WO2008077986A1

WO2008077986A1 - Improvement of the aromatic content of wines and other alcoholic beverages by use of microorganisms which, during fermentation, produce monoterpene synthase

Info

Publication number: WO2008077986A1
Application number: PCT/ES2007/070207
Authority: WO
Inventors: Margarita Lourdes Orejas Suarez; Daniel RAMÓN VIDAL; Oscar Herrero Madrid
Original assignee: Consejo Superior De Investigaciones Científicas
Priority date: 2006-12-27
Filing date: 2007-12-07
Publication date: 2008-07-03
Also published as: ES2324508B1; ES2324508A1

Abstract

The present invention describes a novel procedure for producing alcoholic beverages with increased and/or different terpene content by using microorganisms genetically modified in order to express genes which code monoterpene synthase during fermentation, which enables same to produce de novo aromatic monoterpenes from any starting plant material. Specifically, microorganisms are transformed with the the gene lys, coding for linalol synthase, by increasing the secretion of linalol. Although the microorganisms will preferably be yeasts of the species Saccharomyces cerevisiae, the method would also be applicable to other microorganisms useful in winemaking such as other yeasts and bacterias. One specific use will be to give a more favourable and/or different aroma to fermented alcoholic beverages such as wines, including cava and champagne, beer, cider, sake, etc.

Description

TITLE

IMPROVEMENT OF THE AROMATIC CONTENT OF WINES AND OTHER ALCOHOLIC BEVERAGES THROUGH THE USE OF MICROORGANISMS THAT, DURING FERMENTATION, PRODUCE SYNTHETIC MONOTERPEN

SECTOR OF THE TECHNIQUE

Wine sector It is about solving the problem of wines (and eventually other alcoholic beverages obtained by fermentation) with low terpenic content.

STATE OF THE TECHNIQUE

The aroma of the wines is one of the characteristics that will be important in assessing their quality. It can be divided into three main groups: (i) aromas from the grape variety or primary aromas, produced by volatile substances, transferred by the grape grains to the must, mainly monoterpenes, which are largely lost during the process; (ii) aromas produced during fermentation or secondary aromas, including two types of major aromatic compounds, higher alcohols and esters; and (iii) the "bouquet", also known as tertiary aroma, produced by the final transformation of the former during aging.

The role played by monoterpenes - a very varied group of volatile compounds of low molecular weight - in the fruity and floral aroma is well defined (Williams et al., 1980) and qualitative and quantitative variations in them are responsible for characteristic varietal nuances ( Marais, 1983). More recently it has been established that some monoterpenes can also be considered nutraceuticals (Crowell, 1999; Wise and Croteau, 1999; Croteau et al., 2000), so their presence could cause not only improvements in the characteristics Organoleptic wines but also in their functional characteristics.

Monoterpenes will be abundant in some grape varieties, and that greatly contribute to the varietal character of the wines, are linalool, geraniol, nerol α-terpineol and citronellol (Ribéreau-Gayon et al., 1975; Park et al., 1991). These are present in two distinct fractions: a free one that contributes to the aroma, and another linked, forming non-aromatic diglycosides, fundamentally 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside, 6-O-α-L-arabinopyranosyl -β-D- glucopyranoside, 6-O-α-L-ramnopyranosyl-β-D-glucopyranoside, 6-O-β-D-xylopyranosyl-β-D-glucopyranoside, 6-O-α-L-apiofuranosyl-β -D-glucopyranosides and 6-0-β-D-glucopyranosyl-β-D-glucopyranosides, with aglycone always linked to β-D-glucopyranoside (reviewed in Günata, 2003). This second fraction is quantitatively higher than the first (Günata et al., 1985a; 1985b), and hardly undergoes changes during the fermentation process carried out by Saccharomyces cerevisiae; therefore it is a potentially useful source to increase the aroma of the wines. It has been shown that the enzyme enzyme hydrolysis of these precursor compounds occurs in two stages, in the first the union between sugars is broken by the action of an exoglycosidase: an arabinofuranosidase, a ramnosidase, a β-xylosidase or an apiosidase and subsequently, by The action of a β-glucosidase releases aglycone (usually a monoterpene or a higher alcohol to a lesser extent) with aromatic capacity (Günata et al., 1988; 1990). More recently it has been shown that the unique action of an exoglucanase, acting as an endoglycosidase, is sufficient for the release of monoterpene (Gil et al., 2005).

