CN104131005B - High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain - Google Patents
High-ester-produced saccharomyces cerevisiae strain and method for seamlessly inserting promoter of high-ester-produced saccharomyces cerevisiae strain Download PDFInfo
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Abstract
The invention discloses a high-ester-produced saccharomyces cerevisiae strain and a construction method thereof. The high-ester-produced saccharomyces cerevisiae strain is obtained by accurately inserting a strong promoter PGK1p into the 5' end of a parent yeast strain alcohol acetyltransferase gene (ATF1) ORF, and accurate insertion of the promoter is realized by fusing PCR and yeast integrative plasmid YIplac211. According to the constructed high-ester-produced yeast, the mRNA expression quantity of the ATF1 and alcohol acetyltransferase yield of the high-ester-produced saccharomyces cerevisiae strain are respectively 40 times and 5 times those of an original strain without influencing other fermenting performances; the corn thick mash is fermented for 4 days, and the total ester yield is 2.7 times that of a parent strain, and the yield of ethyl acetate is 3.1 times that of the parent strain. No exogenous gene is remained on the genome in the constructed saccharomyces cerevisiae, so that the saccharomyces cerevisiae can be used in industrial production safely.
Description
Technical field：
The present invention relates to genetic engineering field, it is specifically related to one plant of high ester yield Wine brewing yeast strain and its promoter is seamless
Insertion method.
Background technology：
Ester, as one of most fragrance component of content in Chinese liquor, has fragrance and the taste of fruit, imparts
The fruit aroma that Chinese liquor enriches, plays an important role in terms of the style and quality of Chinese liquor.With acetic acid in China's famous liquor
(Fructus Mali pumilae is fragrant for ethyl ester (fruital and Fructus Ananadis comosi are fragrant), ethyl hexanoate (happy fragrant, brandy perfume and fruital), ethyl lactate and ethyl n-butyrate.
And fruital) etc. based on acetass, reach more than the 80% of total ester content.Wherein, ethyl acetate is delicate fragrance type, rice-fragrant type, medicine odor type,
The main body flavor component of the Chinese liquor such as Laobaigan-flavour.Nordstrom carried out a series of research it was demonstrated that ester to the formation of esters
The living things catalysis synthesis in brewing yeast cell mainly by alcohol acyltransferase or other Lipase absobed enzyme.Yoshioka and
Hashimoto research confirms, alcohol acetyltransferase (AATase) is the synthesis of the acetate esters such as ethyl acetate, isoamyl acetate
Key enzyme, Fujii et al. has carried out refined and examining order to this enzyme, and result has obtained three kinds of different AATase：
AATaseI, Lg-AATaseI and AATaseII, are encoded by ATF1, Lg-ATF1 and ATF2 respectively.ATF1 and ATF2 is in wine brewing ferment
All exist in mother and beer yeast, but Lg-ATF1 exists only in beer yeast.
Source for esters and regulation, Lilly etc. passes through to express ATF1 and ATF2 gene, acetic acid in wine yeast
The content of ethyl ester, isoamyl acetate, phenethyl acetate and Exceed 600 all accordingly increases.And the effect of ATF1 gene is more than ATF2
Gene.Wherein, the overexpression of ATF1 makes the concentration of ethyl acetate, isoamyl acetate and phenethyl acetate increased 10 respectively
Times, 3.8-12 times and 2-10 times.Verstrepen etc. is lacked by the research discovery to beer yeast, ATF1 and ATF2 Gene Double
Yeast strain of beer (atf1 △ atf2 △) does not almost have isoamyl acetate to generate (the 4% of≤wild Yeast strain of beer).With
When confirm ATF1 gene effect be more than ATF2 gene.In China, Zhang Cuiying etc. passes through mistake in beer yeast and yellow wine yeast
Expression ATF1, ethyl acetate yield has been respectively increased 11.5 and 20.9 times.For by improving coding alcohol acyl transferase gene
(ATF1) expression is improving not yet reporting containing quantifier elimination of ester in Chinese liquor.
