CN100358998C

CN100358998C - Beer yeast engineering strain and its construction method

Info

Publication number: CN100358998C
Application number: CNB2005100777815A
Authority: CN
Inventors: 张博润; 张吉娜; 何秀萍; 郭雪娜; 傅秀辉
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2005-06-24
Filing date: 2005-06-24
Publication date: 2008-01-02
Anticipated expiration: 2025-06-24
Also published as: CN1699552A

Abstract

The present invention discloses a beer yeast engineering strain and a construction method thereof. The engineering stain is prepared by that the coding gene of gamma-glutacid aminothiopropionic acid synthetase is led into saccharomyces cerevisiae YSF-31 to obtain a strain of which the homoglutathion content is lower than the content of the diacetyl. The 5' end of the coding gene of the gamma-glutacid aminothiopropionic acid synthetase is connected with a 70-705 bp nucleotide fragment of a 5' end sequence selected from an ILV2 gene, and the 3' end of the coding gene of the gamma-glutacid aminothiopropionic acid synthetase is connected with a 70-739 bp nucleotide fragment of a 3' end sequence selected from the ILV2 gene; the 5' end sequence of the ILV2 gene is a nucleotide sequence from a 1-705 basic group at the 5' end with the GenBank number of X02549, and the 3' end sequence of the ILV2 gene is a nucleotide sequence from a 2983-3522 basic group at the 5' end with the GenBank number of X02549. As the yield of diacetyl is reduced, the fermentation period of the engineering strain is shortened, and the freshness preservation time of produced beer is prolonged obviously. As the formation of the glutathione is increased, the beer flavour is improved, and the ageing resistance of beer is enhanced.

Description

Beer yeast engineering bacteria and construction process thereof

Technical field

The present invention relates to a kind of beer yeast engineering bacteria and construction process thereof in the microbial fermentation industrial technology field.

Background technology

The flavor stability of beer is an important quality index of beer.Because the existence of hydrogen sulfide influence the local flavor of beer, therefore, thereby beer needs storage wine time of growing to reach the maturation of the local flavor of beer to remove these materials.In the metabolism stream of cereuisiae fermentum, hydrogen sulfide is the upstream metabolite of gsh, can reduce the formation of hydrogen sulfide by the activity that strengthens gamma-glutamic acid cysteine synthase system, increases the formation of gsh, to improve the local flavor of beer, strengthen the resistance of aging of beer.

Two hexanoyls are another important flavour substancess in the beer, but its taste thresholding very low (0.1mg/L).When the content of two hexanoyls meets or exceeds the taste thresholding, can produce a kind of sour meal flavor, thereby destroy the local flavor of beer.Two hexanoyls are that the intermediate product α-O-Decanoyllactic acid of synthetic Xie Ansuan of cereuisiae fermentum and leucic pathways metabolism forms through non-enzymatic pathway enzymatic oxidation.And ILV2 genes encoding hexanoyl hydroxy acid synthetic enzyme can be converted into O-Decanoyllactic acid to pyruvic acid.

Summary of the invention

First purpose of the present invention provides the beer yeast engineering bacteria of the low diacetyl content of a kind of homoglutathion content.

Beer yeast engineering bacteria provided by the present invention, be the encoding gene of gamma-glutamic acid cysteine synthase to be imported yeast saccharomyces cerevisiae YSF-31 (purchase in Chinese common micro-organisms DSMZ, original number: CGMCC 2.420) in, the bacterial strain of the low diacetyl content of the homoglutathion content that obtains; 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 5 ' terminal sequence that is selected from the ILV2 gene of 70-705bp, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 3 ' terminal sequence that is selected from the ILV2 gene of 70-539bp; 5 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 1-705 bit base, and 3 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 2983-3522 bit base.

The GenBank of the amino acid residue sequence of described gamma-glutamic acid cysteine synthase number is CAA59393; The GenBank of the nucleotide sequence of described ILV2 gene number is X02549; GenBank number of the nucleotide sequence of described gamma-glutamic acid cysteine synthase encoding gene is D90220.

5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number 263bp nucleotide fragments for 5 of X02549 ' end 443-705 bit base, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number nucleotide fragments for the 379bp of 5 of X02549 ' end 2983-3361 bit base.

For the ease of screening, in the encoding gene that imports described gamma-glutamic acid cysteine synthase, also copper resistant gene C UP1 is together imported yeast saccharomyces cerevisiae YSF-31; GenBank number of the nucleotide sequence of described copper resistant gene C UP1 is K02204.

