METHOD OF PRODUCING YEAST MUTANTS AND THE USE THEREOF

Abstract

A method of producing yeast mutants and the use thereof. In order to provide yeasts which, at a given sugar content, produce a low ethanol content and a relatively high glycerol content in ethanolic fermentation and which are simultaneously obtainable rapidly, at least one yeast strain is contacted in a first mutagenesis step with a first mutagen and in a second mutagenesis step with a second mutagen. The first and second mutagens are different from one another and are selected from the following groups: nucleotide-alkylating agent, nucleotide-deaminating agent, and UV radiation. The method further includes a first selection step executed between the first and second mutagenesis steps and a second selection step being executed after the second mutagenesis step, in which the mutants that originate from the prior mutagenesis step in each case are exposed to a selection factor selected from the following groups: (a) hypertonic medium and (b) alcohol dehydrogenase inhibitor.

Claims

1. A method for producing yeast mutants, comprising: contacting at least one yeast strain a first mutagenesis step with a first mutagen and in a second mutagenesis step with a second mutagen, wherein the first and the second mutagen are different from each other and are selected from the group consisting of: nucleotide-alkylating agent, nucleotide-deaminating agent, and UV radiation; and performing a first selection step between the first and second mutagenesis step and a second selection step after the second mutagenesis step, in which the mutants resulting from the preceding mutagenesis step are exposed to a selection factor selected from the groups consisting of: (a) hypertonic medium and (b) alcohol-dehydrogenase inhibitor.

2. The method according to claim 1, wherein the nucleotide-alkylating agent is selected from the group consisting of: dimethylsulphate (DMS), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS), 1methyl-3-nitro-1-nitrosoguanidine (MNNG), methylnitrosocyanamide (MNC), methylnitrosourea (MNU), and DNA methyltransferases.

3. The method according to claim 1, wherein the nucleotide-deaminating agent is selected from the group consisting of: anorganic nitrite salt, organic nitrite salt, and nitrous acid.

4. The method according to claim 1, wherein the first mutagen is a nucleotide-alkylating agent or a nucleotide-deaminating agent.

5. The method according to claim 1, wherein the first mutagen is a nucleotide-alkylating agent and the second mutagen is a nucleotide-deaminating agent or UV radiation.

6. The method according to claim 1, wherein the hypertonic medium is obtained by addition of a substance selected from the group consisting of: chlorides and sulphates of sodium, potassium, magnesium, calcium and sugar, including fructose and glucose and sugar alcohol, including sorbitol and mannitol.

7. The method according to claim 1, wherein the alcohol dehydrogenase inhibitor is selected from the group consisting of: pyrazole, 3-methylpyrazole, 4-methylpyrazole, and acetyl salicylic acid.

8. The method according to claim 1, wherein one of the selection factors is selected from hypertonic medium and one of the selection factors from alcohol-dehydrogenase inhibitor.

9. The method according to claim 1, further comprising performing a test step after the first selection step, in which intermediate yeast mutants obtained after the first selection step are tested for whether they produce more glycerol during an ethanolic fermentation than the initially used yeast strain under the same conditions, whereby only such intermediate yeast mutants to which this applies are subjected to the second mutagenesis step and the second selection step.

10. A yeast mutant obtained by a method according to claim 1 and deposited at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH under accession number DSM 29822.

11. A yeast mutant obtained by a method according to claim 1, wherein during fermentation of must with 90 Oe (21.6% Brix) and a NOPA value=107 mg/l at a dosing of 4106/ml, after 35 days at a fermentation temperature of 15-25 C. the mutants produce a wine with the following proportions of ethanol and glycerol: TABLE-US-00006 Ethanol 70-150 g/l Glycerol 10-20 g/l

12. A method comprising producing an alcoholic beverage using the yeast mutant according to claim 10.

13. The method according to claim 12, wherein the alcoholic beverage is produced from grape must.

Description

[0042] Other advantages, features and potential applications of the present invention are clear from the following description of preferred embodiments and examples.