Changing promoter intensity is to realize the important channel of gene expression regulation.Traditional promoter one step insertion, inserts
Enter fragment to be made up of the flanking sequence at marker gene and two ends, by the homology between flanking sequence and insertion point two sequencing row
The insertion of promoter is realized in restructuring.Because marker gene can not be reused and low recombination efficiency, the application of a step insertion
It is restricted.The system of recombinase-mediated is widely used in gene knockout and promoter insertion, such as Cre/ due to high efficiency
Loxp system etc..Carry the promoter insertion element in recombinase site by transformation and selection labelling both sides in yeast, a step is whole
Close the insertion realizing genes of interest, then the plasmid of one coding recombinase of conversion, to realize the removal of resistance marker.In industry
In the transformation of yeast, this system has very high efficiency, but it can leave exogenous array (single loxp site), and
When carrying out polygenes operation, these sites staying increase the probability that chromosome rearrangement occurs.Fusion DNA vaccine can be realized
Genetic fragment seamless spliced, and do not selected to be limited by limited restriction enzyme site, thus avoiding traditional plasmid construction mistake
The enzyme action repeating in journey and attended operation.Strong promoter PGK1p (yeast phosphoglycerate kinases, phosphoglycerate
Kinase) it is conventional constitutive promoter in saccharomyces cerevisiae.Therefore, the two step integration sides that the present invention is mediated by fusion DNA vaccine
Method achieves the 5 ' ends that PGK1p is accurately inserted into genes of interest ATF1, plasmid no any exogenous gene residual after ejecting, structure
Commercial production can be used safely in from clone's high ester yield Wine brewing yeast strain.Two step integration methods of fusion DNA vaccine mediation simultaneously can be wide
General be applied to yeast and the regulation and control of other Microbial promoters, promote the development that gene is accurately modified.
Content of the invention：
Present invention solves the technical problem that one of there is provided one plant of high ester yield Wine brewing yeast strain.
Described high ester yield yeast strain is that PGK1p promoter is accurately inserted into the yeast strain-saccharomyces cerevisiae that sets out
5 ' end gained of the alcohol acetyltransferase encoding gene (ATF1) of (Saccharomyces cerevisiae) CICC32315.
Its Gene ID of described ATF1 gene is：854559, SEQ ID NO in nucleotide sequence such as sequence table:Shown in 1；
Its Gene ID of described promoter PGK1p is：850370, SEQ ID NO in nucleotide sequence such as sequence table:2 institutes
Show.
Another technical problem solved by the invention is to provide a kind of high ester yield Wine brewing yeast strain promoter seamless slotting
Enter construction method, comprise the steps：
(1) PCR obtains mutation ura3 fragment；This fragment is imported and sets out in yeast strain, obtain deficient strain；
(2) with the genome of bacterial strain CICC32315 as template, PCR expands strong promoter PGK1p respectively, in insertion point
Trip and downstream homology arm sequence, and by fusion DNA vaccine, three fragments are seamlessly connected, it is cloned into yeast integration matter by merging fragment
On grain, obtain and integrate overexpression plasmid；
(3) in the upstream homology arm sequence integrating overexpression plasmid, select single restriction enzyme site, plasmid enzyme restriction is linear
Change；
(4) linear plasmid that step (3) obtains is imported the deficient strain in (1), without uracil (uracil)
Yeast synthetic medium screening, obtain the first step integrate restructuring yeast mutant；
(5) by the mutant sifting out in step (4), through 5- fluororotic acid (5-FOA) synthetic medium flat board, reversely screening obtains
Obtain the yeast mutant that second step integrates restructuring；
(6) with the genome of bacterial strain CICC32315 as template, PCR obtains normal URA3 genetic fragment, is conducted into
In step (5), two steps are integrated in the yeast mutant of restructuring, with the ura3 marker gene of back mutation, obtain final product described high ester yield
Wine brewing yeast strain.