The encoding gene of described gamma-glutamic acid cysteine synthase and copper resistant gene C UP1 are by using KpnI and PstI to import yeast saccharomyces cerevisiae YSF-31 from the dna fragmentation of the 6.0kb of recombinant plasmid pICG cutting-out; The physical map of described recombinant plasmid pICG such as Fig. 7.

Described beer yeast engineering bacteria is preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2 CGMCC № 1377.

Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2 CGMCC № 1377 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 05 23rd, 2005, preserving number is CGMCC № 1377.

The cell of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2 CGMCC № 1377 is oval, and colony morphology characteristic is bacterium colony projection, smooth, oyster white, neat in edge.30 ℃ of optimum growth temperatures.The growth optimal pH is 5.5.Its fermention medium and culture condition and industrial saccharomyces cerevisiae YSF-31 (purchasing in Chinese common micro-organisms DSMZ, original number: CGMCC 2.420) are identical.

Second purpose of the present invention provides a kind of method that makes up above-mentioned beer yeast engineering bacteria.

The method of the above-mentioned beer yeast engineering bacteria of structure provided by the present invention, be the encoding gene of gamma-glutamic acid cysteine synthase to be imported yeast saccharomyces cerevisiae YSF-31 (purchase in Chinese common micro-organisms DSMZ, original number: CGMCC 2.420) in, the bacterial strain of the low diacetyl content of the homoglutathion content that obtains is beer yeast engineering bacteria; 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 5 ' terminal sequence that is selected from the ILV2 gene of 70-705bp, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 3 ' terminal sequence that is selected from the ILV2 gene of 70-539bp; 5 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 1-705 bit base, and 3 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 2983-3522 bit base.

5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number 263bp nucleotide fragments for 5 of X02549 ' end 443-705 bit base, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number nucleotide fragments for the 539bp of 3 of X02549 ' end 2983-3522 bit base.

For the ease of screening, when importing the encoding gene of described gamma-glutamic acid cysteine synthase, also copper resistant gene C UP1 is together imported yeast saccharomyces cerevisiae YSF-31 (purchasing) in Chinese common micro-organisms DSMZ, original number: CGMCC 2.420; GenBank number of the nucleotide sequence of described copper resistant gene C UP1 is K02204.

The encoding gene of described gamma-glutamic acid cysteine synthase and copper resistant gene C UP1 are that the dna fragmentation by the 6.0kb that downcuts from recombinant plasmid pICG with KpnI and PstI imports yeast saccharomyces cerevisiae YSF-31 (purchasing in Chinese common micro-organisms DSMZ, original number: CGMCC 2.420); The physical map of described recombinant plasmid pICG such as Fig. 7.

Whether the gene constructed engineering bacteria that derives from non-use industrial microorganism is suitable for drinking, can also to need to be accepted facts have proved for a long time by the human consumer, again because cereuisiae fermentum itself just contains the gamma-glutamic acid cysteine synthase gene, so the present invention adopts gene from clone's mode the gamma-glutamic acid cysteine synthase gene to be inserted in the ILV2 gene, it is destroyed and the gamma-glutamic acid cysteine synthase gene has increased the engineering bacteria of a copy to have made up the ILV2 gene.The present invention combines traditional breeding technique with the molecular breeding technology, at first according to the physio-biochemical characteristics of different industrial cereuisiae fermentum bacterial classifications, by the copper resistant screening, obtain containing 3.0mmol/lCuSO ₄The YEPD flat board on the industrial saccharomyces cerevisiae YSF-31 that can not grow as recipient bacterium.Adopt then from clone technology, cut the dna fragmentation that recombinant plasmid pICG obtains a 6.0kb size with KpnI and PstI enzyme.This fragment contains and derives from saccharomycetic copper resistant gene and gamma-glutamic acid cysteine synthase gene, and 5 of recipient bacterium ILV2 gene ' end and 3 ' terminal sequence contained at two ends.Transform industrial saccharomyces cerevisiae YSF-31 with this dna fragmentation, homologous recombination takes place in the ILV2 on this fragment and the YSF-31 genome, and copper resistant gene and gamma-glutamic acid cysteine synthase gene are integrated on the karyomit(e) as a result.Like this, in restructuring yeast strains, the ILV2 gene is destroyed, and while copper resistant gene and gamma-glutamic acid cysteine synthase gene have all increased a copy.Containing 4mmol/l CuSO ₄The YEPD flat board on screen transformant.Test, reach the lab scale experiment and the pilot scale fermentation of simulating the beer fermentation small test, carrying out in brew-house by genetic stability and test, acquisition homoglutathion content hangs down the industrial beer yeast engineering bacteria YSF31 (pICG)-2 (CGMCC № 1377) of diacetyl content.Compare its physiology, biochemical characteristic no significant difference with recipient bacterium; The local flavor of the beer that lab scale and pilot scale fermentation experiment are produced is indifference also; Fermentation equipment and condition are not had particular requirement, and the equipment and the condition of general brew-house all can be used.Beer yeast engineering bacteria of the present invention shortens fermentation period because of the di-acetyl generation reduces, and the beer refreshing time of production rises appreciably; Because of increasing the formation of gsh, improved the local flavor of beer, strengthened the resistance of aging of beer.