Until now the use of maceration enzymes, which facilitate the release of glycosidic precursors, and of glycosidases, which facilitate the release of aglycone, both added to wine and produced by the yeast itself that carries out the fermentation (reviewed by Günata, 2003; Manzanares and Orejas, 2005; Ramón et al., 2005; Schuller and Casal., 2005; Verstrepen et al., 2006; as well as protected by several patents: Günata et al., 1989; Ramón et al., 1994; Rosi and Paolo 1996; Fowler et al., 1999; Vilanova de la Torre et al., 2004) is one of the strategies already described to increase the aroma of wine. However, with this strategy it would only be possible to increase the aromatic components of those wines from grape varieties that have a high level of glycosylated terpenes, for example Moscatel; however, most of the varieties used in winemaking, for example Palomino, Xarel.lo, Bobal etc., have a more neutral aromatic profile due to the shortage of terpenes both in free and glycosylated form (López-Tamames et al ., 1997; Gil and Valles 2001; Estévez et al., 2004).

In these cases, an alternative to the addition of enzymes or the use of recombinant yeasts that express these activities, would be that the yeast itself that carries out the fermentation was able to synthesize de novo monoterpenes as a result of its metabolism. In this sense, although S. cerevisae does not normally produce monoterpenes naturally, in an attempt to obtain yeasts that produced them, two autotrophic mutants for ergosterol, of complicated genotype, were isolated (after UV mutagenesis of the laboratory strain FLlOO) , blocked in the farnesyl diphosphate synthetase enzyme, encoded by ERG20, and which also carry additional mutations in other genes of the pathway (erg! 2-2 and / or erg9, encode mevalonate kinase and squalene synthase respectively) that allowed them to reduce geraniol levels to survive; the VL134 mutant was able to excrete farnesol (0.220 mg / L) and geraniol (0.450 mg / L), produced from its phosphorylated forms possibly by nonspecific phosphatases (Chambón et al., 1990a; 1990b). The ability to produce terpenes was introduced into the wine strain L116 (Fermivin) by crossings, and mutants descended from the cross were capable of producing large quantities of some terpenes, even higher than the parental strain (up to 6 ppm). However, the ability to consume glucose and produce ethanol was lower in all isolated offspring, which made them unfeasible for certain technological applications (Javelot et al., 1991). As in the previous case, this method has been protected by several patents (Karst et al., 1989; 1992; 1994, and Watanabe et al., 2002 for use in Sake).

More recently, Carrau et al. (2005) have described that some wine strains other than L116 were capable of producing monoterpenes in synthetic must; specifically the strain

KUl (Uruguay) produced ~ 5 μg / L of linalool (amount that borders the detection threshold, which is 4-10 ppb) and undetectable amounts of other monoterpenes, such as geraniol. However, in the same study, in other industrially used wine strains, Montrachet 522 (University of California, Davis), CIVC8130 (France) and two Uruguayan strains, the linalool produced was below the detection threshold.

On the other hand, in plants, monoterpenes are present in the essential oils of flowers and fruits and are produced, specifically, by monoterpene synthases, which use geranyl diphosphate (GPP) as a substrate (Bohlmann et al., 1998). This substrate is a common metabolite in microorganisms and higher organisms, which opens up the possibility of applying metabolic engineering techniques to provide a given microorganism with new steps in the isoprenoid pathway, which include the missing genes for the manufacture of a determined monoterpene (Carter et al., 2003 and Reiling et al., 2004 in Escherichia coli; Oswald et al., 2006 in laboratory strains of S. cerevisiae). In the last ten years, different laboratories have begun to characterize the genes and enzymes responsible for the production of the specific components of the aroma and flavor of flowers, fruits and vegetables. Thus, among the genes whose products determine the smell of flowers of the Clarkia breweri species, the Lis gene has been characterized

(Dudareva et al., 1996; patented by Pichersky, 1997) that encodes the enzyme S-linalool synthase (LIS) and catalyzes the production of monoterpene S-linalol, one of the important components of the aroma of wines, in a single reaction from the GPP. Since in GPPS yeast is an intermediary of ergosterol synthesis, the present strain S. cerevisiae T ₇₃ (CECT 1894; Querol et al., 1992) has been endowed with the Lis gene of C. breweri and both its ability to produce linalool throughout winemaking and its fermentative characteristics have been studied.