Described recombinant bacterial strain can be built by said method.Concrete operation method involved by each step is with reference to existing document report
Road, such as Joseph Sambrook etc.,《Molecular Cloning:A Laboratory guide》The second edition, Science Press, 1995.
Wine brewing yeast strain of the present invention can be applicable in liquor production.
Saccharomyces cerevisiae engineered yeast strain of the present invention, in the case that other fermenting properties are unaffected, mRNA expresses
Amount and alcohol acetyltransferase are 40 and 5 times of starting strain respectively；Semen Maydiss thick mash fermentation 4 days, total ester content is parent strain
2.7 times, the yield of ethyl acetate is 3.1 times of parent strain.
Beneficial effect：
1st, the present invention provides a kind of Wine brewing yeast strain of high ester yield, overcomes common saccharomyces cerevisiae and draws because ester yields poorly
The inharmonic problem of fragrance rising.
2nd, the Wine brewing yeast strain of this high ester yield is on the premise of keeping excellent fermenting property, and alcohol acetyltransferase is compiled
The expression of code Gene A TF1 significantly improves, and has established theoretical basiss for producing the Chinese liquor with excellent flavor.Have important
Market value.Promote the production of the Chinese liquor of excellent organoleptic quality, simultaneously for research ethyl acetate to liquor flavor harmony
Impact is laid a good foundation.
3rd, the efficient promoter seamless insertion method that the present invention provides, it is to avoid yeast tradition gene promoter inserted
In journey selection markers residual problem, meet from clone condition, can safety for commercial production.And this method is in base
Because accurate modification aspect has broad application prospects.
Brief description：
Fig. 1 is the structure schematic flow sheet integrating overexpression plasmid YIplac211-UPD
Fig. 2 plasmid YIplac211-UPD integrates restructuring schematic flow sheet with two steps of Yeast genome
Fig. 3 is the electrophoresis proof diagram of strains A Y14-u3-Y occurring the first step to integrate restructuring：
Swimming lane M is DNA maker；
Swimming lane 1,2,3 is using YIP-UPD-F and YIP-UPD-R PCR the result, and swimming lane 1,2,3 template is respectively
AY14-u3, AY14-u3-Y and plasmid YIP-UPD；
Swimming lane 4,5,6 is using primer pair YIP-UD-F and YIP-UD-R PCR the result, and template is followed successively by AY14-
U3, AY14-u3-Y and plasmid YIP-UPD；
Fig. 4 is the electrophoresis proof diagram of strains A Y14-u3-P occurring second step to integrate restructuring：
Swimming lane M is DNA maker；
Swimming lane 1,2 is the PCR the result using primer pair UD-F and UD-R, and swimming lane 1 template is AY14-u3, swimming lane 2 mould
Plate is AY14-u3-P；
Swimming lane 3,4 is the PCR the result using primer pair UP-F and UP-R, and swimming lane 3 template is AY14-u3, swimming lane 4 mould
Plate is AY14-u3-P；
Swimming lane 5,6 is the PCR the result using primer pair PD-F and PD-R, and swimming lane 5 template is AY14-u3, swimming lane 6 mould
Plate is AY14-u3-P；
Fig. 5 is the sequencing result comparison chart of starting strain and mutant target position
Fig. 6 is mrna expression amount and the alcohol acetyltransferase enzyme activity of strains A TF1
Fig. 7 is the yield of bacterial strain ester
Specific embodiment：
Describe the present invention below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, and the unrestricted present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, the material component in these embodiments and consumption are carried out various changes or are changed
Belong to protection scope of the present invention.
Embodiment 1：The structure of high ester yield saccharomyces cerevisiae
Starting strain CICC32315 used by this example.Described escherichia coli DH5a is purchased from Takara company.Described YPD
Culture medium is general complete medium, and described yeast synthetic medium (SD) composition is：2% glucose, 0.67%YNB,
0.13% ispol not containing uracil, solid medium contains 2% import agar powder.