Description of drawings

Fig. 1 is the electrophorogram of the PCR product of CUP1 gene

Fig. 2 is the building process synoptic diagram of recombinant plasmid pMCUP and pYCUP

Fig. 3 cuts the checking collection of illustrative plates for the enzyme of plasmid pMCUP

Fig. 4 is the auxotroph screening of transformant YS58 (pYCUP)

Fig. 5 is the copper resistant screening of transformant YS58 (pYCUP)

Fig. 6 is the building process synoptic diagram of plasmid pICG

Fig. 7 cuts the checking collection of illustrative plates for the enzyme of plasmid pICG

Fig. 8 is that copper resistant gene and gamma-glutamic acid cysteine synthase gene are incorporated into the synoptic diagram on the YSF-31 genome

Fig. 9 is for carrying out the pcr analysis electrophoretogram of acceptor and transformant with primer I LV2-1 and CUP1-P2

Figure 10 is recipient bacterium YSF-31 and the glutathione content of engineering bacteria YSF31 (pICG)-2 and the comparison of diacetyl content in the shake flask fermentation experiment

Figure 11 is that the diacetyl content of recipient bacterium YSF-31 and engineering bacteria YSF31 (pICG)-2 in the hectolitre fermenting experiment detects

Figure 12 is the pol detected result of recipient bacterium YSF-31 and engineering bacteria YSF31 (pICG)-2 in the hectolitre fermenting experiment

Figure 13 is recipient bacterium YSF-31 and the glutathione content of engineering bacteria YSF31 (pICG)-2 and the comparison of diacetyl content in two tons of fermenting experiments

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Embodiment 1, structure beer yeast engineering bacteria-yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2 CGMCC № 1377.

One, the structure that contains the expression vector pICG of the encoding gene of glutamyl-cysteine synthetase and copper resistant gene C UP1

1, the copper resistant of industrial saccharomyces cerevisiae is measured

Industrial saccharomyces cerevisiae is generally on the low side to the resistance of copper, therefore, can effectively screen yeast transformant according to the raising of copper resistant, and copper resistant generally is used as the selective marker of industrial cereuisiae fermentum.Measure by copper resistant, most of industrial beer bacterium is containing 2.5mmol/l CuSO ₄The YEPD flat board on can not grow, mensuration screens YSF-31, YSF-35 and YSF-41 and (purchases in Chinese common micro-organisms DSMZ, original number is respectively: CGMCC 2.420, CGMCC 2.412, CGMCC 2.241) three strain yeast are containing on the YEPD flat board of 3.0mmol/lCuSO4 and can not grow, and determine the recipient bacterium as next step transformation experiment with YSF-31.

2, the pcr amplification of copper resistant gene C UP1

PCR primer according to gene order (GenBank of CUP1 gene order number is K02204) the design CUP1 gene clone of the CUP1 of reported in literature:

P1：5′-CCA AGATCTCGCTATACGTGCATATGTTC-3′

P2：5′-CGA GTCGACATCTGTTGTACTATCCGCTT-3′

The line part is respectively BglII and SalI restriction enzyme site, the PCR reaction is a primer with P1, P2, genomic dna with YSF-31 is a template, in 50 μ l PCR reaction mixtures, contain 25 μ l Taq enzyme Mix, 12.5 each 2 μ L of μ mol/l primer, template 10 μ L add sterilized water 11 μ l and are adjusted to 50 μ L.Condition is 94 ℃, sex change 5min, loop parameter: 94 ℃ of sex change 40s; 56 ℃ of annealing 1min; 72 ℃ are extended 2min; 30 circulations; 72 ℃ are extended 15min product are extended fully.The PCR product that obtains is carried out 0.8% agarose gel electrophoresis, and electrophoresis result obtains the specific band of a treaty 1.1kb as shown in Figure 1, and its size and expection are quite.Among Fig. 1, swimming lane M is that (8576,7427,6106,4899,3639,2799,1953,1882,1515,1412,1164,992,718,710,492,359bp), swimming lane 1 is the PCR product of CUP1 to dna molecular amount standard sppI DNA/EcoRI.Reclaim the dna fragmentation of this about 1.1kb, be cloned into the pGEM-T carrier, check order, the result shows that the CUP1 that obtains of amplification has the nucleotide sequence from 5 ' end 1098-2189 bit base of GenBank Access No.K02204.