Recently, new genes encoding other monoterpene synthases are being characterized. These genes and others that are discovered can also be expressed in suitable strains of wine yeasts, similar to the example here collected. The GES gene, which in basil encodes the enzyme geraniol synthase (Iijima et al.,

2004), the gene that encodes α-terpineol synthase in grapes (Martin and Bohlmann, 2004) and the QHl gene that encodes a 3R-linalo synthase in Artemis (Jia et al., 1999) are some examples.

Probably S. cerevisiae is one of the living organisms with the greatest molecular knowledge. For almost thirty years it has been possible to genetically transform laboratory strains of this species (Beggs 1978). To all this we must add the availability of the complete nucleotide sequence of the genome of this organism (Goffeau et al., 1997) and a plethora of molecular techniques that have allowed and allow progress in its manipulation. Currently, transgenic wine yeasts (YMGs) are available to improve the process of vinification (for example facilitating filterability) and YMGs that serve to improve the organoleptic and / or functional characteristics of the wines (reviewed by: Manzanares and Orejas, 2005; Ramón et al., 2005; Schuller and Casal., 2005; Verstrepen et al., 2006). Recently in Japan the use of a recombinant yeast, which does not need to be qualified as YMG, has been approved for the preparation of Sake (Akada, 2002).

The proposed invention consists in the improvement of the fermentation process of alcoholic beverages, preferably wines, by the use of a microorganism that, having been modified to produce heterologous monoterpene synthases, leads to an improvement in the aromatic content of the beverage.

DESCRIPTION OF THE INVENTION

brief description

The object of the present invention is the use of a microorganism useful for the production of alcoholic beverages made from fruit musts or juices, preferably wines, cereal extracts or other food products of plant origin, genetically manipulated, hereinafter used of the present invention, comprising DNA sequences encoding enzymes with monoterpene synthase activity that allow the production of monoterpenes during fermentation.

A particular object of the present invention is the use of the invention where the alcoholic beverage belongs, by way of illustration and without limiting the scope of the invention, to the following group: wines, (including non-sparkling wines, as well as cava, champagne and others sparkling), beer, cider, sake, etc.

A particular embodiment of the present invention is the use of the invention where the alcoholic beverage is preferably white, red or rosé wines from grape musts of any variety. Another object of the present invention is the use of the invention where DNA sequences encoding enzymes with monoterpene synthase activity include any gene encoding enzymes that catalyze the formation of monoterpenes: linalool synthase, geraniol synthase, α-terpineol synthase, etc .; as well as any nucleotide sequence analogous to these that encrypts enzymes with said activity, in isolation or in their possible combinations.

Another particular embodiment of the present invention is the use of the invention where a DNA sequence encoding an enzyme with monoterpene synthase activity includes the sequence of the C. breweri Lis gene (SEQ ID NO1), which encodes a linalool synthase. 0, as explained above, the protein that encodes, the enzyme with S-linalool synthase activity (SEQ ID N02), as well as any nucleotide or amino acid sequence (aas) analogous to them.

Another particular object of the present invention is the use of the invention in which the microorganism is of interest in oenology: yeasts or bacteria.

A particular embodiment of the present invention is the use of the invention where the microorganism is a wine yeast of the species S. cerevisiae, preferably strain S. cerevisiae YR64 (pGl-Lis-URA) (CECT 12032), which comprises the DNA sequence of the gene that in C. breweri encodes S-linalol synthase (SEQ ID NO1).

A last particular embodiment of the present invention is the use of the present invention in the production of alcoholic beverages together with other microorganisms, recombinant or not, to produce different combinations of aromatic monoterpenes during fermentation.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a totally different method from those mentioned above (see status of technique) to maintain, increase and / or modify the terpenic content of an alcoholic beverage and thus achieve a more favorable and / or different aroma. The proposed improvement method focuses on the fermentation of musts, preferably grapes, for the production of wines; being an extensible method to the production of other alcoholic beverages obtained by fermentation from different vegetable raw materials, by way of illustration and without limiting the scope of the invention, for example: fruit juices, cereal extracts, etc.

Said method is based on the genetic manipulation of wine yeasts so that they are capable of producing heterologous monoterpene synthases and as a consequence they produce and excrete de novo monoterpenes during alcoholic fermentation. The present invention is based on the observation that the expression of a gene, which in plants encodes an S-linalool synthase, in a wine yeast (derived from the industrial strain S. cerevisiae T ₇₃ , Lallemand), which apparently naturally It does not produce linalool (see figures 2 and 5), it induces the concomitant presence of this aromatic monoterpene in wines produced with said recombinant yeast. More specifically, it is based on the cDNA sequence of the C. breweri Lis gene, which encodes an S-linalo synthase. This gene is cloned between the TDH3 gene promoter and the S. cerevisiae PGKl gene terminator, in a yeast expression vector. The transformation of said wine strain with the previous vector yields the corresponding wine yeast of improved characteristics.