According to the Yeast genome data in Genebank and integrated plasmid sequence, devise following primer.
Used primer in table 1. the present embodiment
Note：Lowercase letter restriction enzyme site, underscore represents the overlapping sequence of fusion DNA vaccine the primer.
(1) structure of the saccharomyces cerevisiae AY14-u3 with defective ura3 gene
PCR amplification mutation ura3 fragment, after test kit reclaims, fragment is imported by lithium acetate chemical transformation
To setting out in saccharomyces cerevisiae CICC32315, by being mutated the homologous recombination between ura3 and normal URA3, realize starting strain
URA3 gene mutation.Bacteria suspension after conversion, coats and has added 5- fluororotic acid (5-FOA) and uracil (uracil) yeast
On synthetic medium (SD) flat board, 30 DEG C of culture 48 h, obtain the saccharomyces cerevisiae AY14-u3 with defective ura3 gene.Obtain
Mutant determine through phenotype checking correct.
(2) integrate the structure knocking out plasmid YIplac211-UPD
First, with the genome of saccharomyces cerevisiae CICC32315 as template, obtained by PCR using primer UF and UR respectively
The gene order of insertion point upstream 1048bp, PCR reaction condition：95℃5 min；94 DEG C of 40 s, 54 DEG C of 1 min, 72 DEG C 140
S, 30 circulations；72 DEG C of 10 min, product is named as U；
Primer PF and PR amplification target promoter sequence (1479bp), PCR reaction condition：95℃5 min；94 DEG C of 40 s, 55
DEG C 1 min30s, 72 DEG C of 140 s, 30 circulations, 72 DEG C of 10 min, product is named as P；
Primer DF and DR expands the gene order of insertion point downstream 1046bp, PCR reaction condition：95℃5 min；94℃
40 s, 54 DEG C of 1 min, 72 DEG C of 140 s, 30 circulations；72 DEG C of 10 min, product is named as D；
Then, with U, the mixture of P and D is template, adds primer UF and DR to carry out fusion DNA vaccine, obtains seamless fusion piece
Section, fusion DNA vaccine reaction condition, first, with U after purification, the mixture of P and D is template, is added without primer, 50 DEG C of 45s, 72
DEG C 2 min, 10 circulations.PCR primer template as fusion DNA vaccine after cutting glue reclaim (test kit), with UF and DR for drawing
Thing, PCR reaction condition：95℃5 min；94 DEG C of 40 s, 57 DEG C of 1 min, 72 DEG C of 140 s, 30 circulations；72 DEG C of 10 min, warp
Cross after cutting glue reclaim (test kit), with BamHI and KpnI double digestion, be finally subcloned into the corresponding of integrated plasmid YIplac211
In restriction enzyme site, integrate knockout plasmid YIplac211-UPD and successfully construct, build flow process as shown in Figure 1.
Above-mentioned fusion DNA vaccine method be known in the art using the primer with spacer end, formed and there is overlapping sequence PCR
Product, is extended by PCR primer overlapping sequence, thus the method that any DNA fragment is coupled together, this technology does not need inscribe
So that it may realize the Ligation in vitro of DNA fragmentation, this makes integrated plasmid integrate weight through two steps for the process of the digestion of enzyme and ligase
After group, Yeast genome will not remain external source restriction enzyme site.
(3) the seamless insertion of promoter PGK1p
NruI enzyme action integrated plasmid YIplac211-UPD；With lithium acetate transformation method by linearizing integrated plasmid
YIplac211-UPD imports in defective yeast strains A Y14-u3, integrates after restructuring through two steps, obtains constitutive promoter
PGK1p is accurately inserted into the saccharomyces cerevisiae at ATF15 ' end, and two steps integrate regrouping process such as Fig. 2.