[0043] FIG. 1: Diagram of mutagenesis and selection steps

[0044] FIG. 2: Fermentation progress after a first mutagenesis and a first selection step

[0045] FIG. 3: Fermentation progress after a second mutagenesis and a second selection step

[0046] FIG. 4: DNA profile of the yeast mutant deposited under access number: DSM29822

EXAMPLES

[0047] Various mutation tests were performed to produce yeast mutants, which produce a low ethanol concentration and increased glycerol concentration in a wine or a medium at a specified initial glucose concentration.

[0048] Preliminary Tests

[0049] In the initial tests performed using ethidium bromide, which produces mutations through frameshifts, it was possible to show that in the process only a few viable yeasts could be produced, which are in particular compromised with regard to their respiratory chain and appear to show general changes to the mitochondrial DNA. Such yeasts are unsuitable for a fermentation.

[0050] It was further possible to show that a repeated use of the same mutagen leads to only a few or no yeast mutants at all being obtained after the second mutagenesis step and subsequent selection.

[0051] The mutagenesis schemes shown in FIG. 1 were performed, whereby ++ means that more than 100 yeast mutants with the desired properties were obtained, + means that between 1 and 99 yeast mutants with the desired properties were obtained, means that no yeast mutants with desired properties were obtained and 0 means that no yeast mutants at all were obtained. By desired properties it is understood here that the mutants produce more glycerol and less ethanol during an ethanolic fermentation than the aforementioned yeast strain used under the same conditions.

[0052] It was possible to determine during test fermentations that the production of glycerol generally takes place immediately after fermentation starts and customarily lasts up to 10 days. The majority of the glycerol is produced by fermentation of the first 100 g sugar per litre of medium, thereafter glycerol production slows, but in principle does not stop completely.

[0053] In contrast, it has been seen that the ethanolic fermentation which produces ethanol lasts up to three weeks, so that ethanolic fermentation and glycerol production slightly overlap during fermentation.

[0054] The different mutagenesis and/or selection steps and test methods are explained below in detail.

[0055] General Overview

[0056] The mutagenesis and selection steps were selected such that they are based on the production method for wine.

[0057] After the first mutagenesis and first selection 10,000 mutants were selected and tested for their glycerol production. Of the 10,000 mutants screened in this way, 400 were selected which have the highest glycerol concentration and tested again for their glycerol production.

[0058] Mutants which have a reproducible increased glycerol production were subjected to a small-scale wine fermentation and then organoleptically analysed. Ethanol determination was also carried out.

[0059] The mutants with the best organoleptic profile, the highest glycerol production, the lowest ethanol concentration and the best fermentation capacity were selected for the second mutagenesis step. In the process, the organoleptic profile was subjectively determined by tasting and evaluation of oxidative note, acidity, bitterness and overall impression. The fermentation capacity ensues from the fermentation speed, measured by a weight loss during fermentation and the fermentation's duration until the sugar present, in particular glucose, is consumed.

[0060] A mutant was then regarded as suitable when it had converted at least 70% of the sugar present after 35 days.

[0061] Mutagenesis Step With a Nucleotide-Alkylating Agent

[0062] Ethyl methanesulphonate (EMS) was used as the nucleotide-alkylating agent. A colony of a yeast strain was inoculated from agar plates into 5 ml YPD medium and cultured overnight at 28 C. The cells were then pelleted by centrifugation and washed twice with 100 mM potassium phosphate buffer with a pH value of 7. The cells were then resuspended in 10 ml of a 100 mM potassium phosphate buffer at pH value 7. 37.5 l of pure EMS was added to 500 l of the cell suspension. The suspension was incubated for an hour at 30 C. on a shaker. The reaction was stopped by the addition of 1 ml 5% sodium thiosulphate (% by weight). The yeast cells were then washed once with a 5% aqueous solution of sodium thiosulphate. After centrifugation the pellet was resuspended into 500 l YPD medium.