3, the structure of recombinant plasmid pYCUP

The PCR fragment reclaims through low melting point glue and directly is connected with the plasmid pBluescriptM13 that cuts through the HincII enzyme (precious biotechnology (Dalian) company limited), constitutes plasmid pMCUP.Obtain the CUP1 fragment with KpnI and SalI digested plasmid pMCUP, then with linear plasmid YEp352 (the Hill JE that cuts with KpnI and SalI enzyme, Meyers AM, Koerner TJ, Tzagoloff A.Yeast/E.coli shuttle vectors with multiple unique restriction sites.Yeast, 1993,9:163-167.) connect, obtain plasmid pYCUP (Fig. 2).And utilize EcoRI, SacI, KpnI and XbaI carry out single endonuclease digestion checking pMCUP, the result as shown in Figure 3, pMCUP obtains the dna fragmentation (swimming lane 2) of 4060bp after the EcoRI enzyme is cut; PMCUP obtains the dna fragmentation (swimming lane 3) of 4060bp after the SacI enzyme is cut; PMCUP obtains the dna fragmentation (swimming lane 4) of 4060bp after the KpnI enzyme is cut; PMCUP obtains the dna fragmentation (swimming lane 5) of 3200bp fragment and 660bp behind XbaI enzyme cutting.Enzyme is cut the checking result and is shown that the structure of pMCUP is correct.Among Fig. 3,1 is dna molecular amount standard sppI DNA/EcoRI (8576,7427,6106,4899,3639,2799,1953,1882,1515,1412,1164,992,718,710).

With shuttle plasmid pYCUP by complete yeast conversion method (Adams A.Gottschling DE, Kaiser CA, Stearns T.Methods in Yeast Genetics:a cold spring harbor laboratory course manual.New York:Cold Spring Harbor; 1997.Cold transformation experiment chamber yeast strain YS58 (Teunissen ARH Spring Harbor Laboratory), Holub E, Hucht JVD.Physical localization of theflocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae.Yeast, 1993,9:1-10), during conversion with containing 20mg/L Leu, the YNB substratum of Ura and Trp screens, because of containing the URA3 gene in the plasmid, so the transformant YS58 (pYCUP) that contains plasmid pYCUP also can grow not containing on the YNB substratum of Ura, and YS58 can not grow (Fig. 4).Among Fig. 4, five bacterium colonies of first row in two flat boards are recipient bacterium, and five bacterium colonies of all the other each row are transformant YS58 (pYCUP).Substratum in the flat board of left side is the YNB substratum that contains 20mg/L Leu and 20mg/L Trp, and the right side is dull and stereotyped for containing 20mg/LLeu, the YNB substratum of 20mg/L Ura and 20mg/L Trp.

Obtain transformant YS58 (pYCUP) back with containing different Cu ²⁺The YEPD substratum checking of concentration.The result is containing 3mM Cu ²⁺Plate in YS58 and transformant YS58 (pYCUP) can both grow, and at 5-9mM Cu ²⁺Plate in have only transformant YS58 (pYCUP) to grow, YS58 can not grow (Fig. 5).Among Fig. 5, five bacterium colonies of first row in four flat boards are recipient bacterium, and five bacterium colonies of all the other each row are transformant YS58 (pYCUP).Experimental result shows that the CUP1 gene has the copper resistant function, can be used as selection markers.

