WO1999050422A2

WO1999050422A2 - Method for reduction of fusel oils in alcoholic beverages and food products

Info

Publication number: WO1999050422A2
Application number: PCT/IL1999/000174
Authority: WO
Inventors: Nissim Benvenisty; Amir Eden
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 1998-03-27
Filing date: 1999-03-26
Publication date: 1999-10-07
Also published as: AU3051699A; EP1066386A2; WO1999050422A3; JP2002509726A

Abstract

A method for reduction of fusel oils in alcoholic beverages by substituting wild type yeast strains with genetically engineered yeast strains which lack one or both of the genes encoding the branched-chain amino acid aminotransferases (BCAT). A method for production of alcoholic beverages containing a low amount of fusel oils, using genetically engineered Saccharomyces cerevisiae eca39Δ, eca40Δ and eca39Δ.40Δ strains is preferred.

Description

METHOD FOR REDUCTION OF FUSEL OILS IN ALCOHOLIC BEVERAGES AND FOOD PRODUCTS

FIELD OF THE INVENTION This invention concerns a method for reduction or manipulation of levels of fusel oils and related substances in alcoholic beverages by substituting wild type yeast strains, commonly used in the alcoholic fermentation process, with genetically engineered strains which reduce and even eliminate the formation of fusel oils during the fermentation. In particular, this invention concerns the use of engineered yeast strains, that lack either one or both of the gene(s) encoding the branched-chain amino acid aminotransferases (BCAT) found in yeasts. These yeast strains are useful in the production of alcoholic beverages containing a lesser amount of fusel oils and related undesirable substances.

BACKGROUND AND RELATED DISCLOSURES Fusel oils are considered to be impurities produced during the fermentation process in the production of alcoholic beverages. These fusel oils and likewise formed related substances are more toxic than ethanol and adversely affect the smell and taste of alcoholic beverages, particularly wine and beer. Fusel oils are higher alcohols, such as iso-amyl alcohol, iso-butyl alcohol, etc. These higher alcohols are more toxic than ethanol and consequently may cause adverse effects such as hangovers or aftertaste. In addition to fusel oils, alcoholic beverages produced by fermentation often contain other undesired metabolites, for example 2,3-butanedione (diacetyl) and 2,3- pentanedione. The presence or absence of all substances of this kind may also affect the smell and taste of beer, wine or other alcoholic beverage or food products. Despite numerous attempts to minimize or change levels of the fusel oils in alcoholic beverages, for example, by modifying the temperature of the fermentation medium, the problem has to date remained unsolved.

It would, therefore, be extremely desirable to provide a method for reducing and even completely eliminating the formation of fusel oils during yeast fermentation of alcoholic beverages.

It is therefore a primary object of this invention to provide modified, genetically engineered yeast strains, which are useful in manipulation of the fermentation process for production of alcoholic beverages with reduced and even eliminated content of fusel oils and related substances. The genetically engineered strains S. cerevisiae eca39Δ, eca40Δ and eca39Δ.40Δ are specific strains which are useful in carrying out this invention. These strains lack at least one of the two amino transferase BCAT, present in yeast cells, which are responsible for production of fusel oils.

All patents, patent applications and references cited herein are hereby incorporated by reference.

Summary of the Invention

One aspect of the current invention concerns a method for the reduction and/or elimination of the formation of fusel oils in alcoholic beverages. The term fusel oils as used herein is to be taken to mean also related undesirable fermentation metabolites, including, for example, various alkane diones.

The present invention particularly relates to use of yeast cells lacking or having reduced activity of one or both of the branched-chain amino acid aminotransferase (BCAT) isoenzymes in the production by fermentation of alcoholic beverages containing low concentrations of fusel oils.

In a preferred embodiment, the use according to the invention employs cells of the Saccharomyces cerevisiae eca39Δ, eca40Δ or eca39Δ.40Δ yeast strain. Use according to the invention results in the reduction of the concentration of iso-amyl alcohol and/or iso-butyl alcohol in alcoholic fermentation products.

