

Molasses mainly provides the carbon source essential for the growth of yeast, as well as various organic compounds, minerals and trace elements. It is made up of around 50% sucrose.

To be sure to bring all the essential minerals and trace elements, we add the culture medium of YBN (Yeast Nitrogen Base) without amino acids (Difco). (Note that it has been observed that with or without YNB the same results are obtained).

The pH of the medium with a value of 8-9 is brought to pH 5 (industrial pH condition) by the addition of H2SO4. It was checked beforehand that this acidification of the medium did not cause inhibition of the growth of yeasts. Different molasses concentrations are used for the different tests. Composition of the culture media used

40 to 500 g molasses

6.7 g YNB without amino acids

10 g agar (for solid media) qs 1 liter distilled water pH 5 The medium is sterilized at 110 ° C for 30 minutes.

Isolation of the RTM1 gene

The presence of a gene which, overexpressed, allows the improvement of the growth of Saccharomyces cerevisiae on a culture medium containing molasses has been sought.

- Construction of a genomic DNA library The genomic DNA was extracted from the FL100 strain

(Lacroute 1968). The DNA underwent partial digestion with the restriction enzyme Sau3A. The DNA fragments from this digestion, the size of which is between 5 and 8 kb, have been purified. These were cloned into the multicopy vector pFL44L (Bonneaud N. 1991) at the BamHI site of the multi-cloning site.

The F100 ura ^' strain, derived from the FL100 strain, auxotrophic for uracil, was transformed by these recombinant plasmids.

- Selection

The selection medium consists of a culture medium containing a concentration of molasses higher than the MIC (Minimum Inhibitory Concentration in molasses) of the strain FL100 which is 16%.

The library was spread on the selection medium (containing 40% molasses) agar and poured into a petri dish (in total 10 ⁸ transformed cells are spread per dish). After 2 to 4 days of culture at 30 ° C., a few colonies appeared on a carpet of dead cells.

Among the positive clones, 10 were analyzed. All contained a plasmid carrying the same 4.5 kb DNA fragment.

This fragment carried by the multicopy vector pFL44L was reduced (by digestion-religion) into a 2.8 'kb Sphl-Kpnl fragment exhibiting the capacity during its overexpression to improve the growth of FL100 on molasses. This vector is present in a few tens of copies per cell. This construction is named pFL44L-2.8 and presented in Figure 3.

- EXAMPLE 1: Evaluation of the sensitivity of the strains

The number of loci carrying the RTM1 gene is determined by hybridization of the chromosomes of Saccharomyces cerevisiae strains separated by pulsed fields, with a marked probe. This probe corresponds to the fragment of 1045 HindIII-BamHI base pairs of sequence I.

The evaluation of the growth capacity of these strains is carried out on solid and liquid medium containing different concentrations of molasses.

The results (Table 1) show that the strains having no RTM1 gene or only a copy have their growth totally inhibited at 16% molasses. On the other hand, strains containing 4 or even 5 copies of the RTM1 gene are much more resistant to molasses: they are able to grow on a medium containing 40% molasses. Table 1

number of copies of the CMI gene

RTMl by strains (% molasses)

0 16

1 16 4 or 5> 40

Thus these results clearly demonstrate that there is, in most cases, a correlation between the number of copies of the RTM1 gene and the capacity for growth on molasses.

The MIC, or minimum inhibitory concentration, corresponds to the minimum concentration of molasses in the culture medium which inhibits 100% the growth, preferably on solid medium, of the strain concerned.

EXAMPLE 2: Identification / Characterization of strains

A hybridization profile is presented in FIG. 5 representing the hybridization of the chromosomes of 3 strains of Saccharomyces cerevisiae separated by pulsed fields, by the labeled probe used in Example 1.

It is observed that the three profiles, corresponding to three strains of different origin, are very distinct.

Thanks to the high degree of variability concerning the localization of the RTM1 gene, it is possible to determine a hybridization profile characteristic of a strain. This is confirmed by obtaining a specific profile for 11 different strains (results not shown).

EXAMPLE 3 Measuring the Toxicity of Molasses Specifically The use of laboratory yeast LG16 containing no RTM1 gene (Crouzet et al. 1991), as well as this same strain transformed by the multicopy vector pFL44L-2.8 described in FIG. 3, made it possible to measure the difference in toxicity between 2 batches of molasses. This strain thus transformed was deposited under the reference LG16-2.8 at the CNCM on November 23, 1993 under the number 1-1376.

The results are shown in Table 2, in which "+" means growth of the strain, "-" non-growth and "+/-" intermediate growth, depending on the behavior of the strain on agar media (drop test).