4, the structure of recombinant plasmid pICG

Obtain the CUP1 fragment with BglII and SalI digested plasmid pYCUP, with SalI and XbaI enzyme cutting plasmid pGF-2 (Fan X, He X, Guo X, Qu N, Wang Ch, Zhang B.Increasing glutathioneformation by functional expression of the γ-glutamylcysteine synthetasegene in Saccharomyces cerevisiae.Biotechnol.Lett.2004,26:415-417) obtain fragment GSH1, with BglII and XbaI enzyme cutting plasmid pLZ-2 (Li Yan, the iron kingfisher is beautiful, Wang Zhengxiang, Zhang Borun, Zhu Gejian.The structure of low di-acetyl saccharomyces cerevisiae.Wine brewing, 2002,29:77-79), three fragments are connected, constitute plasmid pICG (Fig. 6).And utilize SalI, BamHI, EcoRI, BglII or SacI single endonuclease digestion, SalI and SacI double digestion checking pICG, the result as shown in Figure 7, pICG obtains 7000bp fragment and 4300bp fragment (swimming lane 2) after the SalI enzyme is cut; PICG obtains the 6100bp fragment after the BamHI enzyme is cut, 3600bp fragment and 1600bp fragment (swimming lane 3); PICG obtains the 6200bp fragment after the EcoRI enzyme is cut, 2900bp fragment and 2200bp fragment (swimming lane 4); PICG obtains 9820bp fragment and 990bp fragment and 490bp fragment (swimming lane 5) after the BglII enzyme is cut; PICG obtains 5700bp and 5600bp fragment (swimming lane 6) after the SacI enzyme is cut; PICG obtains the 5200bp fragment behind SalI and SacI double digestion, 4600bp fragment and 1500bp fragment (swimming lane 7).Enzyme is cut the checking result and is shown that the structure of pICG is correct.Among Fig. 7,1 is dna molecular amount standard sppI DNA/EcoRI (8576,7427,6106,4899,3639,2799,1953,1882,1515,1412,1164,992,718,710).

Two, the structure of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2 CGMCC № 1377

1, the structure of engineering bacteria YSF31 (pICG)-2

Cut the dna fragmentation that recombinant plasmid pICG obtains a 6.0kb size with KpnI and PstI enzyme.This fragment contains copper resistant gene and gamma-glutamic acid cysteine synthase gene, 5 of gamma-glutamic acid cysteine synthase gene ' end is connected with from the GenBank number 263bp nucleotide fragments for 5 of X02549 ' end 443-705 bit base, and 3 of the encoding gene of gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number nucleotide fragments for the 379bp of 5 of X02549 ' end 2983-3361 bit base.Transform (Adams A.Gottschling DE with this dna fragmentation, Kaiser CA, Stearns T.Methods in Yeast Genetics:a cold spring harbor laboratory course manual.New York:Cold Spring Harbor; 1997.Cold industrial saccharomyces cerevisiae YSF-31 Spring Harbor Laboratory) screens transformant containing on the YEPD culture medium flat plate of copper sulfate, obtains the transformant YSF31 (pICG)-2 that the copper sulfate resistance obviously improves.Homologous recombination takes place in the ILV2 on this 6.0kb fragment and the YSF-31 genome, the result, and copper resistant gene and gsh gene are integrated on the karyomit(e).Like this, in restructuring yeast strains, the ILV2 gene is destroyed, and while copper resistant gene and glutamyl-cysteine synthetase gene have all increased a copy (Fig. 8).

Primer P2 with primer I LV2-1 (5 '-GCAGGATCCTGGCTTCAGTTGCTGTCT-3 ') and CUP1, genomic dna with engineering bacteria YSF31 (pICG)-2 is a template, carry out the pcr amplification checking, the result generates without any fragment in recipient bacterium YSF-31, is the fragment of 1.3kb (Fig. 9) and amplified size in the engineering bacteria YSF31 (pICG)-2 that selects.Among Fig. 9,1 is 1kb ladder marker; 2-4: the PCR product of transformant YSF31 (pICG)-2; 5: the PCR product of recipient bacterium YSF-31; 6: positive control pICG is the PCR product of template.

2, gamma-glutamic acid cysteine synthase (AHAS) enzyme activity determination of engineering bacteria YSF31 (pICG)-2

Bacterial strain is connected in the YEPD substratum, cultivated 36 hours at 28 ℃, centrifugal collecting cell, measure AHAS enzyme (the Magee PT alive of engineering bacteria YSF31 (pICG)-2 and recipient bacterium YSF-31 according to the penetrating assay method of cell, Robichon-Szulmajster DH.The regulation of isoleucine-valine biosynthesisin Saccharomyces cerevisiae.2.Identification and characterization ofmutants lacking acteohydroxyacid synthase.Eur.J.Biochem.1968,3:502-506).Measurement result shows that the activity of the enzyme of AHAS is 50% (table 1) of recipient bacterium in engineering bacteria YSF31 (pICG)-2, proves in engineering bacteria YSF31 (pICG)-2 that the ILV2 gene is destroyed to fall an allelotrope.