[0063] Mutagenesis Step With a Nucleotide-Deaminating Agent

[0064] Sodium nitrite was used as a nucleotide-deaminating agent. Colonies of yeast strains were inoculated from agar plates into 5 ml YPD medium and cultured overnight at 28 C. The cells were pelleted by centrifugation and washed twice with 2 ml water. After washing, the cells were resuspended in a mixture of 2 ml water, 2 ml of a 0.6 M sodium acetate buffer with a pH of 4.5 and 2 ml of a 55 ml sodium nitrite solution. The cell suspension was incubated on a shaker for 8 minutes at 30 C. After incubation, 1 ml of the suspension was mixed with 9 ml of a 0.67 M potassium phosphate buffer with a pH value of 7 to stop the reaction.

[0065] Mutagenesis Step With UV Radiation

[0066] Colonies of yeast strains were inoculated from agar plates into 5 ml YPD medium, which contained 0.3 M sodium chloride and cultured overnight at 28 C. The cells were pelleted by centrifugation and washed once in 10 ml KP medium without glucose and fructose and resuspended. The optical density (OD) was measured at 600 nm and the suspension was diluted to an optical density of 0.025 and transferred to a Petri dish. The cell suspension in the petri dish was then irradiated with UV radiation of 254 nm wavelength at an intensity of 2000 J/cm.sup.2 for 45 seconds in a Hoefer UVC 500 Crosslinker.

[0067] Selection Step With Alcohol Dehydrogenase Inhibitor

[0068] Pyrazole was used for selection with an alcohol dehydrogenase inhibitor.

[0069] For this selection step, the yeast cells obtained from a mutation step were spread on plates with KP medium which also contained 5 g/l pyrazole, whereby approximately 2,000 to 3,000 cells were transferred to a plate measuring 3030 cm. The plates were incubated for 10 days at 18 C. under microaerophilic conditions. Microaerophilic conditions designates that the gas mixture (atmosphere) surrounding the plates had only 2 to 10% by volume of oxygen instead of the 20% by volume of oxygen which is otherwise normal for air.

[0070] Selection Step With Hypertonic Medium

[0071] In this selection step, sodium chloride was used to cause osmotic stress. The yeast cells obtained from a mutagenesis step were transferred to plates with KP medium, which also contained 17.53 g/l sodium chloride, whereby approximately 2,000 to 3,000 cells were applied to a plate measuring 3030 cm. It is generally assumed that mutants which have a growth advantage on hypertonic media produce more glycerol. The plates were incubated for 10 days at 18 C. under microaerophilic conditions. Microaerophilic conditions designates that the gas mixture (atmosphere) surrounding the plates had only 2 to 10% by volume of oxygen instead of the 20% by volume of oxygen which is otherwise normal for air.

[0072] Media

[0073] The KP medium used, a medium which is also designated as artificial must, which was used, inter alia, for the selection steps, contained 115.5 g glucose monohydrate, 105 g fructose, 3 g tartaric acid, 0.3 g citric acid, 0.3 g malic acid, 0.3 g (NH.sub.4).sub.2SO.sub.4, 2 g KH.sub.2PO.sub.4, 0.2 g MgSO.sub.47H.sub.2O, 4 mg MnSO.sub.4H.sub.2O, 4 mg ZnSO.sub.47 H.sub.2O, 0.5 mg CuSO.sub.45 H.sub.2O, 0.5 mg Kl, 0.2 mg CoCl.sub.26 H.sub.2O, 0.5 mg (NH.sub.4).sub.6Mo.sub.7O.sub.24, 0.5 mg H.sub.3BO.sub.3, 300 mg myoinositol, 1 mg nicotinic acid, 1 mg calcium pantothenate, 1 mg pyridoxine hydrochloride, 0.04 mg biotin, 1 mg p-aminobenzoic acid, 247 mg L-glutamine, 183 mg L-arginine, 87.7 mg L-tryptophan, 71 mg L-alanine, 58.9 mg L-glutamic acid, 38.4 mg L-serine, 37.1 mg L-threonine, 23.7 mg L-leucine, 21.8 mg L-aspartic acid, 21.8 mg L-valine, 18.6 mg L-phenylalanine, 16 mg L-isoleucine, 16 mg L-histidine, 15.4 mg L-methionine, 9 mg L-tyrosine, 9 mg L-glycine, 8.3 mg L-lysine and 6.4 mg L-cysteine per litre.