Table 2: lot 1 lot 2

% molasses 16 30 30 50

strain LG16 - - + +/-

LG16 strain transformed by + + + +/- pFL44L-2,8

At the same molasses concentration in the culture medium (30%), the strain LG16 is capable of growing on an medium containing lot 2 but not lot 1. The latter is much more toxic than lot 2 and its presence at 16 % in the culture medium is sufficient to inhibit the growth of the LG16 laboratory strain.

In comparison, the growths observed for the transformed LG16 strain are given. Present in multicopy, the RTM1 gene makes it possible to restore high growth on the medium containing toxic molasses. Its overexpression specifically responds to inhibition of growth by the toxic element (s) present in lot 1.

These inhibitory elements are in much lower concentration in batch 2 and the growth of the transformed strain is not much greater than that of the untransformed strain. Furthermore, at high molasses concentration (50%), there is equivalent inhibition for the two strains. This growth inhibition seems in this case rather linked to the osmotic pressure.

Thus the use of differential growth between a "sensitive" strain and this same strain transformed by the vector pFL44L-2,8, makes it possible to assess the degree of concentration of specific toxic element (s).

EXAMPLE 4 Manufacture of reference batches for measuring the sensitivity of the strains of microorganisms

The use of LG16 strains as well as of this same strain transformed by the multicopy vector pFL44L-2.8, makes it possible to define the minimum inhibitory concentration (MIC) of a batch of molasses (Table 3). Table 3:

lot 1 (% molasses) 4 8 12 16 20 30 40 50

LG16 + + + / "- - - - - LG16 transformed by pFL44L-2,8 + + + + + + + + +/-

Thus for batch 1 of molasses, this MIC corresponds to 16% or 80g of sucrose / 1 of medium.

We can define that either the batch of molasses, its limiting dilution where we observe growth discrimination between LG16 and LG16 transformed by pFL44L-2,8. From this limit value used as a reference, the sensitivity to molasses of any strain can be defined (Table 4).

Table 4:

lot 1 (% molasses) 4 8 12 16 20 30 40 50

strain D + _ _ _ _ _ _ _ _

Y + + + + + - - - z + + + + + + + + +/-

The strain D is the strain ATCC 4126. The latter, transformed by the plasmid pFL44L-2.8 14DM described in FIG. 4, is named 4126-2.8DM deposited at the C.N.C.M. on November 23, 1993 under the number 1-1377.

The MICs are 8%, 30% and more than 50% for strains D, Y and Z respectively.

EXAMPLE 5 Improvement of growth on molasses

The multicopy vector (presented in FIG. 2) associated with an appropriate selection marker can be used to improve the growth of industrial microorganisms, in particular yeast.

The 4.6 kb BamHI-HindIII fragment from pFD384 containing the mutated C14 lanosterol demethylase gene (p450 14DM) (Doignon et al 1993), is ligated into the multicopy vector pFL44L-2.8 at the single site Kpnl. The ends of these two molecules are made blunt by the activity of the Klenow enzyme. The resulting construction is named pFL44L-2,8-14DM (Figure 4).

The presence in a yeast of the mutated C14 gene has lanosterol demethylase, confers on it resistance to the inhibitor: fluzilazol.

An industrial yeast D is transformed by this construction and selected on a medium containing a concentration of fluzilazol (2μg / ml) higher than the MIC for this strain. As a control, this same strain is transformed by pFD384.

The clones selected for their resistance to this inhibitory concentration of fluzilazol are tested for their growth capacity on a culture medium containing a toxic concentration of molasses for the strain (Table 5).

Table 5:

Solid medium liquid medium culture medium fluzilazol molasses molasses 20%

2 μg / ml 20% 0 16 40 48h

strain:

D - - 0.01 0.01 0.01 0.03

D transformed by pFD384 + - 0.01 0.01 0.02 0.03

D transformed by pFL44L-2.8-14DM + + 0.01 0.2 9.0 20.0

Table 5 shows, in diagrammatic form, the growth on solid medium (molasses and fluzilazol) and the growth, measured in optical density at different culture times, on liquid medium containing 20% molasses from batch 1. These measurements were carried out for the industrial strain D, this same strain transformed by the vector pFD384 and the industrial strain D transformed by the construction pFL44L-2, 8-14DM. The same results are obtained for different clones corresponding to these three types of yeast.

These results allow us to conclude that the overexpression of the RTM1 gene in industrial yeast gives it the ability to multiply on a medium. containing a normally toxic concentration of molasses.