In a further aspect, the invention relates to a method for producing an alcoholic beverage having substantially reduced content of fusel oils during yeast fermentation of alcoholic beverages by substituting a wild type S. cerevisiae yeast used for fermentation of alcoholic beverages with a genetically engineered Saccharomyces cerevisiae eca39Δ, Saccharomyces cerevisiae eca40Δ, Saccharomyces cerevisiae eca39Δ.40Δ yeast strains lacking or having reduced activity of one or both of the two branched-chain amino acid aminotransferases, preferably Saccharomyces cerevisiae eca39Δ.40Δ strains. The alcoholic beverages produced by the method of the invention are also encompassed. In yet a further aspect, the invention relates to a DNA sequence of Schizosaccharomyces pombe EC A39 gene identified as SEQ LD NO: l encoding a protein identified by the amino acid sequence SEQ LD NO :2.

Brief Description of Figures

Figure 1 is a comparative scheme showing isobutanol production by wild type yeast strain S. cerevisiae (Figure 1A) and by genetically engineered strains S. cerevisiae eca39Δ.40Δ (Figure IB) lacking one or both of the branched-chain amino acid aminotransferase (BCAT) isoenzymes. Figure 2 is a complete DNA coding sequence SEQ ID NO: l of S. pombe ECA39Δ available at GenBank Accession Number U88029 and the deduced amino acid sequence SEQ LD NO.2 of BACT encoded thereby. Figure 3 shows amino acid sequence of S. pombe ECA39/BCAT (SEQ LD NO:2), compared to amino acid sequences of two strains of S. cerevisiae producing BCAT cytosolic isoenzyme cBCAT (SEQ LD NO:3) and mitochondrial mBCAT (SEQ LD NO:4). Figure 4 illustrates a genetic assay for detection of BCAT activity. Figure 5 shows BCAT activity on the three branched-chain amino acids measured in total yeast cell extract. Figure 5A shows BCAT activity (act.) of eca39Δ.40Δ S. cerevisiae cells, transformed with EC A39, ECA40 or with a control (C) vector, in units (U). Figure 5B shows BCAT activity (act) in units (u) of eca39Δ.40Δ S. cerevisiae cells transformed with S. pombe EC A39 gene.

Detailed Description of the Invention

This invention has been developed in order to solve problems encountered during the production of alcoholic beverages using yeast fermentation.

During fermentation, yeast strains produce ethanol and several other higher alcohols. This fact is widely utilized for production of alcoholic beverages, such as beer, wine, hard alcohohc beverages and other food products Among the alcohols produced duπng fermentation are higher alcohols, known as fusel oils, such as iso-amyl alcohol, iso-butyl alcohol, etc These higher alcohols are more toxic than ethanol, may have adverse effects, such as hangover, on the consumer, may result in the beverage aftertaste which tends to rum the taste of beer or wine or may affect the smell of the beverage or the food product In addition to fusel oils, other related undesirable substances are formed during the fermentation process, mainly alkane diones such as dyacetyl and 2,3-pentanedιone The current invention intends to minimize or change the production and level of fusel oils in beer, wine, hard alcoholic beverages or other food products

Alcoholic beverage production is based on the process of yeast fermentation When the wild type, unmodified, yeast is used for fermentation of grain or fruit, as discussed above, high levels of ethanol and alcohols known as fusel oils result

Figure 1A shows a schematic description of the production of isobutyl alcohol from branched amino acid vahne This production of isobutanol is based on the fact that yeast may utilize and metabolize branched-chain ammo acids such as vahne, leucine or lsoleucine Catabohsm of one branched chain amino acid, vahne, illustrated in Figure 1A, is initiated by a transamination reaction catalyzed by the branched-chain amino acid transferase (BCAT, EC 2 6 1 42) As a result of this reaction, the level of produced isobutyl alcohol increases When the enzyme BCAT is absent, such as when the yeast strain is modified so as not to contain transaminating enzymatic activity, conversion of va ne or other branched-chain amino acid is not possible, does not occur and the level of produced isobutyl alcohol decreases, as seen in Figure IB