[0074] The YPD medium contained 10 g yeast extract, 20 g peptone and 20 g glucose per litre and was set at a pH value of 5.5 to 6.0.

[0075] 15 g/l agar was added to the media for plates with the media described.

[0076] Investigation of Glycerol Production

[0077] Yeast colonies from an agar plate were inoculated into YPD medium to determine glycerol production. After three days' growth at 30 C., 1 ml KP medium was inoculated with 20 l of the culture grown in this way. There was no adjustment of the cell density. The KP cultures were then incubated at 18 C. under microaerophilic conditions. After ten days the excess culture was removed by centrifugation and filtration. The excess was then analysed with regard to the glycerol concentration. Determination of the glycerol concentration was carried out photometrically. A kit from R-Biopharm AG was used for the measurement, whereby the test was adjusted for measurement in a microtitration plate to a total sample volume of 155 l.

[0078] Fermentation Tests

[0079] A real must was used for the fermentation tests, not an artificial must, whereby a 2013 vintage Riesling with 70 Oe (17.1% Brix) enriched by addition of saccharose (52 g) to 90 Oe (21.6% Brix) with a NOPA value of 107 mg/l (+/5 mg/l) was used.

[0080] For the fermentation, whose results are shown in FIG. 2, a 2013 vintage Riesling must was used, which was enriched to 91 Oe (21.7% Brix) and which had a NOPA value of 124 mg/l (+/5 mg/l).

[0081] The media pH value was between 3.1 and 3.2. In every case, the fermentation temperature was 18 C. The fermentation trials were not stirred.

[0082] Determination of Glycerol, Ethanol, Glucose and Fructose Produced and Total Sugar/Organoleptic Analysis/Fermentation Capacity

[0083] Analysis of the test fermentations was carried out by HPLC with the following parameters:

[0084] Equipment: Ultimate 3000 ThermoScientific

[0085] Column REZEX ROA Organic Acid H+ Phenomenex

[0086] RI Detector & UV detector at 210 nm

[0087] Solvent mixture: 0.005 N H.sub.2SO.sub.4 (isocratic)

[0088] Temperature: 75 C.

[0089] Flow rate: 0.5 ml/min

[0090] The organoleptic profile, which also results from the wine's composition, was also subjectively determined by tasting and evaluation of the oxidative note, acidity, bitterness and overall impression.

[0091] The fermentation capacity ensues from the fermentation speed, measured by a weight loss during fermentation and the fermentation duration, until the sugar present, in particular glucose, is consumed.

Example 1

[0092] To determine the various effects of different mutagenic stimuli, tests were first carried out in which a Saccharomyces cerevisiae subsp. bayanus strain was exposed to UV radiation, EMS or sodium nitrite. In the case of the strain used in this example, it was the yeast strain available worldwide under the designation Oenoferm Freddo F3.

[0093] This was followed by selection either through a hypertonic sodium chloride medium or on a pyrazole medium.

[0094] FIG. 2 shows the results of investigation of some strains which resulted from the mutation with EMS or sodium nitrite. FIG. 2 shows in a graph the weight loss during a test fermentation over a total of 35 days, whereby the total weight of the must used was determined by weighing and the weight-loss data relates to the weight of the must with yeast originally used. The greater the weight loss, the better the respective strain's fermentative capacity.