The AHAS enzyme of table 1 transformant YSF31 (pICG)-2 and recipient bacterium YSF-31 is lived relatively

Bacterial strain	AHAS enzyme (Umg alive ^-1Albumen)
Bacterial strain	AHAS enzyme (Umg alive ^-1Albumen)	Recipient bacterium YSF-31 engineering bacteria YSF31 (pICG)-2	4.18 2.33

3, shake flask fermentation experimental result

Recipient bacterium YSF-31 and engineering bacteria YSF31 (pICG)-2 are inserted the 5ml malt extract medium respectively, cultivated 12 hours for 25 ℃, the inoculum size with 10% is inoculated in the 10ml malt extract medium, cultivates 36 hours for 25 ℃, all be connected in the triangular flask that contains the 270ml wort, 9 ℃ leave standstill cultivation 10 days.Sampling in per two days, get the 10ml nutrient solution with aseptic transfer pipet at every turn, with two-layer 3MM filter paper filtering, detect glutathion inside cell content (Fan X respectively, He X, Guo X, Qu N, Wang Ch, Zhang B.Increasing glutathioneformation by functional expression of the γ-glutamylcysteine synthetasegene in Saccharomyces cerevisiae.Biotechnol.Lett.2004,26:415-417) and filtrate in diacetyl content (Guan Dunyi.The brewing industry handbook, middle volume, p234, light industry press).The result shows the content of gsh of engineering bacteria than the height of recipient bacterium, and in fermented liquid, the diacetyl content of engineering bacteria is lower than the recipient bacterium, and particularly the peak value of engineering bacteria is 75% of YSF-31.This proves absolutely in engineering bacteria that the GSH1 gene has obtained high expression level and the ILV2 gene is destroyed falls a copy (Figure 10).Among Figure 10, the GSH content (■) of YSF-31; The GSH content () of engineering bacteria YSF31 (pICG)-2; The diacetyl content (▲) of YSF-31; The diacetyl content (△) of engineering bacteria YSF31 (pICG)-2.

4, lab scale (hectolitre) fermenting experiment

Yeast is inserted the 5mL malt extract medium, cultivated 12 hours for 25 ℃, inoculum size with 10% is inoculated in the 10mL malt extract medium, cultivated 36 hours for 25 ℃, nutrient solution all is connected in the 270mL wort, cultivates 36 hours, and then nutrient solution is inserted the big triangular flask of 2000ml for 25 ℃, cultivated 36 hours, and be connected in the 100L fermentor tank (the 80L malt extract medium is housed) for 20 ℃.Inspire back 10 ℃ of cultivations, when pol drops to 2.6, cool to 0 ℃ after the storage.

(1) glutathione content detects

With the centrifugal collection thalline of fermented liquid, survey the GSH content in the cell then, the result shows that the GSH content in transformant YSF31 (pICG)-2 cell is 1.38 times of acceptor YSF31 cell, illustrates that the GSH1 gene has obtained high expression level (table 2) in transformant YSF31 (pICG)-2 cell.

The comparison of the glutathione content of table 2 recipient bacterium and engineering bacteria

Bacterial strain	GSH content (mg/g stem cell)
Bacterial strain	GSH content (mg/g stem cell)	Recipient bacterium YSF-31	1.5032
Engineering bacteria YSF31 (pICG)-2	2.0704	Recipient bacterium YSF-31	1.5032

(2) diacetyl content detects

With the fermented liquid filter paper filtering, survey the diacetyl content in the filtrate then, in the hectolitre fermenting experiment, the fermenting speed of engineering bacteria YSF31 (pICG)-2 is more faster than the recipient bacterium, engineering bacteria YSF31 (pICG)-2 entered the back storage phase since the 10th day, recipient bacterium YSF-31 entered the back storage phase since the 14th day, engineering bacteria entered the back storage phase in 4 days in advance.Low (Figure 11) of the peakedness ratio acceptor of the diacetyl content of engineering bacteria.Among Figure 11, the diacetyl content (▲) of recipient bacterium YSF-31; The diacetyl content of engineering bacteria YSF31 (pICG)-2 detects (△).

(3) pol detects

From fermentor tank, get the 500ml fermented liquid,, detect the pol of fermented liquid with saccharometer by toppling over the eliminating carbonic acid gas repeatedly.The pol test experience shows that the sugar-fermenting speed of engineering bacteria YSF31 (pICG)-2 is faster than recipient bacterium YSF-31's, reaches 2.5 (Figure 12) in advance in six days.Among Figure 12, the pol of recipient bacterium YSF-31 detects (▲); The pol of engineering bacteria YSF31 (pICG)-2 detects (△).