Beer, for example, is made by alcohol fermentation of the carbohydrates present in cereal grains, such as barley The carbohydrates have to be first converted into disacchaπdes or monosacchaπdes, such as glucose Yeast then ferment glucose to alcohol and carbon dioxide Glucose is then converted to pyruvate by glycolysis and the pyruvate is converted by a sequence of reactions to ethanol and carbon dioxide When branched amino acids, vahne, leucine or isoleucine, are present in the fermentation medium, the fermentation pathway includes production of α-keto isovalerate for vahne or other -keto acids for leucine or isoleucine α-Keto acids are then converted to fusel oils, such as isobutyl alcohol The enzyme involved in conversion of the branched amino acid into α-keto acid is branched amino acid aminotransferase The detailed description of the process may be found, for example, in Principles of Biochemistry, Ed Lehninger, et al (1993)

For the purpose of manipulating, l e , increasing and particularly decreasing the formation of fusel oils during fermentation, mutated yeast strains were genetically engineered These bioengineered yeast lack genes encoding enzymes responsible for production of high alcohols, namely the branched-chain amino acid aminotransferases (BCAT) These branched-chain amino acid transferases are key enzymes in the metabolism of branched-chain amino acids (BCAA) such as vahne, leucine, isoleucine, etc Branched amino acids are the main source of fusel oils produced during fermentation and, therefore, when the yeast strains lacking genetic apparatus for production of BCATs are used, the level of fusel oils is significantly decreased or is eliminated On the other hand, should it become desirable for smell or taste improvement to increase production of one or more higher alcohols, the yeast may be manipulated to overexpress the BCAT encoding genes

The inventors utilized the bioengineered strains of S cerevisiae lacking the EC A39 and/or ECA40 BCATs genes, encoding BACTs, and showed that a specific higher alcohol (iso-butyl alcohol) is indeed dramatically reduced during the fermentation process using the yeast strains Also the formation of diacetyl and 2,3-pentane dione was substantially reduced

I. Isolation and Characterization a Homolog of ECA39 BCAT S. pombe In order to produce alcoholic beverages of higher quality and better taste, a common yeast, S cerevisiae, was genetically modified to eliminate its BCAT enzymatic apparatus for its one or two BCAT isoenzymes This yeast strain is identified as eca39Δ, eca40Δ or eca39Δ eca40Δ

The mutated S cerevisiae eca39Δ, eca40Δ or eca39Δ eca40Δ were prepared essentially using methods described below These mutated yeast strains are preferred strains for use in alcohol beverage or food production by alcoholic fermentation The Saccharomyces cerevisiae genes for the cytosohc (cBcAT) and mitochondπal (mBCAT) branched-chain amino-acid aminotransferases (BCAT) show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene Two ECA39-hke genes were identified in yeast, as well as in man [J Biol Chem , 271 20242 (1996)] All ECA39 genes show significant similarity to LLVE genes coding for the BCAT in prokaryotes [J Biochem , 97 993 (1985), Biochem , 28 5306 (1989)]

Because gene c-Myc had been shown to control ECA39/BCAT in mammals [Oncogene, 13 1859 (1996)] and because myc oncogenes had been found to be absent in S cerevisiae, search for possible regulators of ECA39/BCAT in yeast was initiated It was demonstrated that ECA39 BCAT expression in S cerevisiae is controlled by the general transcription factor Gcn4, a c-Jun/c-Fos homo log EC A39 has several Gcn4 binding sites in its promoter region Moreover, expression of EC A39 depends on the number of these Gcn4 binding sites and is greatly reduced in gcn4 strains All other genes in the biosynthetic pathway of branched-chain amino acids are regulated by Gcn4