The first step integrates the generation of restructuring, is because the analogous parts of the linearization plasmid importing and Yeast genome occur
Integrate, thus whole plasmid is brought into genome.Bacteria suspension after conversion, coats the yeast synthetic medium without uracil
On flat board, 30 DEG C of culture 48 h, obtain the yeast strain AY14-u3-Y occurring the first step to integrate restructuring.List obtained by selecting
Bacterium colony, using YIP-UPD-F and YIP-UPD-R, YIP-UD-F and YIP-UD-R is checking primer, carries out bacterium colony PCR screening.
PCR primer agarose gel electrophoresiies result is shown in Fig. 3.Transformant shows with comparing result, and linearizing plasmid integration is to mesh
Site.
Through the restructuring yeast strains AY14-u3-Y screening and identify, a ring is taken to be connected in 5ml liquid YPD medium, 30
After 200rpm vibrates 24 h at DEG C, dilute 10 times of yeast synthetic medium flat boards coated containing 5- fluororotic acid and uracil
On, 30 DEG C of culture 48 h, obtain the yeast strain occurring second step to integrate restructuring.As shown in Fig. 2 after second step recombination and integration,
Two kinds of results occur：One, if integrated between the repetitive sequence that formed of upstream homologous sequence (U), yeast reverts back to out
Send out strains A Y14-u3；Two, if integrated between the repetitive sequence that formed of downstream homologous sequence (D), in the middle of all sequences bullet
Go out, realize the accurate insertion of PGK1p, any exogenous gene is not introduced on target position simultaneously.Single bacterium colony obtained by selecting, adopts
UD-F and UD-R, UP-F and UP-R, PD-F and PD-R tri-, to primer, carries out bacterium colony PCR, filters out turning of the accurate insertion of PGK1p
Beggar.PCR primer agarose gel electrophoresiies result is shown in Fig. 4.Transformant shows with comparing result, and promoter PGK1p is inserted
To before ATF1.
(4) integrate the reply that recombinant bacterial strain AY14-u3-P is mutated ura3 gene
Method described in same step (1), using primer pair URA3-F and URA3-R, expands normal URA3 genetic fragment, leads to
Cross chemical conversion, to reply the ura3 gene of AY14-u3-P bacterial strain mutation, finally construct and do not remain any exogenous gene sequence
Promoter accurately insert strains A Y14-P.
In order to verify the sequence situation of promoter on position further, extract the genome of strains A Y14-P, using drawing
Thing PS-F and PX-R enters performing PCR amplification, and the fragment obtaining 1829bp is sent in Huada gene company sequencing, result such as Fig. 5.Knock out
Context contrast is it may be determined that the seamless 5 ' ends being inserted into ATF1 of PGK1p successfully construct.
Embodiment 2：The fermenting experiment of high ester yield saccharomyces cerevisiae
(1) fermenting property of high ester yield saccharomyces cerevisiae
Monoploid parent and its corresponding recombinant bacterial strain are carried out Semen Maydiss thick mash fermentation experiment, fermentation technology road respectively simultaneously
Line：Semen Maydis powder → immersion → liquefaction → saccharifying → cool down → connect bacterium → fermentation → steaming wine → testing index；
Process conditions：
Soaking conditionses：60～70 DEG C, impregnate 20 min；Liquefaction condition：85～90 DEG C, add Thermostable α-Amylase, liquid
Change 90 min；
Saccharifying condition：55～60 DEG C, add saccharifying enzyme, saccharifying 20 min；
Dispensing：Semen Maydis powder 60 g, water 180 mL, Thermostable α-Amylase 2 × 105U/mL, 30 μ L, saccharifying enzyme 200 U/
ML, 90 μ L, 2.5 × 103U/mL acid protease, 1.2 mL；Nutritive salt 1 mL (MgSO4150 g/L、KH2PO475 g/L, urine
Plain 81 g/L, filter, 4 DEG C of preservations)；Inoculum concentration：7.5%, 30 DEG C, ferment 4 days；
Fermentation took 100 mL mash after 4 days, plus 100 mL water, steamed 100 mL wine samples；
Fermenting property index determining is carried out to wine sample, the results are shown in Table 2.As shown in the results, described engineered strain and the bacterium that sets out
Fermenting property (the CO of strain2Weightlessness, wine degree, residual sugar) basically identical, the amount that titrimetry records total ester reaches 1.46 g/L, is to set out
2.7 times of bacterial strain.