[0095] Table 1 below shows the results of the HPLC analysis of this test fermentation after 35 days, whereby two independent fermentation trials were investigated for each mutant.

TABLE-US-00003 TABLE 1 Glucose Fructose Total sugar Ethanol Glycerol Sample g/l g/l g/l g/l g/l F EMS16 NaCl D7 2.53 14.83 17.36 100.2 8.97 F EMS16 NaCl D7 0.26 4.28 4.54 105.0 9.75 F EMS16 NaCl B7 0.29 4.62 4.91 105.7 6.57 F EMS16 NaCl B7 0.02 0.23 0.25 107.1 6.57 F Nitrite 8 NaCl H10 0.07 1.38 1.45 107.1 6.64 F Nitrite 8 NaCl H10 6.66 26.25 32.91 92.6 6.24 F Nitrite 8 NaCl A12 0.04 1.31 1.35 106.4 6.75 F Nitrite 8 NaCl A12 0.38 4.82 5.20 107.1 6.51 F Nitrite 6 Pyra B9 12.04 39.09 51.13 85.2 5.84 F Nitrite 6 Pyra B9 0.03 0.60 0.63 111.4 6.88 F Nitrite 7 Pyra F10 3.59 19.85 23.44 100.2 6.54 F Nitrite 7 Pyra F10 3.56 19.31 22.87 98.1 6.57 F EMS 3 Pyra A9 18.37 45.85 64.22 79.8 5.72 F EMS 3 Pyra A9 0.09 2.34 2.43 109.2 6.93 F EMS 4 Pyra D11 0.26 4.03 4.29 105.7 8.06 F EMS 4 Pyra D11 0.00 0.31 0.31 108.5 8.14 F EMS 3 Pyra E10 0.00 0.77 0.77 109.2 7.09 F EMS 3 Pyra E10 0.00 2.55 2.55 110.7 7.01 Reference 0.00 0.21 0.21 112.1 7.17 Reference 0.00 0.19 0.19 109.9 7.25

[0096] In FIG. 2 and Table 1 the yeasts which were first subjected to a mutagenesis step with EMS and then a selection with NaCl are designated F EMS 16 NaCl E7 and F EMS 16 NaCl B7. Strains which were first exposed to a mutagenesis step with nitrite and a selection with NaCl are designated F Nitrite 8 NaCl H10 and F Nitrite 8 NaCl A12, strains which were first subjected to a mutagenesis step with nitrite and then a selection with pyrazole are designated F Nitrite 6 Pyra B9 and F Nitrite 7 Pyra F10 and strains which were first subjected to a mutagenesis step with EMS and then a selection with pyrazole are designated F EMS 3 Pyra A9, F EMS 4 Pyra D11 and F EMS 3 Pyra E10. The designation Reference refers to the Saccharomyces cerevisiae subsp. bayanus strain used as a comparison yeast, which was also used for the aforementioned first mutagenesis step.

[0097] It can be seen that as a result of this first mutagenesis step and subsequent selection step mutants are obtained which both produce less and more glycerol than the comparison yeast under the same conditions. Basically, it must be taken into consideration, though, that a part of the intermediate mutants had not converted all the sugar after 35 days, so that the glycerol values in these tests are comparable only to limited extent.

Example 2

[0098] In this specimen test the F EMS 16 NaCl D7 strain obtained from Example 1, which showed a particularly high glycerol concentration in the test fermentation, was selected for a second mutagenesis step and selection step. EMS, sodium nitrite and UV radiation were also used for the second mutagenesis step. A subsequent selection in each case was done with sodium chloride or pyrazole. It was seen that in the case of the strain previously mutated with EMS, no viable mutants were obtained in a further mutation with EMS.

[0099] The results of a fermentation test with different mutants obtained after the second mutagenesis step and the second selection are shown as an example in FIG. 3.

[0100] F EMS 16 NaCl D7 and F EMS 4 Pyra D11 designate intermediate mutants. In this example, they represent comparison values.