(4) detection of other indexs after the fermentation ends

The hectolitre experiment detects pH, flocculence, α-N assimilation ratio and fermentation degree in back storage phase (the back storage phase of engineering bacteria YSF31 (pICG)-2 and recipient bacterium YSF-31 is 14 days) sampling, the result shows pH, flocculence, α-N assimilation ratio and fermentation degree, recipient bacterium YSF-31 and engineering bacteria YSF31 (pICG)-2 are basic identical, illustrate that the integration on the karyomit(e) does not change other characteristics (table 3) of zymic.

The detection of other fermentation indexs of table 3

Test item	Recipient bacterium YSF-31	Engineering bacteria YSF31 (pICG)-2T2
Test item	Recipient bacterium YSF-31	Engineering bacteria YSF31 (pICG)-2T2	pH	4.42	4.42
Flocculence (%)	84.9	88.9	pH	4.42	4.42
Flocculence (%)	84.9	88.9	α-N assimilation ratio (%)	53.4	53.7
Fermentation degree (%)	61.83	62.04	α-N assimilation ratio (%)	53.4	53.7

5, pilot scale (two tons) fermenting experiment

Yeast is inserted the 5mL malt extract medium, cultivated 12 hours for 25 ℃, inoculum size with 10% is inoculated in the 10mL malt extract medium, cultivated 36 hours for 25 ℃, nutrient solution all is connected in the 270mL wort, cultivated 36 hours for 25 ℃, then nutrient solution is inserted the big triangular flask of 2000ml, cultivated 36 hours for 20 ℃, be connected in the Ka Shi jar of 15L, cultivated 36 hours for 20 ℃, bacterium liquid all is connected in two tons of fermentor tanks (the 1600L malt extract medium is housed), 10 ℃ of fermentations, when pol drops to 2.6, cool to 0 ℃ after the storage.

(1) glutathione content and diacetyl content detect

In two tons of fermenting experiments, the content of the gsh of engineering bacteria YSF31 (pICG)-2 is than the height of recipient bacterium YSF-31, and the content of the di-acetyl of engineering bacteria YSF31 (pICG)-2 low than recipient bacterium YSF-31.With lab scale experimental result and hectolitre fermenting experiment result consistent (Figure 13).Among Figure 13, the diacetyl content of recipient bacterium YSF-31 (■); The diacetyl content () of engineering bacteria YSF31 (pICG)-2; Recipient bacterium YSF-31 glutathione content (zero); The glutathione content (●) of engineering bacteria YSF31 (pICG)-2.

(2) anti-aging experiment

The examining report that thiobarbituricacid (TBA) method is measured carbonyl compound shows, the fresh keeping time of the finished wine of producing with engineering bacteria YSF31 (pICG)-2 is about 1.5 times (table 4) of the finished wine fresh keeping time of recipient bacterium YSF-31 production.

The anti-aging experiment of table 4 recipient bacterium YSF-31 and engineering bacteria YSF31 (pICG)-2

Sample	Analysis project and result
	Analysis project and result						TBA	ΔTBA ₁₂	ΔTBA ₂₄	ΔTBA ₃₆	ΔTBA ₄₈	RSV
	1	0.222	0.111	0.205	0.284	0.391	TBA	ΔTBA ₁₂	ΔTBA ₂₄	ΔTBA ₃₆	ΔTBA ₄₈	RSV	237.352
1’	1	0.222	0.111	0.205	0.284	0.391	0.225	0.125	0.252	0.333	0.374	213.844	237.352
1’	2	0.220	0.163	0.322	0.491	0.567	0.225	0.125	0.252	0.333	0.374	213.844	153.065
2’	2	0.220	0.163	0.322	0.491	0.567	0.215	0.171	0.341	0.441	0.608	150.568	153.065

Annotate: 1 is two tons of pilot scale plant bacterium YSF31 (pICG)-2 and parallel sample thereof with 1 ' sample, and 2 and 2 ' is two tons of pilot scale recipient bacterium YSF-31 and parallel sample thereof.Illustrate: the RSV value is big more, and the fresh keeping time of expression finished beer is long more.

(3) after the beer process ageing that recipient bacterium and engineering bacterium fermentation are produced, it is as follows to judge the result:

10 judging panels judge the result and are: think that the beer of engineering bacteria YSF31 (pICG)-2 fermentative production is better than 7 judging panels that have of beer that recipient bacterium YSF-31 produces; Think that the beer of engineering bacteria YSF31 (pICG)-2 fermentative production is same as 1 judging panel that has of beer that recipient bacterium YSF-31 produces; Think that the beer of engineering bacterium fermentation production is inferior to 2 judging panels that have of beer that recipient bacterium YSF-31 produces.