Improved vinification process proposed here has been performed in a mutant generated from the wine yeast S. cerevisiae T ₇₃ (T ₇₃ -4 mutant, uracil auxotroph;. Puig et al, 1998) and the gene Lis, which in C. breweri encodes the enzyme S-linalool synthase (Example 1). It has been proven that the transformed wine strain is capable, without further manipulation of the route and without the contribution of precursors, to produce and excrete free linalool throughout of winemaking While the wines resulting from the fermentation of Parellada musts with strains T ₇₃ and T ₇₃ -4 transformed with a control platinum that does not carry the expression cassette of the Lis gene - the so-called pGl-URA - do not have detectable amounts of linalool , those fermented with the yeasts that carry the plastic pGl-Lis-URA present quantities of linalool relevant from the sensory point of view, since they exceed the detection threshold thereof (Burdock, 2002) (Example 2). Therefore, in the present invention it has been shown that, unlike what happens with other strategies already described (see prior art), (i) the expression of cDNAs encoding monoterpene synthases in wine yeasts is sufficient for de novo production of monoterpenes, regardless of the raw material; (ii) that it is possible to change metabolic pathways in wine strains of Saccharomyces, specifically the isoprenoid route, for the production of linalool and possibly other monoterpenes; and (iii) that it is possible to achieve a continuous production of linalool in yeasts without this concentration being toxic.

In addition, it has been found that both the production of alcohol and the fermentation rate of these recombinant yeasts are comparable to those of a commercial yeast, a result of great importance for their applicability, indicating that the expression of the Lis gene in the modified wine yeast does not change its fermentative characteristics (Example 3).

It should be noted that with the aforementioned genetically modified yeasts, there are also no technical problems, mentioned above, that arise when vinified with yeasts carrying mutations in the ERG genes. As for its comparison with yeasts that naturally produce monoterpenes, not all wine yeasts are capable of producing them in detectable quantities, and also with this invention better yields are achieved and Doors open to the directed modification of volatile composition.

Therefore, the object of the present invention is the use of a microorganism useful for the production of alcoholic beverages made from fruit musts or juices, preferably wines, cereal extracts or other food products of plant origin, genetically manipulated Hereinafter, use of the present invention, comprising DNA sequences encoding enzymes with monoterpene synthase activity that allow the production of monoterpenes during fermentation.

A particular embodiment of the present invention is the use of the invention where the alcoholic beverage is preferably white, red or rosé wines from grape musts of any variety.

Another object of the present invention is the use of the invention where DNA sequences encoding enzymes with monoterpene synthase activity include any gene encoding enzymes that catalyze the formation of monoterpenes: linalool synthase, geraniol synthase, α-terpineol synthase, etc .; as well as any nucleotide sequence analogous to these that encrypts enzymes with said activity, in isolation or in their possible combinations. As used in the present invention, the term "monoterpene synthase" refers both to the nucleotide sequence of the gene in question, as well as to the protein it encodes, an enzyme with monoterpene synthase activity, as well as to any nucleotide or nucleotide sequence. amino acids (aas) analogous to these of other species. In the sense used in this description, the term "analogous" is intended also include any nucleotide or amino acid sequence that can be isolated or constructed based on the nucleotide or aas sequences contemplated herein, for example, by introducing nucelotide or aas substitutions, conservative or non-conservative, including insertion of one or more nucleotides or aas, the addition of one or will be nucleotides or aas in any part of the molecule or the deletion of one or will be nucleotides or aas at any end or within the sequence, and that constitutes a coding sequence or peptide with activity similar to the sequence of the invention, that is, is capable of synthesizing monoterpenes.

In general, a nucleotide or analogic amino acid sequence is substantially homologous to the amino acid sequence discussed above. In the sense used in this description, the expression "substantially homologous" means the nucleotide sequences or aas. in question they have a degree of identity of at least 40%, preferably of at least 85%, or you will preferably be at least 95%.

Nucleotide sequences encoding enzymes with monoterpene synthase activity are known today in such detail, which can be obtained by an expert by employing techniques widely known in the state of the art (Sambrook and Russel, 2001). Said nucleotide sequences can be in a linear DNA fragment integrated in the genome, or integrated in a vector that allows their expression, under suitable conditions, in different microorganisms in such a way that at the same time they carry out the alcoholic fermentation and other related fermentations can produce linalool or other monoterpenes from any must, juice, or wine, starting.