[0101] Nitrite Pyra 12 B8, Nitrite Pyra 12 H3 and Nitrite Pyra 25 Al designate strains for which the second mutagenesis step was done with sodium nitrite and the second selection step with pyrazole. UV Pyra 17 A8 designates a strain for which the second mutagenesis step was done with UV radiation and the second selection with pyrazole.

[0102] Table 2 shows the results of the HPLC investigations of these strains. Here too it can be seen that some strains were obtained which produce much more glycerol than the intermediate mutants obtained in Example 1 and some strains were obtained which produce less glycerol. In total only a few mutants were produced which produce much less ethanol than the aforementioned intermediate mutants used at the start of the second mutagenesis step. The Nitrite Pyra 12 H3 strain is characterised by neither glucose nor fructose being fully converted, which indicates an incomplete fermentation.

[0103] The data shown in Table 2 were obtained after 35 days of test fermentation, whereby two fermentation trials for each mutant were performed and evaluated independently of each other.

TABLE-US-00004 TABLE 2 Glucose Fructose Total sugar Ethanol Glycerol Sample g/l g/l g/l g/l g/l F EMS 16 NaCl D7 0.03 1.25 1.28 102.9 8.23 F EMS 16 NaCl D7 0.01 0.24 0.25 101.6 8.48 F EMS 4 Pyra D11 0.06 1.49 1.55 105.0 6.65 F EMS 4 Pyra D11 0.95 8.02 8.97 100.9 6.38 Nitrite Pyra 12 B8 0.18 3.58 3.76 98.8 11.31 Nitrite Pyra 12 B8 0.42 5.55 5.97 96.7 11.08 Nitrite Pyra 12 H3 21.02 51.82 72.84 85.8 10.55 Nitrite Pyra 12 H3 10.90 34.44 45.34 77.2 10.86 Nitrite Pyra 25 A1 0.70 4.77 5.47 101.6 8.73 Nitrite Pyra 25 A1 0.76 4.85 5.61 100.9 8.74 UV Pyra 17 H8 0.02 0.72 0.74 103.6 8.52 UV Pyra 17 H8 0.15 3.20 3.35 102.2 8.42

[0104] In conclusion, it was seen in the tests that after 35 days the mutant Nitrite Pyra 12 B8 provides almost complete fermentation and in both fermentation trials produced more than 11 g/l glycerol. This mutant corresponds to the strain deposited with the Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH under accession number DSM 29822.

TABLE-US-00005 Printout (original in electronic format) (This sheet does not count as a sheet in the International Application and is not part thereof) PCT 0-1 Form PCT/RO/134 Indications Relating to Deposited Microor- ganism or Other Biological Material 0-1-1 Prepared with PCT Online Filing Version 3.5.000.244e MT/FOP 20141031/0.20.5.20 0-2 International application No. PCT/EP2016/052437 0-3 Applicant's or agent's file reference 140557WO-ERS 1 The indications made below relate to the microorganism and/or other biological material referred to in the description on 1-1 Page 6 1-2 Line 19-24 1-3 Identification of deposit 1-3-1 Name of depositary institution DSMZ Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) 1-3-2 Address of depositary institution Inhoffenstr. 15,38124 Braunschweig, Germany 1-3-3 Date of deposit Dec. 16, 2014 (16.12.2014) 1-3-4 Accession number DSMZ 29822 1-4 Additional indications The expert solution is applied for in accordance with Rule 13bis. 6 PCT for all countries/bureaux for which this is applicable. 1-5 Designated states for which indications are All designated states made FOR RECEIVING OFFICE USE ONLY 0-4 This sheet was received with the Interna- Yes tional Application (iYes or No) 0-4-1 Authorized Officer Van Dooren, Luc FOR INTERNATIONAL BUREAU USE ONLY 0-5 This sheet was received by the International Bureau on 0-5-1 Authorized officer