6, the Detection of Stability of engineering bacteria YSF31 (pICG)-2

Engineering bacteria YSF31 (pICG)-2 there is being selective pressure (YEPD+3mmol/L CuSO4) and is not having in the liquid nutrient medium of selective pressure (YEPD) continuous passage and cultivate 120h (24 hours pass once generation) respectively, every day, sampling detected the loss situation of the dna fragmentation that inserts, the result shows, no matter having under the selective pressure condition, still under no selective pressure condition, behind the cultured continuously 120h, 100% engineering bacteria cell has the insertion dna fragmentation, does not lose phenomenon.

By above-mentioned experimental procedure, obtain the industrial beer yeast engineering bacteria YSF31 (pICG)-2 of the low di-acetyl growing amount of homoglutathion content.Beer yeast engineering bacteria YSF31 (pICG)-2 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 05 23rd, 2005, preserving number is CGMCC № 1377.

Claims

1, a kind of beer yeast engineering bacteria is among the encoding gene importing yeast saccharomyces cerevisiae YSF-31 with the gamma-glutamic acid cysteine synthase, the bacterial strain of the low diacetyl content of the homoglutathion content that obtains; 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 5 ' terminal sequence that is selected from the ILV2 gene of 70-705bp, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 3 ' terminal sequence that is selected from the ILV2 gene of 70-539bp; 5 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 1-705 bit base, and 3 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 2983-3522 bit base.

2, beer yeast engineering bacteria according to claim 1 is characterized in that: the GenBank of the aminoacid sequence of described gamma-glutamic acid cysteine synthase number is CAA59393; The GenBank of the nucleotide sequence of described ILV2 gene number is X02549; The nucleotide sequence of described gamma-glutamic acid cysteine synthase encoding gene such as GenBank D90220.

3, beer yeast engineering bacteria according to claim 2, it is characterized in that: 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number 263bp nucleotide fragments for 5 of X02549 ' end 443-705 bit base, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number nucleotide fragments for the 379bp of 5 of X02549 ' end 2983-3361 bit base.

4, according to claim 1,2 or 3 described beer yeast engineering bacterias, it is characterized in that: in the encoding gene that imports described gamma-glutamic acid cysteine synthase, also copper resistant gene C UP1 is together imported yeast saccharomyces cerevisiae YSF-31; The nucleotide sequence of described copper resistant gene C UP1 such as GenBank K02204.

5, beer yeast engineering bacteria according to claim 4 is characterized in that: described beer yeast engineering bacteria is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2, CGMCC № 1377.

6, a kind of method that makes up the described beer yeast engineering bacteria of claim 1, be that the bacterial strain of the low diacetyl content of the homoglutathion content that obtains is beer yeast engineering bacteria among the encoding gene importing yeast saccharomyces cerevisiae YSF-31 with the gamma-glutamic acid cysteine synthase; 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 5 ' terminal sequence that is selected from the ILV2 gene of 70-705bp, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with the nucleotide fragments of the 3 ' terminal sequence that is selected from the ILV2 gene of 70-539bp; 5 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 1-705 bit base, and 3 ' terminal sequence of described ILV2 gene is from the GenBank number nucleotide sequence for 5 of X02549 ' end 2983-3522 bit base.

7, method according to claim 6 is characterized in that: the GenBank of the aminoacid sequence of described gamma-glutamic acid cysteine synthase number is CAA59393; The GenBank of the nucleotide sequence of described ILV2 gene number is X02549; The nucleotide sequence of described gamma-glutamic acid cysteine synthase encoding gene such as GenBank D90220.

8, method according to claim 7, it is characterized in that: 5 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number 263bp nucleotide fragments for 5 of X02549 ' end 443-705 bit base, and 3 of the encoding gene of described gamma-glutamic acid cysteine synthase ' end is connected with from the GenBank number nucleotide fragments for the 379bp of 5 of X02549 ' end 2983-3361 bit base; In the encoding gene that imports described gamma-glutamic acid cysteine synthase, also copper resistant gene C UP1 is together imported yeast saccharomyces cerevisiae YSF-31; GenBank number of the nucleotide sequence of described copper resistant gene C UP1 is K02204.

9, according to claim 6,7 or 8 described methods, it is characterized in that: described beer yeast engineering bacteria is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) YSF31 (pICG)-2, CGMCC № 1377.