EXAMPLE 6: Selection marker

The vector pFL44L-2.8 defined in FIG. 2 can be used for the cloning of a given gene. The RTM1 gene used as the dominant marker will allow the strain transformed by such a plasmid to be positively selected on molasses.

BIBLIOGRAPHY

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- Bonneaud N., 0. Ozier-Kalogeropoulos, G. Li, M. Labouesse, L. Minvielle-Sebastia and F. Lacroute. A Family of Low and High Copy Replicative, Integrative and Single-Stranded S. cerevisiae / E. coll. Shuttle Vectors. Yeast (1991) 7,609-615.

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Doignon F., M. Aigle and P. Ribéreau-Gayon: Résistance to Imidazole and Triazole Fungicide in Saccharomyces cerevisiae as a new dominant marker. Plasmid (1993) in press.

Fiedler A.: Research of the inhibitory compounds contained in molasses. Thesis (Berlin) 1981.

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Claims

1. Nucleic acid sequence, characterized in that it allows in a cell using molasses as a substrate in a culture medium, the expression of a protein or a peptide capable of conferring on said cell resistance increased to the toxicity of molasses, said nucleic acid sequence being chosen from the group consisting of: a) the sequence shown in FIG. 1 or its complementary strand, b) the sequence between nucleotides 664 and 1591 in the figure 1 or its complementary strand, said sequence being the coding region, c) the sequence between nucleotides 664 and 2235 of the sequence of Figure 1 or its complementary strand, d) the sequence between nucleotides 1 and 1591 of Figure 1 or its complementary strand, e) the sequences derived from one or other of the sequences defined in (a), (b) or (c) by substitution of one or more of their nucleotides, or even addition or deletion of certain codons, since this sequence continues to code for a protein conferring the above-mentioned property on the host in which it is expressed. 2. Nucleotide probe, characterized in that it comprises a nucleic acid sequence of at least 10 nucleotides capable of hybridizing with all or part of the sequence as defined in FIG. 1. 3. Probe according to claim 2, characterized in that it is chosen from the group consisting of a) the sequences defined in (a) to (e) of claim 1, b). the restriction fragments of the sequence represented in FIGS. 1 and 2, in particular the fragment between nucleotides 492 and 1538 of the sequence of FIG. 1, - the fragment between nucleotides 1537 and 2167 of the sequence of Figure 1. c) any sequence homologous or derived from a sequence defined in (a) or (b) by substitution, deletion or addition of one or more nucleotides since this sequence specifically hybridizes with all or part of the nucleotide sequence represented in FIG. 1. 4. A probe according to claims 2 and 3, characterized in that it is covalently associated with a labeling substance detectable directly or indirectly in particular by an optical or luminescent radioactive signal. 5. Pair of DNA polymerization primers constituted by probes according to one of claims 2 to 4 chosen in such a way that it allows the amplification of a fragment included in the sequence of FIG. 1. 6. A pair of primers according to claim 5, characterized in that it consists of the following two sequences: 5ΑTGTCAAATGACTCTAGTGG3 'and 5'GGTTTCAAGGACTGACACTC3' 7. Biologically functional vector, characterized in that it contains one or more copies of a nucleic acid sequence according to claim 1. 8. Vector according to claim 7, characterized in that it is a multicopy vector. 9. Vector according to claims 7 and 8, characterized in that it is the vector pFL44L-2.8 used to transfect a strain of yeast S. Cerevisiae LG16 and deposited at the CNCM on November 23, 1993 UNDER n ° 1-1376. 10. Recombinant cell host, characterized in that it comprises one or more nucleotide sequences according to claim 1 or one or more copies of a vector according to one of claims 7 to 9, which host may exhibit increased resistance of the toxicity of molasses used in the composition of its culture medium. 11. Host cell according to claim 10, characterized in that it is yeast, in particular baker's yeast, or wine or distillery yeast. 12. Host cell according to claim 10, characterized in that it is a bacterium, in particular E. coli, Streptomyceε spp, Bacillus spp, Coryneba'ccerium spp or Zymomonaε mobiliε. 13. Host cell according to claim 10, characterized in that it is a fungus, in particular Aεpergilluε, Penicillium OR Trichoderma reseii. 14. Host cell according to claims 10 and 11, characterized in that it is the yeast strain Saccharomyces cerevisiae 4126-2,8 DM deposited at the C.N.C.M. November 23, 1993 under No. 1-1377. 15. Polypeptide characterized in that it contains the amino acid sequence represented in FIG. 6, said sequence being capable of conferring on the cell which contains it an increased resistance to the toxicity of molasses. 16. A polypeptide according to claim 15, characterized in that it comprises an amino acid sequence ^' modified with respect to the sequence of Figure 6 by deletion, insertion, replacement or addition of one or more amino acids when the polypeptide thus modified is involved in resistance to the toxicity of molasses entering the culture medium of a microorganism of industrial interest. 