The expression of two S cerevisiae BCAT isoenzymes in the S cerevisiae strain appears to be inversely regulated during cell proliferation The cytosohc isoenzyme (cBCAT) is expressed at low levels in growing cells with a slight increase in stationary phase, while the mitochondπal isoenzyme (mBCAT) is highly expressed during growth and is down-regulated in stationary cells

Since c-myc proto-oncogene plays a role in cell proliferation and apoptosis with ECA39 serving as its target [Curr Opm Genet De , 4 102 (1994)], yeast strains lacking the ECA39 BCAT homologs were prepared and cell growth was examined Results show that in these strains the cell cycle was impaired in that eca39ΔmBCAT deficient cells grew faster than wild type cells and displayed a 25% shorter Gl stage of the cell cycle There was no effect on other stages One of the ways to understand the significance of the cell cycle impairment caused by altering BCAT activity is to study its relation to other, well defined, cycle mutants The S pombe cell cycle mutants are a good system for this purpose Therefore, the S pombe homologs of EC A39 and ECA40 were isolated and characterized

A Isolation of Homologs EC A39 S pombe

Based on the alignment of seven sequences of eukaryotic BCAT proteins, two highly conserved regions were identified and degenerative o gonucleotides coding for these regions were designed The ohgonucleotides were used as primers in PCR on S pombe genomic DNA A single PCR product of 502bp was cloned and sequenced The sequence obtained showed high homology to BCAT sequences This DNA fragment was used as a probe to screen an S pombe genomic library From approximately 9000 colonies screened three positive clones were isolated after successive hybridizations All three clones gave a similar restriction pattern One of these clones was sequenced to obtain the complete coding sequence of ECA39 from S pombe The complete sequence (SEQ ID NO 1) is shown in Figure 2

Figure 2 shows the complete DNA coding sequence (SEQ ID NO 1) of S pombe ECA39 BCAT (GenBank accession number U88029) The TATA box is marked by a box and the putative transcription start site is marked by an arrow Regions corresponding to the conserved domains, used to design the degenerative oligo- nucleotides are underlined An open reading frame (ORF) was identified with a TATA box upstream to it The gene isolated codes for a protein of 381 amino acids with a calculated size of 42 5 kDa This protein shows about the same degree of identity to S cerevisiae EC A39 and ECA40 (51-52%)

Figure 2 shows amino acid sequences of S pombe ECA39/BCAT (SEQ LD NO 2) compared to two S cerevisiae BCAT isoenzymes, namely S cerevisiae cytosohc cBCAT (SEQ ID NO 3) and S cerevisiae mitochondπal mBCAT (SEQ LD NO 4) Black background indicates conserved residues in all three proteins When compared to all seven eukaryotic BCAT sequences, the S pombe protein shows 47-52% identity which is distributed throughout the protein with a slight decrease in the N-terminus B Genetic Assay for BCAT Activity

To confirm the catalytic activity vis-a-vis branched amino acids of the isolated S pombe gene a genetic assay for presence of the BCAT activity was devised

Since BCAT enzymes are responsible for the last step of leucine, vahne and isoleucine biosvnthesis, a haploid S cerevisiae strain lacking both cytosohc and mitochondπal BCAT isoenzymes was constructed eca39Δ 40Δ This yeast strain loses the ability to grow when any of the branched-chain amino-acids is absent, as shown in Figure 4 for vahne This phenotype can be rescued by transformation with a plasmid containing a genomic fragment with either EC A39 or ECA40 from S cerevisiae, as seen in Figure 4

Figure 4 represents genetic assay devised for determination of BCAT activity S cerevisiae lacking both cytosohc and mitochondπal BCAT (eca39Δ 40Δ) cannot grow on a medium lacking vahne This phenotype is rescued either by EC A39 or ECA40 on a TRP1 vector, but not by the vector alone The S pombe genomic clone containing ECA39 [pSpECA39] can functionally replace the S cerevisiae genes