Table 2 fermenting performance compares
Note：Shown data is the meansigma methodss of three parallel test results.
(2) mrna expression amount of described high ester yield saccharomyces cerevisiae ATF1 and alcohol acetyltransferase enzyme activity
Measure the mrna expression amount of ATF1 with RT-qPCR.Extract examination using Yeast RNAiso Kit yeast Total RNA
Agent box extracts total serum IgE, carries out the synthesis of cDNA using TIANScript RT Kit (Tiangeng is biochemical) PCR kit for fluorescence quantitative,
Reverse transcription reaction system：Total RNA, 1 μ g；Oligo dT (10 μM), 2 μ l；DNTP (2.5 mM), 2 μ l；RNase-
Free ddH2O polishing is to 14.5 μ l；70 DEG C, water-bath 5 min, rear ice bath 2 min；Add：5×First-Strand
Buffer, 4 μ l；RNasin (40 U/ μ l), 0.5 μ l；TIANScript M-MLV (200 U/ μ l), 1 μ l；Continue reverse transcription
Program：42 DEG C, 50 min；95 DEG C, 5 min.Real-time PCR reaction system：SYBR FAST Qpcr Kit Mast er
Mix (2 ×) Universal, 5 μ l；Primer F (10 μM), 0.2 μ l；Primer R (10 μM), 0.2 μ l；CDNA, 1 μ
l；ROX correction dye, 0.2 μ l；H2O, polishing to 10 μ l.PCR reaction condition：95℃3 min；95 DEG C of 3 s, 60 DEG C of 120 s,
30 circulations；95 DEG C of 15 s, 60 DEG C of 15 s, 95 DEG C of 15 s.
Measure the vigor of the produced alcohol acetyltransferase of each bacterial strain using substrate reactions method.Add in 10 mL centrifuge tubes
A certain amount of fresh yeast thalline, 1.0 mL Tris-HCl, 20 μ L ethanol (chromatograph alcohol) and 20 μ L 10 mg/mL acetylcoenzyme
A, will be centrifuged the seal of tube with sealed membrane after mixing, and put in 25 DEG C of rotating speeds 150 r/min shaking table and reacted.Question response is certain
After time (generally 2～6 h), 12000 r/min are centrifuged 5 min, take supernatant gas chromatogram measured reaction acetic acid product second
The growing amount of ester.Alcohol acetyltransferase vigor defines：At 25 DEG C, 1 g saccharomyces cerevisiae thalline 1 h catalysis ethanol and acetyl are auxiliary
Enzyme A generates the amount (μm ol) of ethyl acetate.
The mrna expression amount of described high ester yield saccharomyces cerevisiae ATF1 and alcohol acetyltransferase enzyme activity are shown in Fig. 6.ATF1's
Mrna expression amount and alcohol acetyltransferase yield are 40 and 5 times of starting strain respectively.
(3) gas chromatography measures the yield of ester and alcohol
Gas chromatograph：Agilent 7890C；Chromatographic column：Chinese liquor dedicated columns, AT.LZP-930,230 DEG C, 50 m × 320
μm×1μm；Detector：Fid detector, detector temperature：200℃；Carrier gas：High Purity Nitrogen, flow velocity 5 mL/min；Testing conditions：
Temperature programming, 50 DEG C of holding 8 min, 5 DEG C/min are raised to 120 DEG C, keep 8 min；Injector temperature：200℃；Sample size：1.0
μL；Shunting mode：Shunting, split ratio is 5：1；Result such as Fig. 7, the yield of isobutyl acetate and isoamyl acetate does not significantly increase
Plus, the yield of ethyl acetate is 3.1 times of parent strain.