17. Peptide fragment characterized in that it corresponds to the amino acid sequence between positions 1 and 309 of FIG. 6 or to any part of this sequence functionally associated with resistance to the toxicity of molasses. 18. Monoclonal or polyclonal antibodies, characterized in that they specifically recognize a polypeptide, defined according to one of claims 15 to 17 or a fragment thereof. 19. A method of detecting and quantifying the presence, in a culture of industrial microorganisms, of a nucleic acid sequence according to claim 1, characterized by the following sequence of steps: the DNA of the microorganism is made accessible for hybridization either by extraction or by cell permeabilization, the DNA is hybridized with a probe labeled directly or indirectly as defined in claims 2 to 4, if necessary, the DNA is amplified by PCR using a pair of primers according to claims 5 or 6, - the revelation of the presence and if necessary of the marking quality. 20. Detection method according to claim 19, characterized in that it uses the method of PCR with a pair of primers according to claim 5 or 6. 21. Method for measuring the specific toxicity of a molasses on the growth of a microorganism, consisting in comparing the relative growths on said molasses of a microorganism before and after transformation with a multicopy plasmid containing at least 1 copy of the RTM- ,, gene and measure the increase the minimum inhibitory concentration for the transformed strain relative to the untransformed strain. 22. Method according to claim 21, characterized in that the control strain is Saccharomyces Cerevisiae LG16 and the transformed strain LG16-2, 8 deposited at the C.N.C.M. November 23, 1993 under No. 1-1376. 23. Kit for measuring the specific toxicity of a molasses on the growth of a microorganism, in particular a yeast of industrial interest, containing - an ampoule of a control microorganism, - an ampoule of the same microorganism transformed and containing at least 4 copies of the gene conferring resistance to the toxicity of molasses to said micro¬ organism, these two ampoules being in a form allowing their conservation, in particular lyophilization, if necessary molasses to prepare a reference medium. 24. A method of improving the production growth of a microorganism of industrial interest, the growth of which is carried out on a culture medium containing molasses, consisting in transforming said strain with a vector containing at least one copy of 'A sequence as defined in claim 1. 25. Use of a nucleotide sequence according to claims 1, as a selection marker for a microorganism of industrial interest whose growth takes place on a medium containing molasses. 26. Use of a probe according to one of claims 2 to 4, as a selection marker for microorganisms of industrial interest whose growth takes place on a medium containing molasses. 27. Method for detecting and measuring the presence of a polypeptide, the presence of which confers increased resistance to the toxicity of molasses to industrial microorganisms, the culture medium of which contains molasses, characterized in that it uses antibodies according to claim 17 in an immunometric test, in particular a method of the sandwich type, or of competition. 28. Kit for detecting the presence of a polypeptide, the presence of which confers increased resistance to the toxicity of molasses to industrial microorganisms whose culture medium contains molasses, characterized in that it comprises: - antibodies according to claim 18, optionally labeled, - a reagent for the detection of an immunological reaction of antibody-antigen type, - where appropriate, reagents to perform lysis of the cells of the sample tested. 29. Kit for detecting the presence of a gene, the presence of which confers increased resistance to the toxicity of molasses to industrial microorganisms whose culture medium contains molasses, characterized in that it comprises - at least nucleotide primers according to one of claims 5 or 6, where appropriate marked, - nucleotide triphosphates dATP, dCTP, dTTP, dGTP, - a DNA polymerization agent. 30. A method of screening molasses for entering into the composition of culture media for industrial micro¬ organisms, characterized in that the minimum inhibitory concentration of said molasses is measured on a cell line comprising at least four nucleotide sequences according to claim 1 compared to the MIC obtained with the same cell line not containing them. 31. Method for identifying the constituent (s) of molasses responsible for toxicity on the growth of industrial microorganisms, characterized by the implementation of the following steps: - fractionation of molasses by gentle fractionation techniques, separation of the constituents of the fraction (s) for which the toxic effect is preserved, measurement of the toxic effect of each of these constituents, the toxicity measurements at each of the stages being carried out by measurement of the MIC of each of the fractions on a reference line and on the same line having incorporated at least 4 copies of one of the sequences of claim 1, the index of a specific toxicity being provided by the differential effect of the constituent on the reference strain and the transformed strain. 31. Method according to claim 30, characterized in that the reference strain is the strain LG16 and the transformed strain LG16-2.8 deposited at the C.N.C.M. November 23, 1993 under No. 1-1376.