The catalytic activity of the isolated S pombe gene was first confirmed using the genetic assay as described above For this purpose, the gene isolated from the S pombe genomic library was transformed into the S cerevisiae strain which lacks both cytosohc and mitochondnal BCATs The S pombe gene did allow growth of this strain on a medium lacking vahne (Figure 4) or isoleucine (data not shown) indicating that the S pombe gene restored BCAT activity to this strain It was not necessary to add an S cerevisiae promoter to the S pombe gene in order to obtain its expression Consequently, S pombe ECA39/BCAT is suitable to be used as a trans-species selection marker, similarly to the S pombe URA4 gene

C Activity of S pombe BCAT

The manipulation of the yeast genetic system is further illustrated in S pombe BCAT system The results obtained with genetic assay were confirmed with biochemical assay for BCAT activity on total cell extracts from these strains is shown in Figure 5

Figure 5 shows BCAT activity on three branched-chain ammo acids measured in total cell extract Results shown are the average of three measurements with standard deviation Specific activity was calculated in units defined as micromoles of branched- chain keto acids produced x min^"1 x protein^'1 Figure 5A shows S cerevisiae cells eca39Δ 40Δ, lacking both cytosohc and mitochondnal BCAT, transformed with EC A39, ECA40 or a control vector As seen in Figure 5 A, the control cells did not show any BCAT activity, resulting in small or nonexistent levels of branched-chain keto acids When the control cells were transformed with ECA39 or ECA40, BCAT activity was restored

Figure 5B shows eca39Δ 40Δ S cerevisiae cells transformed with the S pombe EC A39 gene As seen in Figure 5B, the yeast strain S cerevisiae eca39Δ 40Δ transformed with S pombe ECA39 had restored BCAT activity with respect to all three branched-chain amino acids as substrates with leucine being the best substrate the activity of the S pombe protein in the current assay was fairly high, probably since the gene was introduced on a multicopy plasmid

The results shown in Figure 5 illustrate genetic manipulating of the yeast When the EC A39 genes or ECA40 encoding BCAT protein are deleted, the BCAT protein production is disrupted and so is production of branched-chain keto acids This process, however, may be reversed as seen in Figures 5 A and 5B

The catalytic activity was further confirmed by directly measuring BCAT activity in cytosohc and mitochondnal fractions from the different strains As shown in Figure 5B, the S pombe gene restored transamination activity using all three branched-chain amino acids as substrates with leucine being the best substrate The activity of the S pombe protein in the cunent assay was fairly high, probably since the gene was introduced on a multicopy plasmid S pombe ECA39/BCAT has a calculated lsoelectnc point pi of 9 0, suggesting that it is localized in the mitochondria, although it does not harbor the classical signal peptide which directs proteins to mitochondria

π. A Method For Reduction of Fusel Oils and related substances in Alcoholic Beverages

S cerevisiae eca39Δ, eca40Δ or S cerevisiae eca39Δ 40Δ yeast strains are suitable to be used and replace generally utilized S cerevisiae in fermentation processes for production of alcoholic beverages such as beer or wine or for use as baker's yeast for baking Analysis of alcohol content of the fermentation media used with these bio-engineered strains revealed a marked decrease in the content of high alcohols (fusel oils) contaminants, with no decrease in production of ethanol Results are seen in Table 1

Table 1 Analysis of Alcohols in Fermented Yeast Media

As seen in Table 1, wild type S cerevisiae or other unmodified yeast commonly used for fermentation produced significantly higher amount, 1 56 and 1 21 mg%, of isobutanol than S cerevisiae eca39Δ 40Δ, BCAT-deficient strains, which produced less than 0 5 mg% or nondetectable amount of isobutanol altogether

This experiment was repeated also with malt as a substrate for fermentation Accordingly, wild type S cerevisiae cells and mutant cells lacking both exa39 and exa40 were used for the preparation of beer by anaerobic fermentation of malt Samples were collected in the specified time points and were analyzed using gas chromatography Ethanol, isobutyl alcohol, diacetyl and 2,3-pentanedione levels are reported The results presented in Table 2 show that production of these compounds is abolished in the mutant strain Table 2 Analysis of Alcohols, Diacetyl and 2.3-Pentanedione in Beer

mu an

These results clearly show that the replacement of wild type S. cerevisiae or other yeast strain, typically used for brewing alcoholic beverages, with the yeast lacking gene(s) for BCAT, results in production of high quality alcoholic beverages without or with minimal amounts of high alcohol and other related contaminants.