Claims (5)
1. the Wine brewing yeast strain of one plant of high ester yield, is, in yeast starting strain, strong promoter PGK1p is accurately inserted alcohol second
5 ' the ends of acyl transferase gene ATF1 obtain it is characterised in that described starting strain is saccharomyces cerevisiae (Saccharomyces
Cerevisiae) CICC32315, promoter is that the seamless insertion of the two step integration method being mediated by fusion DNA vaccine is realized, including such as
Lower step：
(1) PCR obtains mutation ura3 fragment；This fragment is imported in starting strain, obtains deficient strain；
(2) with the genome of bacterial strain CICC 32315 as template, PCR expands strong promoter PGK1p respectively, insertion point upstream and
Downstream homology arm sequence, and by fusion DNA vaccine, three fragments are seamlessly connected, it is cloned into Yeast Integrating plasmids by merging fragment
On, obtain and integrate overexpression plasmid；SEQ ID NO in described upstream homology arm sequence such as sequence table:Shown in 3, downstream homology arm
SEQ ID NO in sequence such as sequence table:Shown in 4；
(3) in the upstream homology arm sequence integrating overexpression plasmid, select single restriction enzyme site, by plasmid enzyme restriction linearisation；
(4) linear plasmid that step (3) obtains is imported the deficient strain in (1), in the yeast synthesis training without uracil
Foster base screening, obtains the yeast mutant that the first step integrates restructuring；
(5) mutant sifting out in step (4) is reversely screened acquisition second step through 5- fluororotic acid synthetic medium and integrate weight
The yeast mutant of group；
(6) with the genome of bacterial strain CICC 32315 as template, PCR obtains normal URA3 genetic fragment, is conducted into step
Suddenly in (5), two steps are integrated in the yeast mutant of restructuring, with the ura3 marker gene of back mutation, obtain final product the wine of described high ester yield
Brewer yeast bacterial strain；
SEQ ID NO in described its nucleotide sequence of ATF1 gene such as sequence table:Shown in 1；Described its nucleoside of strong promoter PGK1p
SEQ ID NO in acid sequence such as sequence table:Shown in 2.
2. the Wine brewing yeast strain of one plant of high ester yield as claimed in claim 1 is it is characterised in that described Yeast Integrating plasmids are
YIplac211 plasmid.
3. the Wine brewing yeast strain of one plant of high ester yield as claimed in claim 1 is it is characterised in that described single restriction enzyme site is
NruΙ.
4. the Wine brewing yeast strain of one plant of high ester yield as claimed in claim 1 is it is characterised in that the importing of described linear plasmid lacks
The method of swaged bacterial strain is lithium acetate transformation method.
5. application in liquor production for the high ester yield Wine brewing yeast strain as claimed in claim 1.
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| CN107937294A (en) * | 2017-06-14 | 2018-04-20 | 天津科技大学 | The Wine brewing yeast strain and its construction method of one plant of appropriate production ethyl acetate |
| CN107858368A (en) * | 2017-06-30 | 2018-03-30 | 天津科技大学 | The Wine brewing yeast strain and its construction method of one plant of appropriate production higher alcohol |
| CN108384809A (en) * | 2018-03-16 | 2018-08-10 | 亳州学院 | A kind of answering method of polyploid Industrial yeast strain strain URA3 gene defection types |
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| CN110760453B (en) * | 2019-10-23 | 2021-07-30 | 波顿（上海）生物技术有限公司 | Genetically engineered yeast strain for high-yield phenylethyl acetate, construction method thereof and method for producing phenylethyl acetate |
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| ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究;刘玉兰等;《中国酿造》;20121231;第31卷(第1期);第151-153页 * |
| EF059515.1;Hu et al.;《GenBank》;20070424;1-2 * |
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