Another particular embodiment of the present invention is the use of the invention where a DNA sequence encoding an enzyme with monoterpene synthase activity includes the sequence of the C. breweri Lis gene (SEQ ID NO1), which encodes a linalo synthase. 0, as explained above, the protein that encodes, the enzyme with S-linalool synthase activity (SEQ ID N02), as well as any nucleotide or amino acid sequence (aas) analogous to them. Likewise, when talking about "DNA sequences that encode enzymes with monoterpene synthase activity", we also include the sequences already known, and available to a person skilled in the art, of the genes that encode geraniol synthase, α-terpineol synthase, 3R -Linalol synthase (Iijima et al., 2004; Martin and Bohlmann, 2004; Jia et al., 1999), etc .; as well as other monoterpene synthase sequences that can be isolated, characterized, made known and used for the same purpose as the present invention. 0, as explained above, the proteins that encode, as well as any nucleotide or amino acid sequence (aas) analogous to these of other species. These and other sequences can be obtained in public domain databases and can be obtained by an expert by employing techniques widely known in the state of the art.

Since each monoterpene has a peculiar aroma and different sensory threshold, these embodiments could also have a great impact on the sensory properties of the wines, which render it "starters" with different abilities in terms of terpene composition. In this sense, as an alternative to fermentations with one or different microorganisms, it is also possible to co-transform the microorganism for the use of the present invention with several of the above genes. Another particular object of the present invention is the use of the invention in which the microorganism is of interest in oenology: yeasts or bacteria.

This definition of microorganism also includes other microorganisms, other than S. cerevisiae, which while not responsible for the winemaking process itself, they take part in certain circumstances in this process (eg other levaduriform species and also bacteria).

As used in the present invention, the term yeasts refers to yeasts belonging, by way of illustration and without limiting the scope of the present invention, to the following group: Saccharomyces, Hanseniaspora (Kloeckera), Candida, Pichia, Metschnikowia, Kluyveromyces, Zygosaccharomyces, etc.

A particular embodiment of the present invention is the use of the invention where the microorganism is a wine yeast of the S. cerevisiae species, preferably S. cerevisiae YR64 strain (pGl-Lis-URA) (CECT 12032), which comprises the DNA sequence of the gene that in C. breweri encodes S-linalol synthase (SEQ ID NO1). As used herein, the term "bacteria" refers, by way of illustration and without limiting the scope of the present invention, to bacteria belonging to the following group: Lactobacillus, Pediococcus, Oenococcus, etc. On the other hand, the microorganism using the present invention can be used in the production of alcoholic beverages together with other microorganisms, recombinant or not, to produce different combinations of aromatic monoterpenes during alcoholic fermentation.

DESCRIPTION OF THE CONTENT OF THE FIGURES

Figure 1. Diagram of the plastic developed in this invention (The enzymes used for its construction as well as other single-cut ones are indicated): pGl-Lis-URA (Figure IA) and its control pGl-URA (Figure IB)

Figure 2. Evolution of the growth of YR63 (pGl-Lis-URA), YR64 (pGl-Lis-URA; CECT 12032), and YR65 (pGl-URA) transformants in YPD medium (Figure 2A) and their production kinetics of linalool (Figure 2B). Figure 3. Faithful chromatographic profiles of the culture media of strains YR63 (pGl-Lis-URA) (3A), YR64 (pGl-Lis-URA; CECT 12032) (3B) and YR65 (pGl-URA) (3C), after a 24-hour incubation in YPD medium. The arrow indicates the elution time of linalool established by comparison with a commercial pattern. Figure 4. Comparison of the ionic profiles of monoterpene produced by strain YR64 (pGl-Lis-URA) and linalool obtained by GC-MS.

Figure 5. Microvinifications. Growth evolution (5A), sugar consumption (5B), ethanol production (5C) and linalool accumulation kinetics (5D) of strains YR64 (pGl-Lis-URA; CECT 12032) and controls T ₇₃ and YR65 (pGl-URA). The results are the average of three independent experiments.