As described above, new BCAT-deficient yeast strains S. cerevisiae eca39Δ, lacking mBCAT, eca40Δ, lacking cBCAT and eca39Δ.40Δ, lacking both BACTs, are useful in a method for producing alcoholic beverages having lower content of high alcohol and other related contaminants, and to enhance dough to rising in baking.

In addition, as shown above, the S. pombe ECA39/BCAT gene was expressed and was found to be functional in S. cerevisiae transformed therewith, and thus may be used for shuttle vectors designed to work in both species and generally for genetic manipulation of the wild type S. cerevisiae. The isolation of S. pombe ECA39 BCAT gene also opens the way to study the relations between ECA39, a Myc target homo log, and other cell cycle mutants in order to understand the connection between cell cycle regulation and branched-chain amino acid metabolism.

Examples

Example 1 Yeast Strains, Plasmids and Media

This example describes yeast strains, plasmids and media used for development of this invention

Yeast strain AE88 was used This strain-is lsogemc to YPH857 (MATα, ura-3-52, lys2,801, ade2-101, trplΔ63, hιs3Δ200, leu2Δl, cyh2R), with eca39Δ URA3, eca40Δ HIS3, as described in Genomics, 22 118 (1994) Construction of this strain is described in J Biol Chem , 271 20242 (1996) This strain is viable only in rich growth media supplemented with va ne, leucine and isoleucine as described in Yeast 14 189-194 (1998)

Plasmids used were pScECA39-TRPl and pScECA40-TRPl These are

CEN, ARSl, TRP1 plasmids contaming a genomic fragment from S cerevisiae including ECA39 or ECA40, respectively PspECA39 is an ARSl, URA4 plasmid carrying S pombe

EC A39 isolated from a genomic library as described below

Selective media were prepared according to Methods in Yeast Genetics, (1990) Cold

Spnng Harbor Laboratory Press The media used was either with or without valine If added, vahne was in final concentration of 40 μg/ml

Example 2

DNA Manipulation and Analysis

This example describes methods used for DNA manipulation and analysis

Isolation of the S pombe BCAT was performed by PCR according to Short Protocols n Molecular Biology, 2ed, John Wiley & Sons, New York (1992), using the following degenerative ohgonucleotides

5 TT/CGAAGGA/T/GT/ATGAAG/AGC3¹ SEQ ID NO 5

S'TTCATA/GGTA/GCCA/G/CACT/CTCAGTS' SEQ ID NO 6 DNA sequencing was performed automatically using the dideoxynucleotide chain termination method according to PNAS (USA), 74:5463 (1977). Computer analysis of protein and DNA sequences was performed using the GCG Wisconsin package as described in Nucleic Acid Res., 12:387 (1984) and Psort server [Genomics, 14:897 (1992)]. Transformation of yeast strains was performed with lithium acetate as described in J. Bacteriol., 153:163 (1983).

Example 3 Library Screening

This example describes screening of S. pombe genomic library.

The S. pombe genomic library, constructed by methods described in Gene, 114:59 (1992), is based on a pUC19 derived vector carrying the S. pombe ARSl and URA4. The library was screened according to standard techniques described in Short Protocols in Molecular Biology, supra, with the PCR product used as a probe. Radiolabelling of DNA probes was performed by random priming according to Anal. Biochem., 132:6 (1983) using [α-³²P]dCTP (3000 Ci/mM) obtained from Rotem Industries, Israel.