EXAMPLES OF EMBODIMENT Example 1.- Construction of a wine yeast strain that synthesizes de novo linalool

1.1.- Cloning of the Clarkia breweri Lis gene in a yeast expression vector

The coding region of the C. breweri Lis gene (Dudareva et al., 1996) (SEQ ID NO1) was cloned into the pG-1 vector (Schena et al., 1991) between the promoter of the S. cerevisiae TDH3 gene ( encodes glyceraldehyde-3-phosphate dehydrogenase, formerly called GPD _P ) and the terminator of the PGK1 gene of S. cerevisiae (encodes 3-phosphoglycerate kinase) from two fragments, one of 650 bp and another of 2080 bp. The first one was obtained in a PCR reaction using the oligonucleotides LisBgl2 (SEQ ID N03) and LisXbal (SEQ ID N04) and the plasmid pR65 (plasmid pBlueScript II SK + containing the Lis gene cDNA) as the template DNA. The Expand High Fidelity (Roche) enzyme was used for the PCR reaction. The amplification consisted of 10 cycles of 1 minute at 94 ⁰ C, 1 minute at 52.5 ⁰ C and 45 seconds at 72 ⁰ C and then another 25 cycles of 1 minute at 94 ⁰ C, 1 minute at 52.5 ⁰ C and 45 seconds at 72 ⁰ C, with increments of 5 seconds in the extension time for each cycle.

The PCR product, after being purified, was cloned — in the pGEM-T Easy vector (pious smido for the cloning of PCR products, Promega) to yield the pGEM-Lis plasmid. The sequencing of the PCR product revealed the absence of polymerization errors occurred during amplification thereof; although two changes in two amino acids were identified, not occurring during the polymerization reaction, which differ from the sequence of the database (NCBI AAC49395). This plastic was treated with the enzymes BgIII and Xbal and the 650 bp fragment was isolated. The second fragment (2080 bp), necessary to complete the ORF of the Lis gene, was obtained by restriction of the plastic pR65 with the enzymes Xbal and Salí. Both fragments were ligated into the vector pG-1, previously digested with the enzymes Bamñl and Salí, to construct the plasmid pGl-Lis, which carries the TDH3 _P expression cassette:: Lis:: PGK _t and the TRPl selection marker .

For the construction of the plasmid pGl-Lis-URA (Figure IA), which also bears the URA3 selection marker, a fragment of approximately 1500 bp containing the URA3 gene of the URA3 gene was subcloned into the Smal site of the plasmid pGl-Lis S. cerevisiae generated by PCR using genomic DNA from strain T ₇₃ and oligonucleotides URAl (SEQ ID NO5) and URA4 (SEQ ID NO6) as template. The PCR conditions differed only from the previous ones in the banding temperature, which was 55 ⁰ C. The above fragment was digested with the Hindlll enzyme and the protruding ends were filled with the Klenow enzyme; The resulting fragment was cloned into the Smal site of the plastic pGl-Lis-URA.

Similarly, the control vector pGl-URA (FiguralB) was constructed from the PG-I plastic.

1.2.- Construction of a wine yeast strain that synthesizes de novo linalool

Next, the S. cerevisiae T ₁₃ -A (ura3:: 470 / ura3:: 470) wine strain (Puig et al., 1998), derived from the industrial strain T ₇₃ , was transformed by the lithium acetate method (Gietz et al., 1995) improved, (Puig et al., 1998) with the plastic pGl-Lis-URA and the transformants were selected in medium without uracil. Several different transformants were analyzed and all behaved in a similar way regarding the production of linalool; YR63 (pGl-Lis-URA) and YR64 (pGl-Lis-URA; CECT 12032) transformants were isolated for further studies. Strain T ₇₃ -4 s adema transformed with plá smido pGl-URA for a control strain generated recombinant yeast, denominating YR65. The expression of the Lis gene was checked, indirectly, by the detection of linalool by gas chromatography (GC) in the culture media (YPD: 2% glucose; 2% peptone; 1% yeast extract) where the YR63 transformants were grown , YR64 and YR65. See below.

Example 2. Physiology of recombinant yeasts.

Analysis of metabolites in S. cerevisae expressing Lis.

Detection of linalool produced by strains YR63 (pGl-Lis-URA), YR64 (pGl-Lis-URA; CECT 12032) and YR65 (pGl-URA) was carried out using the solid phase, space microextraction technique head, coupled to gas chromatography (HS-SPME-GC). Solid phase microextraction was performed with polydimethylsiloxane (PDMS) fibers. 3 mL aliquots were taken that were introduced into 9 mL vials containing an 8x3 mm magnetic bar and 0.6 g of NaCl (to intensify the response in analyte extraction) (Arthur et al., 1992). Also, for subsequent quantification, a fixed amount of the internal 2-octanol standard (15 μL of a 0.005% w / v solution in ethanol) was added to the samples. The vials were sealed and left for 2 hours, with stirring, at room temperature, to achieve the phase equilibrium (liquid-vapor). PDMS fiber was introduced through the teflon septum of the plugs into the head space of the tube. After an adsorption period of 30 minutes, the fiber was injected into the gas chromatograph (HP 5890 series II), in which the analytes were released for four minutes.

For chromatography an HP innowax capillary column (Hewlett-Packard) of 15 m length, 0.25 mm internal diameter and 0.25 microns of phase thickness was used. The temperature of the injector was 22O ⁰ C and that of the detector of 28O ⁰ C. The oven temperature was initially 6O ⁰ C for 5 minutes and then increased at a rate of 5 ° C / min to 19O ⁰ C, and at a rate 20 ° C / min to 25O ⁰ C, temperature at which it was held for 2 minutes. The identification of linalool and other volatile components was carried out by comparing their retention times with that of the commercial standard. To obtain its concentration, a calibration curve was initially constructed with different concentrations of linalool (Fluka) and subsequently the chromatogram area was related to that.

Strains YR63 (pGl-Lis-URA), YR64 (pGl-Lis-URA) and YR65 were grown for 18 hours in selective SD medium (2% glucose, 0.5% ammonium sulfate, 0.143% YNB) and then in YPD medium ( 10 ⁶ cells / mL in 0.5 L flasks with 100 mL of medium) and at different times, for 97 hours at 3O ⁰ C with a stirring of 100 rpm, samples were taken to make counts (Figure 2). No significant differences were observed in the growth kinetics of the two strains that produce linalool, YR63 (pGl-Lis-URA) and YR64 (pGl-Lis-URA), and the control yeast, YR65 (Figure 2A). Both strain T ₇₃ -4 untransformed (data not shown) and transformants thereof with smido plá pGl-URA (YR65) linalool not detected; only yeasts transformed with pGl-Lis-URA, YR63 and YR64 produced linalool (Figure 2B). The maximum production of linalool (80 μg / L) is reached towards the middle of the exponential phase of growth.

No differences are observed, except for the linalool peak, between the chromatograms of the YR63 strains (pGl-Lis-URA)

(Figure 3A) and YR64 (pGl-Lis-URA) (Figure 3B) and strain YR65 control (Figure 3C), which suggests the absence of bioconversions from linalool produced in the experimental conditions of the study.

In addition, it was confirmed by gas chromatography-mass spectrometry (GC-MS) that the volatile that has been identified by the retention time in GC is linalool. For this, the YR63 and YR64 strains (pGl-Lis-URA) were grown in YPD medium and samples were collected at 20 hours (maximum linalool production). The samples were analyzed by the solid phase microextraction technique, in headspace, coupled to gas chromatography with mass detector. GC-MS was carried out on an Agilent 6890 chromatograph coupled to an Agilent 5973N mass spectrometer (Agilent Technologies, Waldbronn, Germany). The ion spectrum of the compound eluting in the expected time to linalool was the same as the linalool spectrum (Figure 4). In this way it was unequivocally verified that the volatile produced by the recombinant strains YR63 and YR64, (pGl-Lis-URA) was linalool.

Example 3. Microvinification assays with the recombinant wine strain YR64 (CECT 12032).

To find out if the recombinant yeasts previously constructed were able to carry out the vinification until the end, if the physical-chemical parameters of the wine are similar to those of the wine made with a yeast for industrial use and if the genetic manipulations previously carried out carry changes in the volatile profile that could be detected in tastings, microvinifications were carried out with strain YR64 (pGl-Lis-URA; CECT 12032) and controls T ₇₃ and YR65 (PGl-URA). Parellada must from the 2003 harvest from Villafranca del Penedés was used. The must was filtered through cartridges

Sartopure PP2 1.2 μm pore (Sartorius) and subsequently sterilized by adding 0.2 Velcorin (dimethyl dicarbonate)