Example 4

BCAT Activity Assay

This example describes methods used for assaying of BCAT activity in cytosohc and mitochondrial extracts. For assaying BCAT activity, cytosohc and mitochondrial extracts were prepared and BCAT activity was assayed as previously described in J. Biol. Chem., 271:20242 (1998).

Preparation of Cytosohc and Mitochondrial Extracts

Cells were grown on YEPD to A_OOO of about 1.6, collected, and resuspended to 0.2 g/ml in 0.1M Tris-S0₄, pH 9.3, with lOmM dithiothreitol, incubated for 10 min at 30° C, collected again, washed in spheroplasting buffer (1.2M sorbitol, 20mM potassium phosphate, pH

7.4), and resuspended to 0.1 g/ml in spheroplasting buffer containing 7,000 units/ml of yeast lytic enzyme (ICN Immunobiologicals). Cells were incubated for 45 minutes in 30°C, and spheroplasts were collected and washed twice in spheroplasting buffer. Spheroplasts were resuspended in MLB buffer (0.6M manitol, 20mM HEPES, pH 7.4) to a final concentration of 0.5 g of cells/ml and broken in a Dounce homogenizer. The homogenate was centrifuged at 3,000 x g for 5 minutes at 4°C. The pellet contained the crude mitochondrial fraction. The supernatant from this centrifugation was further centrifuged at 200,000 x g for 20 minutes at 4°C, and the supernatant from this spin was frozen and used as the cytosohc extract. The crude mitochondrial fraction was washed three times in MLB buffer and sonicated, and the supernatant was frozen as the mitochondrial extract.

Branched-chain Amino Acid Aminotransferase Activity Assay

Cytosohc or mitochondrial extracts were incubated with 37mM sodium pyro- phosphate buffer, pH 9.2, 6.7mM α-ketoglutarate, 67μM pyridoxal phosphate, and 6.7mM branched-chain amino acid (either leucine, isoleucine, or valine). The reaction was terminated after 10 minutes by the addition of trichloroacetic acid. Samples were centrifuged and clear solution was transferred to a new tube and incubated at 25°C with 2,4-dinitrophenylhydrazine. The hydrazones of the branched-chain keto acids were first extracted with toluene and then with sodium carbonate. The amount of the hydrazone was determined by measuring the optical absorbance at 440 nm. An extinction coefficient of 1.4 A/μmol was found for the hydrazones of all three keto acids. Specific activity was calculated in units defined as micromoles of branched-chain keto acids produced x min^"1 x mg of protein^"1.

Claims

1 Use of yeast cells lacking or having reduced activity of one or both of the branched-chain amino acid aminotransferase (BCAT) isoenzymes in the production by fermentation of alcoholic beverages containing low concentrations of fusel oils

2 Use according to claim 1 wherein said yeast cells are cells of the Saccharomyces cerevisiae ecaΔ39, ecaΔ40 or ecaΔ39 Δ40 yeast strain

Use according to claim 1 or claim 2 wherein said yeasts reduce the concentration of iso-amyl alcohol and/or iso-butyl alcohol during fermentation

4 A method for producing an alcoholic beverage having substantially reduced content of fusel oils during yeast fermentation of alcoholic beverages by substituting a wild type S cerevisiae yeast used for fermentation of alcoholic beverages with a genetically engineered Saccharomyces cerevisiae eca39Δ, Saccharomyces cerevisiae eca40Δ, Saccharomyces cerevisiae eca39Δ, ecaΔ40 yeast strains lacking or having reduced activity of one or both of the two branched-chain amino acid aminotransferases

5 The method of claim 4 wherein said yeast is Saccharomyces cerevisiae eca39Δ 40Δ

6 Alcoholic beverages with low content of fusel oils produced by the method of any one of claims 4 or 5

7 A DNA sequence of Schizosaccharomyces pombe EC A39 gene identified as SEQ ID NO 1 encoding a protein identified by the amino acid sequence SEQ ED NO 2

8 A protein comprising amino acid sequence SEQ ED NO 2