% v / v, and incubated at room temperature overnight. Finally, it was supplemented with 0.3 g / L of fermentation activator (LALVIN NUTRIENT VIT, Lallemand). The microvinifications were carried out in triplicate, at 18 ⁰ C and under standard conditions (Querol et al., 1992). The three previous strains were grown for 18 hours in selective SD medium (2% glucose, 0.5% ammonium sulfate, 0.143% YNB). After this growth, the cells were collected by centrifugation, resuspended in must and 10 ⁶ cells / mL were inoculated in 250 mL bottles with 225 mL of sterile must closed with screw cap. Throughout the experiment, samples were taken at different times and in each of them the number of cells, the consumption of sugars, and the production of ethanol and linalool were determined. The fermentation evolution was monitored by measuring the OD at 600 nm on an Ultrospecc 2000 spectrophotometer (Pharmacia, Biotech, England). Sugar consumption was continued by measuring the BRIX grades with a refractometer (Cari Zeiss, Germany). In the last days of winemaking, the ECHO / ENOSYS automatic multianalyzer (Tecnova SA) was used to determine the end of the fermentation, that is, when the sugar content was less than 2 g / L. Ethanol was measured using an infrared spectrophotometer (Enfrascan, Alliance Instruments, France). In the final wines, the study of volatiles was carried out using the technique described above (example 2).

As seen in Figure 5A, the evolution of the growth of the three strains was similar, although the carrier of the plastic pGl-Lis-URA, YR64 (CECT 12032), has a slightly displaced growth kinetics with respect to the commercial strain . The consumption of sugars (Figure 5B) was also similar in all strains, reaching in all cases a level lower than 2 g / L after 6 days of fermentation. Similarly, the kinetics of ethanol production (Figure 5C) was similar in the three vinifications, reaching in all of them an alcoholic degree close to 8%. This value is slightly lower than that of wines commercial because of the low concentration of starting sugars in the must, which did not exceed 130 g / L. These results indicated that the expression of the Lis gene in the modified wine yeast apparently did not modify its fermentative characteristics.

Regarding the accumulation kinetics of linalool (Figure 5D), it was observed that strain YR64 (CECT 12032), carrier of the smoldering pio PGl-Lis-URA, was capable of producing this monoterpene during vinification. The other two control strains did not accumulate linalool during the trial. The maximum concentration of linalool was reached on the second day of fermentation (26.01 μg / L), at which point it was gradually decreasing (up to 18.60 μg / L in the finished wine). Since the olfactory detection threshold of this compound has been set at 4-10 μg / L, these results validate the proposed invention.

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Claims

1.- Use of a microorganism useful for the production of alcoholic beverages made from fruit musts or juices, preferably grapes, cereal extracts or other food products of plant origin, genetically manipulated and characterized because it comprises DNA sequences that encode monoterpene synthases, which also allow de novo production of monoterpenes during fermentation.

2. Use of the microorganism according to claim 1 characterized in that the fermented beverage belongs to the following group: wines (including non-sparkling wines such as cava, champagne and other sparkling wines), beer, cider, sake, etc.

3. Use of the microorganism according to claim 2, characterized in that the fermented beverage is wine: white, red or rosé wine obtained from grape musts of any variety.

4. Use of the microorganism according to claim 1 characterized in that the sequences, 1 or several, of DNA encoding enzymes with monoterpene synthase activity include any gene encoding enzymes that catalyze the formation of monoterpenes: linalool synthase, geraniol synthase, α-terpineol synthase, etc; as well as any nucleotide sequence analogous to these that encrypts enzymes with said activity, in isolation or in their possible combinations.

5. Use of the microorganism according to claim 4 characterized in that a DNA sequence encoding an enzyme with monoterpene synthase activity includes the sequence of the Lis bre gene of C. breweri (SEQ ID NO1, which encodes a linalool synthase).

6. Use of the microorganism according to claim 1 characterized in that the microorganism is a yeast or bacterium of interest in oenology.

7. Use of the microorganism according to claim 6 characterized in that the yeast belongs to the following Group: Saccharomyces, Hanseniaspora (Kloeckera), Candida, Pichia, Metschnikowia, Kluyveromyces, Zygosaccharomyces, etc.

8. Use of the microorganism according to claim 7 characterized in that the yeast is the species Saccharomyces cerevisiae.

9. Use of the microorganism according to claim 8 wherein the yeast is S. cerevisiae strain YR64, (CECT 12032), ie, the vinous strain T ₇₃ -4 transformed with plá smido pGl-Lys-URA.

10. Use of the microorganism according to claim 6, characterized in that the bacterium belongs to the following group: Lactobacillus, Pediococcus, Oenococcus, etc.

11. Use of the microorganism according to claim 1 characterized in that it is used in the production of fermented alcoholic beverages together with other recombinant microorganisms or not.